Glucagon like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion proteins and uses thereof

Abstract

To provide a GLP1 analogue or derivative thereof and / or a GDF15 analogue or derivative thereof with improved metabolic stability and pharmacokinetic profile relative to GLP1 or GDF15, respectively.SOLUTION: A glucagon-like peptide-1 (GLP1) / growth differentiation factor 15 (GDF15) fusion protein is provided, wherein the GLP1-GDF15 fusion protein comprises a GLP1 peptide, a first linker peptide, a serum albumin protein, a second linker peptide, and a GDF15 protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 62 / 784,603, filed October 22, 2018. Priority is claimed, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention generally relates to novel glucagon-like peptide-1 (GLP1)-growth differentiation factor 15 (GIF1)-binding proteins. The present invention relates to a GLP1-GDF15 fusion protein. The present invention also relates to pharmaceutical compositions and methods of use thereof. The novel GLP1-GDF15 fusion protein is useful for treating, among other things, obesity, type 2 diabetes, , metabolic syndrome, insulin resistance, and dyslipidemia. It is useful for preventing, treating, or ameliorating.

[0003] (Reference to electronically submitted sequence listing) This application is filed under the file name "PRD3474 Sequence Listing" and has a creation date of October 14, 2019. , submitted electronically via EFS-Web as an ASCII sequence listing, 327k The sequence listing submitted via EFS-Web includes a sequence listing having a size of 1.25 MB. No. 6,399,413, filed on Oct. 23, 2003, and is incorporated herein by reference in its entirety. [Background technology]

[0004] GDF15, a member of the TGFβ family, is expressed in plasma as a 25 kDa homodimer. It circulates in the brain and exerts its biological functions through interaction with the brainstem-expressed receptor GFRAL. It is a secreted protein that 150-7(2017), Yang et al., Nat Med.23:1158- 66(2017), Hsu et al., Nature 550:255-9(201 7), Emmerson et al., Nat Med 23:1215-9(201 7) Plasma concentrations of GDF15 range from 150 to 1150 pg / mL in most individuals. (Tsai et al.,J Cachexia Sarcopenia Musc le.3:239-43(2012)). High plasma concentrations of GDF15 are a key factor in the pathogenesis of cancer. It is associated with weight loss due to anorexia and cachexia, as well as weight loss in renal and cardiac failure. Furthermore, GDF15 suppresses weight loss after Roux-en-Y gastric bypass (RYGB) surgery. It is increased in patients experiencing 57:309-16(2011)).

[0005] The correlation between weight loss and GDF15 is conserved in rodents. Overexpression of this gene resulted in reduced food intake, reduced body weight, and impaired the survival of mice on a high-fat diet. Protects against obesity, fatty liver, and glucose intolerance (Baek et al., Gastroe Terology 131:1553-60(2006), Johnen et al. ., Nat Med 13:1333-40 (2007), Chrysovergis et al., Int J Obesity 38:1555-64(2014), Ma cia et al., PloS One 7:e34868(2012), Jones et al.,Cell Reports 22:1522-30(2018),Xi ong et al., Sci Trans Med 9:412(2017)). GD Xenografts of prostate tumor cells transfected with F15 also reduced food intake and body weight. (Johnen et al., Nat Med 13:1333-40(20 07) Conversely, numerous researchers have shown that mice lacking GDF15 gain more weight. reported that mice with IFN-γ-γ 1 (IFN-γ) had a greater fat mass than wild-type animals (Strelau et al. al., J Neurosci 29:13640-8(2009), Casanov as et al.,Haematologica 98:444-7(2013),B onaterra et al.,J Amer Heart Assoc 1:e00 2550(2012),Tsai et al.,PloS one.8:e55174 (2013)).

[0006] Pharmacologically administered GDF15 reduces energy intake and thereby weight loss The potential to induce inflammatory bowel disease has been demonstrated in mice, rats, and monkeys. Non-obese mice treated with 15 eat less and lose weight (Johnen et al. al., Nat Med 13:1333-40(2007), Hsu et al. ,Nature 550:255-9(2017),Mullican et al., Nat Med 23:1150-7(2017),Tsai et al.,Int J Obesity 42:561-71(2018)). Reduction in food intake and body weight was This was also observed in genetically and diet-induced obese rat and mouse models after administration of GDF15. (Johnen et al., Nat Med 13:1333-40 (2007 ), Hsu et al., Nature 550:255-9(2017), Mull ican et al., Nat Med 23:1150-7(2017), Yang et al., Nat Med 23:1158-66(2017), Tsai et al. al.,Int J Obesity 42:561-71(2018),Xiong et al.,Sci Trans Med 9:412(2017)). Diet-induced GDF15 administration-mediated weight loss in obese mice is associated with enhanced glucose homeostasis and These effects include metabolic improvements, including lowering plasma triglycerides and cholesterol. Six weeks of daily treatment with recombinant human GDF15 in spontaneously obese non-human primates The regimen reduced food intake, body weight, and plasma triglyceride concentrations and improved glucose tolerance. This also applies to higher species (Xiong et al., Sci Transl. s Med 9:412(2017)). Furthermore, the half-life of recombinant GDF15 was It has previously been demonstrated that the protein can be extended by fusion with albumin (HSA), and It is predicted that this will be a suitable treatment for once-weekly administration (Mullican et al. , Nat Med 23:1150-7(2017) and U.S. Patent Publication No. 2017 / 03 No. 27560).

[0007] GLP1 is a neurotransmitter derived from enteroendocrine cells that also reduces food intake and leads to weight loss. Its function is via the GLP1 receptor (GLP1R) in the central nervous system. This ligand / receptor interaction in peripheral tissues is mediated by interactions with Enhancement of glucose-stimulated insulin secretion, inhibition of glucagon release, and delayed gastric emptying. (Druker et al. Cell Met 27 :740-56(2018)). Taking advantage of all these biological effects, GLP1R Agonists improve glucose homeostasis and promote weight loss in humans. Administration of GLP1R agonists has been shown to significantly improve cardiovascular outcomes in diabetic patients. Although it has been reported, the exact mechanism has not yet been determined (Lim et al., 2003). nds Endocrinol Metab.29:238-48(2018)). GL P1R agonist agents are native human GLPs with modifications to prevent enzymatic cleavage. 1 or exendin-4, a peptide isolated from the saliva of the Gila monster. GLP1R agonists are peptide sequences based on lipidation, antibody Fc or human It can be delivered via a platform that extends half-life, such as high serum albumin (HSA). (Cheang and Moyle, Chem Med Chem 13:662- 71(2018)).

[0008] Thus, improved metabolic stability and drug safety compared to GLP1 or GDF15, respectively. GLP1 analogs or derivatives thereof and / or GDF15 analogs having kinetic profiles It would be desirable to obtain analogs or derivatives thereof. Such derivatives have a longer duration of action. The present invention provides a method for the regulation of GDF1 and GDF15 receptors having a long duration, and the method includes the steps of: It would be suitable as a therapeutic agent for a subject in need thereof.

[0009] The foregoing discussion has been presented solely to provide a better understanding of the nature of the problems facing the art. and such references are not admitted to constitute "Prior Art" to this application. should not be interpreted as Summary of the Invention [Means for solving the problem]

[0010] In one general aspect, the invention provides novel glucagon-like peptide-1 (GLP1)-proliferative peptides. GLP1-GDF15 fusion protein The quality of life of patients with glaucoma is improved by regulating GLP1R and / or GDF15R (GFRAL).

[0011] As used herein, glucagon-like peptide-1 (GLP1)-growth differentiation factor 15 (GDF1 5) A fusion protein is provided. The GLP1-GDF15 fusion protein is a GLP1 peptide. a peptide, a first linker peptide, a serum albumin protein, and a second linker peptide. , and GDF15 protein.

[0012] In certain embodiments, the GLP1 peptide is selected from the group consisting of SEQ ID NOs: 1-4. Contains the amino acid sequence.

[0013] In certain embodiments, the first linker peptide is selected from the group consisting of SEQ ID NOs: 5-25. The amino acid sequence of the selected polypeptide is

[0014] In certain embodiments, the serum albumin protein is selected from SEQ ID NO:26 and SEQ ID NO:27. The amino acid sequence may be selected from the group consisting of:

[0015] In certain embodiments, the second linker peptide is selected from the group consisting of SEQ ID NOs: 28-30. Contains a selected amino acid sequence.

[0016] In certain embodiments, the GDF15 protein is selected from SEQ ID NO:31 or SEQ ID NO:32. The amino acid sequence of the selected polypeptide is

[0017] In certain embodiments, the GLP1-GDF15 fusion protein is selected from the group consisting of SEQ ID NOs: 33-74 and and 84.

[0018] Also provided is an isolated nucleic acid encoding a GLP1-GDF15 fusion protein of the invention. Also provided is a vector comprising the isolated nucleic acid of the invention. A host cell comprising the same is also provided.

[0019] A pharmaceutical composition comprising the GLP1-GDF15 fusion protein of the present invention and a pharma- ceutical acceptable carrier. Compositions are also provided.

[0020] Methods of treating or preventing a disease or disorder in a subject in need thereof are also provided. The disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, , insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia , hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, and atherosclerosis , diabetic nephropathy, and uncontrolled cholesterol and / or lipid levels Other cardiovascular risk factors, such as hypertension and cardiovascular risk factors associated with osteoporosis, inflammation, non-alcoholic fatty liver disease, and alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), renal disease, and eczema, and the method comprises administering to the patient an effective amount of the pharmaceutical composition of the present invention. Administering to a subject in need thereof.

[0021] Also provided is a method of reducing food intake in a subject in need thereof, the method comprising: The method includes administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present invention.

[0022] Also provided are methods of modulating GLP1 receptor activity in a subject in need thereof. The method comprises administering to a subject in need thereof an effective amount of the pharmaceutical composition of the present invention. Includes:

[0023] The present invention relates to a method for modulating GDF15 receptor (GFRAL) activity in a subject in need thereof. Also provided is a method for carrying out the method comprising administering an effective amount of the pharmaceutical composition of the present invention to a patient in need thereof. Administering the compound to a subject.

[0024] In certain embodiments, the pharmaceutical composition is administered by injection.

[0025] The GLP1-GDF15 fusion protein of the invention, the isolated nucleic acid of the invention, and / or the Kits containing the vector are also provided. The kits may further include, for example, an injection device. This can be done.

[0026] Methods for producing pharmaceutical compositions comprising the GLP1-GDF15 fusion proteins of the invention are also provided. The method includes combining a GLP1-GDF15 fusion protein with a pharma- ceutical acceptable carrier. Combining these to obtain a pharmaceutical composition.

[0027] Methods for producing the GLP1-GDF15 fusion proteins of the invention are also provided. The GLP1-GDF15 fusion protein was generated under conditions that produced the GLP1-GDF15 fusion protein. Culturing cells containing a nucleic acid encoding a GLP1-GDF15 fusion protein; and recovering the protein from the cell or culture. [Brief description of the drawings]

[0028] The above summary and the following detailed description of preferred embodiments of the present application should be taken in conjunction with the accompanying drawings. The present application will be better understood upon reading the accompanying drawings. It should be understood that there is no limitation to the precise embodiment. [Figure 1] 1 shows a graph demonstrating the percentage of body weight change (from day 0) in DIO mice receiving daily liraglutide (lira) and / or alternate day administration of HSA-GDF15 (SEQ ID NO: 81). [Diagram 2] Graph demonstrating the mean percent weight change (from day 0) in DIO mice receiving daily treatment with GLP1-GSA-GDF15 (SEQ ID NO: 72), GLP1-GSA-GDF15(I89R), (SEQ ID NO: 73, I89R mutation abolishes GDF15 activity), GLP1(9-39)-GSA-GDF15 (SEQ ID NO: 74, abolishes GLP1 activity), or GSA-GDF15 (SEQ ID NO: 75). Dulaglutide treatment served as the positive control and vehicle treatment served as the negative control. ±SEM, n=8. [Diagram 3] Schematic diagram of the GLP1-GDF15 fusion protein. [Figure 4] 1 shows a graph demonstrating the effect of subcutaneous administration of SEQ ID NOs: 45, 49, 50, 33, 46, and 44 on food intake in C57BL / 6 mice. Data is presented as the percentage change in food intake compared to pre-dose food intake at 24 hours. [Diagram 5] 1 shows a graph demonstrating plasma concentrations of GLP1-GDF15 fusion proteins (SEQ ID NOs: 45, 49, 50, 33, 46, and 44) ​​24 hours after administration in mice as determined by immunoaffinity capture-trypsin digestion-LC-MS / MS analysis. [Figure 6] Graphs demonstrating the effect of subcutaneous administration of SEQ ID NOs: 45, 50, 46, and 44 on food intake in GFRAL-deficient mice are shown. Data are presented as percent change in food intake (compared to vehicle administration) relative to plasma concentration of test article with intact GLP1 arm at 24 hours. [Figure 7]Graph showing the effect of subcutaneous administration of SEQ ID NO: 45 on food intake in GFRAL-deficient mice. Data is presented as the percentage change in food intake (compared to vehicle administration) relative to plasma concentration of test article with intact GLP1 population at 24 hours. [Figure 8] Graph showing the effect of subcutaneous administration of SEQ ID NO: 33 on food intake in GFRAL-deficient mice. Data is presented as percent change in food intake (compared to vehicle administration) relative to plasma concentration of test article with intact GLP1 population at 24 hours. [Figure 9] 1 shows a graph demonstrating the effect of subcutaneous administration of SEQ ID NOs: 50, 46, and 44 on glucose tolerance in mice. Data is presented as percent change in delta AUC compared to vehicle administration for plasma concentrations of intact GLP1 peptide or GLP1-GDF15 fusion protein with GLP1 peptide variants. [Figure 10] 1 shows a graph demonstrating the effect of subcutaneous administration of SEQ ID NOs: 45 and 49 on glucose tolerance in mice. Data is presented as percent change in delta AUC compared to vehicle administration for plasma concentrations of GLP1-GDF15 fusion protein with intact GLP1 peptide or GLP1 peptide variants. [Figure 11] 1 shows a graph demonstrating the effect of subcutaneous administration of SEQ ID NO: 33 on glucose tolerance in mice. Data is presented as the percentage change in delta AUC compared to vehicle administration for plasma concentrations of GLP1-GDF15 fusion protein with intact GLP1 peptide or GLP1 peptide variants. [Figure 12] 1 shows a graph demonstrating the effect of subcutaneous administration of SEQ ID NO: 45 and SEQ ID NO: 36 on glucose tolerance in mice. Data is presented as percent change in delta AUC compared to vehicle administration for plasma concentrations of GLP1-GDF15 fusion protein with intact GLP1 peptide or GLP1 peptide variants. [Figure 13]Graphs demonstrating the effect of subcutaneous administration of SEQ ID NOs: 45 and 44 on insulin secretion during graded glucose infusion in cynomolgus monkeys. Data are presented as fold change in ISR AUC (relative to baseline) normalized to glucose AUC for plasma concentrations of intact GLP1 peptide or GLP1-GDF15 fusion protein with GLP1 peptide variants. [Figure 14] 1 shows a graph demonstrating the weight loss efficacy of daily subcutaneous administration of GLP1-GDF15 fusion protein (SEQ ID NOs: 45 and 44) ​​in diet-induced obese mice. Data is presented as percentage change in body weight from day 0. [Figure 15] 1 shows a graph demonstrating plasma concentrations of GLP1-GDF15 fusion protein (SEQ ID NOs: 45 and 44) ​​after 7 days of daily dosing in diet-induced obese mice as determined by immunoaffinity capture-trypsin digestion-LC-MS / MS analysis. [Figure 16] 1 shows a graph demonstrating the 3-day percent change in food intake after baseline and 4 weeks of subcutaneous administration of each of GLP1-GDF15 fusion proteins (SEQ ID NOs: 44 and 83) in spontaneously obese non-human primates. Data is presented for individual subjects, and those without detectable levels of test article in their plasma are not included in the figure, as outlined below the x-axis. [Figure 17] Graph showing plasma concentrations of all test articles during 4 weeks of administration of varying concentrations of GLP1-GDF15 fusion protein (SEQ ID NOs: 44 and 83) administered QW in spontaneously obese non-human primates as determined by immunoassay. Data are presented as mean (± SEM). [Figure 18] Graph showing the plasma concentration of GLP1 moiety-containing test article during 4 weeks of administration of varying concentrations of GLP1-GDF15 fusion protein (SEQ ID NO: 44) administered QW in spontaneously obese non-human primates as determined by immunoassay.Data are presented as mean (± SEM). [Figure 19]This figure shows a graph demonstrating absolute daily food intake before, during, and after 21 days of administration of increasing concentrations of GLP1-GDF15 fusion protein (SEQ ID NO: 44, 83, and 84) administered Q3D in spontaneously obese non-human primates. Data are presented as the mean (± SEM) of 10 subjects per group. [Figure 20] Graph showing the change in body weight (relative to baseline) after 21 days of administration of increasing concentrations of GLP1-GDF15 fusion protein (SEQ ID NO: 44, 83 and 84) administered Q3D in spontaneously obese non-human primates. Data are presented as the mean (± SEM) of 10 subjects per group. [Figure 21] Graph showing plasma concentrations of all test articles during and after 21 days of treatment with increasing concentrations of GLP1-GDF15 fusion protein (SEQ ID NO: 44, 83, and 84) administered Q3D in spontaneously obese non-human primates as determined by immunoassay.Data is presented for individual subjects. [Figure 22] Graph showing plasma concentrations of GLP1 moiety-containing test articles during and after 21 days of administration of increasing concentrations of GLP1-GDF15 fusion protein (SEQ ID NOs: 44 and 84) administered Q3D in spontaneously obese non-human primates as determined by immunoassay.Data is presented for individual subjects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Various publications, articles and patents are cited or referenced in the Background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles or the like which is included in the specification is indicative of the context of the present invention. Such discussion is intended to provide a basis for understanding whether any or all of these matters are disclosed or disclosed. is admitted to be part of the prior art to any claimed invention. isn't it.

[0030] Unless otherwise defined, all technical and scientific terms used herein refer to the It has the same meaning as commonly understood by one of ordinary skill in the art to which it pertains. Certain terms used herein have the meanings set forth herein.

[0031] As used in this specification and the appended claims, the singular forms "a," "an," and "the" It should be noted that "includes plural referents unless the context clearly indicates otherwise.

[0032] Unless otherwise indicated, any numerical values, such as concentrations or concentration ranges, described herein are In all cases, the terms "about" should be understood to be modified. Thus, numerical values ​​typically include ±10% of the stated value. For example, A concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration of 1% to 10 The concentration range of % (w / v) includes 0.9% (w / v) to 11% (w / v). When used herein, the use of a numerical range shall include the range unless the context clearly indicates otherwise. All possible subranges, including integers and fractions of values, and all individual numbers within that range Explicitly included.

[0033] Unless otherwise noted, the term "at least" preceding a series of elements refers to every element in the series. It should be understood that the present invention refers to elements of the It will be appreciated that the present invention is not limited to the above embodiment, and that many equivalents to the specific embodiments described herein may be recognized. Such equivalents are intended to be encompassed by this invention. can be.

[0034] As used herein, the terms "comprises" and "comprising" "includes," "including," "has," "having," ng}," "contains," or "containing," or any of these Any other variations are intended to include the stated integer or groups of integers, but this It does not exclude any other integer or group of integers other than these, and is non-exclusive or non-limiting. It will be understood that the present invention contemplates, for example, compositions, mixtures, processes, etc. that include a series of elements. The terms "method, article, or apparatus" are not necessarily limited to those elements, but may be expressly incorporated herein by reference. Any compositions, mixtures, processes, methods, articles, or apparatus not specifically listed herein are not intended to be limiting. In addition, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, For example, condition A or B is true if A is true (or exists) and B is false (or does not exist). ), if A is false (or does not exist) and B is true (or exists), then If both A and B are true (or exist), then it is satisfied by one of .

[0035] As used herein, the conjunctive term "and / or" between multiple listed elements means Both individual and combined options are understood to be encompassing. For example, When elements are connected by "and / or," the first option may be used without the second option. The first element is applicable. The second option is the second element without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is within the meaning and therefore the It is understood that the term "and / or" is used in the specification. It is understood that the simultaneous applicability of two or more of the terms "and" is also included in the meaning, and therefore the terms "and" are used interchangeably. and / or" requirements are met.

[0036] The term "about" is used herein when referring to a size or characteristic of a component of the preferred invention. The terms "approximately," "generally," "substantially," and the like, as will be understood by those of ordinary skill in the art, are used to describe The dimensions / characteristics listed are not precise boundaries or parameters and are functionally the same or similar. It should also be understood that this does not exclude slight variations therefrom. Such references, including value parameters, are based on mathematical expressions accepted in the art. and using industrial principles (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, etc.) The least significant figure will then include the variant that does not change.

[0037] Two or more nucleic acid or polypeptide sequences (e.g., a GLP1 peptide, a linker peptide Polynucleotides encoding the serum albumin protein, the GDF15 protein, and peptides The term "identical" or percent "identity" in relation to a sequence (nucleotide) is used in the following sequence: using one of the string comparison algorithms or known in the art in light of this disclosure Compare and maximize agreement when measured by visual inspection using known methods. When aligned to each other, amino acid residues that are identical or have a specified percentage of identical residues are selected. It also refers to two or more sequences or subsequences having nucleotides.

[0038] For sequence comparison, typically one sequence serves as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer If necessary, subsequence coordinates are specified and the sequence algorithm program is executed. The sequence comparison algorithm is then run according to the designated program parameters. Based on the data, the percent sequence identity of the test sequence relative to the reference sequence is calculated.

[0039] Optimal alignment of sequences for comparison can be performed using, for example, the method of Smith & Waterman. The local homology algorithm of Adv. Appl. Math. 2:482 (1981); Needleman & Wunsch, J. Mol. Biol. 48:443 (197 0) Homology Alignment Algorithm, Pearson & Lipman, Proc. Similarity search method for Nat'l Acad Sci USA 85:2444(1988) , and a computerized implementation of these algorithms (Wisconsin Genet ics Software Package,Genetics Computer G roup, 575 Science Dr., Madison, WI GAP, B ESTFIT, FASTA, and TFASTA), or visual inspection (generally Curre nt Protocols in Molecular Biology,FMAu Subel et al., eds., Current Protocols, a jo int venture between Greene Publishing As sociates,Inc.and John Wiley & Sons,Inc., (1995 Supplement) (Ausubel) It is possible.

[0040] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are Altschul et al. (1990) J. Mol. Biol. 215:4, respectively. 03-410 and Altschul et al. (1997) Nucleic Aci BLAST and BLAST described in ds Res.25:3389-3402, respectively. The BLAST 2.0 algorithm is the software used to perform the BLAST analysis. Publicly available through the National Center for Biotechnology Information. The algorithm first finds all words of the same length in the database sequence. A match or meets some positive threshold score T when aligned with by identifying short words of length W in the query sequence that either: The method includes identifying high-scoring sequence pairs (HSPs). T is the neighborhood word score threshold (Alt These initial neighborhood word hits are then It serves as a seed to initiate searches to find longer HSPs that contain The cumulative alignment score is increased by adding as many word fragments along each sequence as possible. Extends the hit in both directions.

[0041] In addition to calculating percent sequence identity, the BLAST algorithm also Statistical analysis of similarities between columns is also performed (e.g., Karlin & Altschul, Proc. c. Nat'l. Acad. Sci. USA 90:5873-5787(1993) One measure of similarity provided by the BLAST algorithm is the minimum sum probability. The probability that a match between two nucleotide or amino acid sequences occurs by chance (P(N)). For example, the nucleic acid provides an indication of the probability of a minimum repeat occurrence in a comparison between a test nucleic acid and a reference nucleic acid. The small sum probability is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.0 If it is less than 01, it is considered similar to the reference sequence.

[0042] Further indications that two nucleic acid sequences or polypeptides are substantially identical include the following: As described above, the polypeptide encoded by the first nucleic acid is encoded by the second nucleic acid. The objective of the present invention is to provide a method for the preparation of a polypeptide that is immunologically cross-reactive with the polypeptide to be administered. A polypeptide is typically substantially identical to a second polypeptide, e.g., two peptides Another indication that two nucleic acid sequences are substantially identical is It is when two molecules hybridize to each other under stringent conditions.

[0043] As used herein, a "subject" refers to any animal, preferably a mammal, most preferably a mammalian animal. As used herein, the term "mammal" refers to any mammalian animal or human. Examples of mammals include, but are not limited to, cows, Ma, sheep, pig, cat, dog, mouse, rat, rabbit, guinea pig, monkey, human, etc. and more preferably humans.

[0044] The term "administering" with respect to the methods of the invention means administering a conjugate or compound of the invention, or The use of such forms, compositions, or medicaments may be used to treat the syndromes, disorders, and conditions described herein. or a method of therapeutically or prophylactically preventing, treating, or ameliorating a disease. Such methods include administering an effective amount of the conjugate, compound, form, composition, or This includes administering the medicaments in combination form, at different times during the course of treatment, or simultaneously. The methods of the present invention are understood to encompass all known therapeutic treatment regimens. That is why.

[0045] The term "effective amount" refers to the biological effect sought by a researcher, veterinarian, physician, or other clinician. eliciting a therapeutic or medical response in a tissue system, animal, or human (the syndrome, disorder being treated) or to prevent or treat the disease or symptoms of the syndrome, disorder or disease being treated. By "active" is meant the amount of active conjugate, compound, or agent that is capable of inhibiting or ameliorating the effect of a given condition.

[0046] As used herein, the term "composition" refers to a product containing specific ingredients in specific amounts, as well as and any product resulting directly or indirectly from the combination of specific ingredients in specific amounts. This includes:

[0047] The term "isolated" refers to a substance that is free from cellular, bacterial, viral, or Substantially free of culture medium, nucleic acid or polypeptide (produced by recombinant DNA technology) It may refer to a compound (if it is chemically synthesized), or a chemical precursor or other chemical substance (if it is chemically synthesized). Additionally, an isolated polypeptide refers to one that can be administered to a subject as an isolated polypeptide, In other words, when the polypeptide is applied to a column or embedded in a gel, It cannot be considered simply "isolated." Furthermore, "isolated nucleic acid fragments" or " An "isolated peptide" is one that does not naturally occur as a fragment and / or is typically in a functional state. It is a nucleic acid or protein fragment not found in

[0048] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid molecule." ", "nucleotides" or "nucleic acids" and may be unmodified RNA or DNA or modified Any polyribonucleotide or polydeoxyribonucleotide, which may be RNA or DNA. "Polynucleotide" refers to a nucleotide. "Polynucleotide" is not limited to the following: However, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and and double-stranded RNA, as well as RNA that is a mixture of single-stranded and double-stranded regions, DNA and RNA, which may typically be double stranded or a mixture of single and double stranded regions, In addition, "polynucleotide" includes RNA or hybrid molecules containing the RNA. The term polynucleotide refers to a triple-stranded region that contains DNA or both RNA and DNA. is a DNA or RNA that contains one or more modified bases and that may be modified for stability or other reasons. The term "modified" bases also includes DNA or RNA having a backbone that has been modified by a Various modifications to DNA and RNA include substituted and unusual bases, e.g., inosine. Thus, a "polynucleotide" is a polypeptide typically found in nature. Chemically, enzymatically or metabolically modified forms of oligonucleotides, as well as viruses and cells "Polynucleotides" also include chemical forms that have the characteristics of DNA and RNA. , but also includes relatively short nucleic acid chains, often called oligonucleotides.

[0049] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes the transcription of a gene into RNA. This includes translation into polypeptides, as well as all naturally occurring post-transcriptional and post-translational modifications. The expressed polypeptide may be expressed in the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or in a host cell culture medium. It may be present in the cell boundary or anchored in the cell membrane.

[0050] As used herein, the terms "peptide," "polypeptide," or "protein" can refer to molecules composed of amino acids and recognized by those skilled in the art as proteins. In this specification, the conventional one-letter or three-letter codes for amino acid residues are used. The terms "peptide," "polypeptide," and "protein" include those that are conjugated (e.g., fused) to one another. Amino acids of any length, including those containing peptides / polypeptides (e.g., fusion proteins). The term "polymer of amino acids" may be used interchangeably herein to refer to a polymer of amino acids. It may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also refer to amino acids that have been naturally modified or modified by intervention. Polymers, e.g., disulfide bond formation, glycosylation, lipidation, acetylation , phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, one or more analogs of an amino acid (e.g., a non-natural amino acid). Polypeptides containing nucleotides such as nucleotides, ... etc., as well as other modifications known in the art. Included.

[0051] The peptide sequences described herein are written according to the usual convention and include the N-terminal region of the peptide. The C-terminal region is on the left and the C-terminal region is on the right. The isomeric forms of the amino acids are known but are not shown separately. Unless explicitly stated, the L-form of the amino acid is shown.

[0052] Glucagon-like peptide-1 (GLP1)-growth differentiation factor 15 (GDF15) fusion protein Quality Both GDF15 and GLP1 signaling can reduce food intake, These effects appear to be independent of each other. For example, the GDF15 effect in mice In contrast, GLP1 administration still inhibited the expression of GLP1R. (Hsu et al., Nature 550 :255-9(2017), Mullican et al., Nat Med 23: 1150-7 (2017)). Therefore, targeting both mechanisms simultaneously is It is hypothesized that this mechanism may result in greater reduction in food intake and weight loss than either mechanism alone. Independent expression of GDF15 and GLP1 agonists through fusion with human serum albumin Combining complementary pharmacology into a single, fully recombinant molecule suitable for once-weekly administration This results in benefits for weight loss, insulin sensitivity, insulin secretion, and cardiovascular outcomes. A major challenge with this approach is to find enough, but not enough, Achieving a balance between avoiding adverse effects on the target and engaging the corresponding receptor at a desired level The goal of this balance is to deliver each agonist in a controlled manner. By adjusting the efficacy and / or pharmacokinetic properties of either the agonist arm of can be adjusted.

[0053] In one general aspect, the invention provides a glucagon-like peptide-1 (GLP1)-proliferative differentiation factor. The GLP1-GDF15 fusion protein is , a GLP1 or GLP1 variant peptide, a first linker peptide (e.g., amino(N )-terminal linker), human serum albumin (HSA) protein, a second linker peptide a carboxy (C)-terminal linker, and GDF15 or a GDF15 variant. Contains protein.

[0054] Glucagon-like peptide-1 (GLP1) or GLP1 variant peptides Glucagon-like peptide 1 (GLP1) is synthesized in the intestine and released in response to food intake. These are insulin secretagogues that are primarily involved in the synthesis of GLP1-(7-37) and GLP1- (7-36)NH2 is secreted in two forms, both of which are involved in glucose-stimulated inactivation. Many tissues, including pancreatic beta cells, which enhance secretion of insulin and regulate satiety and food intake It binds to specific GLP1 receptors (GLP1R) found in the brain stem.

[0055] Many GLP1 analogs and derivatives are known and are referred to herein as "GLP-1 variants." These GLP1 variant peptides are sometimes called Gila monsters. These exendins are peptides that are expressed in the natural GL It shares sequence homology with GLP1 and binds to the GLP1 receptor, mediating signal transduction cascade responses. You can start answering.

[0056] GLP1 and GLP1 variant peptides have been shown to act in a variety of ways, These include reducing food intake, stimulating insulin release, decreasing glucagon secretion, and inhibiting gastric emptying. These include, but are not limited to, increased glucose utilization and reduced cholesterol levels.

[0057] GLP1R is a seven-transmembrane heterotrimeric G protein-coupled receptor of the class B family. They belong to the α-, β-, and δ-cells of the pancreatic islets, the heart, kidneys, stomach, intestines, and the ganglion nerves of the vagus nerve. , as well as several regions of the central nervous system (CNS), including the hypothalamus and brainstem. GLP1R is expressed in a wide range of tissues, not limited to Gα s , Gα q , Gα i , and Gα o (Montrose-Rafizadeh et al., Endoc rinology 140:1132-40(1999); al., Biochim Biophys Acta 1546:79-86(2001 )), increases in intracellular calcium, adenylate cyclase, and phospholipase C; and Activation of PKA, PKC, PI-3K, Epac2, and MAPK signaling pathways (Drucker et al., PNAS 84:3434-8 (1987 );Wheeler et al., Endrocrinology 133:57-6 2(1993); and Holz et al., JBC 270:17749-57(1 995)).

[0058] Provided herein is a GLP1-GDF15 fusion protein comprising a first component. The first component is a GLP1 or GLP1 variant peptide. In this case, the terms "GLP1 peptide", "GLP1 variant peptide", and "GLP1 variant peptide" are used. The terms "GLP1 variant" and "GLP1 or GLP1 variant peptide" are used interchangeably. Or the GLP1 variant peptide may comprise one of the sequences provided in Table 1. 1 or the GLP1 variant peptide has at least 75% repeat sequence with one of the sequences provided in Table 1. %, 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 GLP1 or a GLP1 variant. The variant peptide sequences were selected based on the following criteria: (i) expression yield and purity, (ii) in vitro stability (iii) in vitro potency; (iv) interaction with GDF15 or GDF15 mutant proteins; (v) retention of in vitro potency in combination; (vi) the lack of silylating potential, and the properties of the GLP1-GDF15 mutant protein ( For example, in vivo stability, in vivo efficacy (i.e., GLP1 or GLP1 variant peptides) and GDF15 or GDF15 mutant proteins are capable of binding to GLP1R and GDF Whether or not it can have agonist activity against the 15R (GFRAL) receptor may be selected based on at least one of

[0059] The first component of the GLP1 fusion peptide is a GLP1 or GLP1 variant peptide. The term GLP1 is intended to encompass peptides having sufficient homology and functionality to native GLP1. GLP1 or GLP1 variant peptides promote the uptake of GLP1 in tissues including the pancreas and brainstem. P1 receptors, resulting in the natural GLP1 being able to bind to the GLP receptors in these tissues. 1 receptor, and induces the same signal transduction pathway as insulin, which is the same or similar to insulin. It is designed to exhibit secretory activity.

[0060] [Table 1]

[0061] First linker peptide: amino-terminal linker (N-terminal linker) Provided herein is a GLP1-GDF15 fusion protein that includes a second component. , the second component is a first linker peptide (i.e., an amino-terminal linker peptide The first linker peptide may include one of the sequences provided in Table 2. The first linker peptide has at least 75%, 70%, or more identical to one of the sequences provided in Table 2. 6%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 8 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 It may comprise 6%, 97%, 98% or 99% identity.

[0062] The first linker peptide may be, for example, about 5 to about 60 amino acid residues, about 10 to about 50 about 10 to about 60 amino acid residues, about 5 to about 50 amino acid residues, About 15 to about 40 amino acid residues, about 12 to about 30 amino acid residues, about 12 to about 42 amino acid residues about 20 to about 25 amino acid residues, about 8 to about 48 amino acid residues, about 10 to about 46 amino acid residues about 12 to about 44 amino acid residues, about 14 to about 42 amino acid residues, about 16 to about 40 Amino acid residues, about 18 to about 38 amino acid residues, about 20 to about 36 amino acid residues, about 20 about 42 amino acid residues, about 22 to about 34 amino acid residues, about 24 to about 32 amino acid residues The amino acid residues may be from about 26 to about 30 amino acid residues, or any value therebetween. The linker peptide of 1 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2 9, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, It may comprise 56, 57, 58, 59, or 60 amino acid residues.

[0063] In certain embodiments, the first linker peptide comprises an alanine-proline repeat (i.e., AP repeats), and the AP dipeptide may be referred to as an AP unit. , 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, or 27 AP units. In certain embodiments, the first linker peptide may be, for example, 2 to 25 AP units. 5-25 units, 4-23 AP units, 6-21 AP units, 8-19 A units P units, 10-17 AP units, 12-15 AP units, 5-10 AP units, 5 ~15 AP units, 10-25 AP units, 15-25 AP units, 20-25 In certain embodiments, the AP repeat may include AP units of: Alanine-serine dipeptide (i.e., AS unit) and glycine-serine dipeptide ( The AS unit may be present in, for example, the amino acid sequence of the first linker peptide. The GS unit may be present, for example, at the carboxyl terminus of the first linker peptide. may exist in

[0064] In certain embodiments, the first linker peptide is glycine-glycine-glycine-glycine It may contain lysine-serine repeats (i.e., G4S repeats), and the G4S pentapeptide may contain G4 G4S repeats can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 In certain embodiments, the first phosphorus may include 2, 13, 14, or 15 G4S units. Car peptides may be, for example, peptides having 2 to 15 G4S units, 4 to 13 G4S units, 、 6~11 G4S units, 8-9 G4S units, 2-8 G4S units, 2-6 G4S units , 6-8 G4S units, 7-8 G4S units, 7-9 G4S units, 7-10 G4S units, or any value in between. In certain embodiments, AS or GS The unit can be, for example, at the amino terminus of the first linker peptide.

[0065] In certain embodiments, the first linker peptide is glycine-glycine-glycine-glycine It may contain lysine-alanine repeats (i.e., G4A repeats), and the G4A pentapeptide may be G G4A repeats can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, In certain embodiments, the first linker may comprise 12, 13, 14, or 15 G4A units. The anchor peptide may be, for example, 2 to 15 G4A units, 4 to 13 G4A units, 、 6~1 1 G4A unit, 8-9 G4A units, 2-8 G4A units, 2-4 G4A units 2-6 G4A units, 4-6 G4A units, 4-8 G4A units, 6-8 G4A units, 6-9 G4A units, 6-10 G4A units, or anything in between In certain embodiments, the dipeptide glycine-alanine (i.e. , GA unit) unit may be present, for example, at the amino terminus of the first linker peptide.

[0066] In certain embodiments, the first linker peptide can be a polyglycine peptide. Polyglycine peptides are peptides that contain from about 6 to about 50 glycine residues, from about 10 to about 45 glycine residues, about 15 to about 40 glycine residues, about 20 to about 35 glycine residues, about 25 to about 30 glycine residues, about 20 to about 25 glycine residues, or any number therebetween. The first linker peptide of polyglycine may comprise 6, 7, 8, 9, 10, 11, 1 2, 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 It may contain lysine residues.

[0067] The first linker peptide sequence should satisfy the following criteria: (i) expression yield and purity; (ii) enzyme activity; (iii) in vitro potency; (iv) serine xylosylation or serine xylosylation; (v) the lack of potential for rosylation, and the properties of the GLP1-GDF15 fusion protein (e.g. For example, in vivo stability and in vivo efficacy (i.e., GLP1 or GLP1 variant peptides) and GDF15 or GDF15 mutant proteins are capable of binding to GLP1R and GDF Whether or not it can have agonist activity against the 15R (GFRAL) receptor may be selected based on at least one of

[0068] [Table 2]

[0069] Serum Albumin Protein Provided herein is a GLP1-GDF15 fusion protein that includes a third component. The third component is a serum albumin protein (e.g., human serum albumin (HSA) ) protein or gorilla serum albumin (GSA) protein). Albumin protein contains 35 cysteines (C Cys, C) residues, with Cys-34 being the only free cysteine ​​in the molecule. This free Cys-34 captures multiple reactive oxygen species (ROS) and reactive nitrogen species (RNS). It has been shown that it functions as a free radical scavenger by capturing Taverna et al., Ann. Intensive Care 3:4(20 13) This free Cys was mutated to serine (Ser) to cause oxidative heterogeneity. Risk minimised.

[0070] The serum albumin protein may include one of the sequences provided in Table 3. The albumin protein has at least 75%, 76% or more identical sequences to one of the sequences provided in Table 3. , 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. The serum albumin protein sequence may include The following criteria: (i) in vitro stability, (ii) in vitro potency, (iii) GLP1- The properties of the GDF15 fusion protein, such as in vivo stability and in vivo efficacy (i.e. , GLP1 or GLP1 variant peptides and GDF15 or GDF15 variant proteins have agonistic activity against the GLP1R and GDF15R receptors, respectively. The method may be selected based on at least one of the following:

[0071] [Table 3]

[0072] Second linker peptide: Carboxy-terminal linker (C-terminal linker) Provided herein is a GLP1-GDF15 fusion protein that includes a fourth component. The fourth component is a second linker peptide (i.e., a carboxy-terminal linker peptide). The second linker peptide may comprise one of the sequences provided in Table 4. The second linker peptide has at least 75% identity with one of the sequences provided in Table 4. , 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.

[0073] The second linker peptide may be, for example, about 5 to about 60 amino acid residues, about 10 to about 50 about 10 to about 60 amino acid residues, about 5 to about 50 amino acid residues, About 15 to about 40 amino acid residues, about 12 to about 30 amino acid residues, about 12 to about 42 amino acid residues about 20 to about 25 amino acid residues, about 8 to about 48 amino acid residues, about 10 to about 46 amino acid residues about 12 to about 44 amino acid residues, about 14 to about 42 amino acid residues, about 16 to about 40 Amino acid residues, about 18 to about 38 amino acid residues, about 20 to about 36 amino acid residues, about 20 about 42 amino acid residues, about 22 to about 34 amino acid residues, about 24 to about 32 amino acid residues The amino acid residues may be from about 26 to about 30 amino acid residues, or any value therebetween. The linker peptides of 2 are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2 9, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, It may comprise 56, 57, 58, 59, or 60 amino acid residues.

[0074] In certain embodiments, the second linker peptide comprises an alanine-proline repeat (i.e., AP repeats), and the AP dipeptide may be referred to as an AP unit. , 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, or 27 AP units. In certain embodiments, the second linker peptide may be, for example, 2 to 25 AP units. 5-25 units, 4-23 AP units, 6-21 AP units, 8-19 A units P credits, 10-17 AP credits, 12-15 AP credits, 5-10 credits, 5-15 units of 10-25 units, units of 15-25 units, units of 20-25 units, or any of these In certain embodiments, the AP repeat can include any value between The dipeptide is a glycine-serine dipeptide (i.e., GS unit) and a glycine-serine dipeptide (i.e., AS unit). The AS unit may be present, for example, at the amino terminus of the second linker peptide. The S unit can be, for example, at the carboxyl terminus of the second linker peptide.

[0075] In certain embodiments, the second linker peptide is glycine-glycine-glycine-glycine It may contain lysine-serine repeats (i.e., G4S repeats), and the G4S pentapeptide may contain G4 G4S repeats can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 In certain embodiments, the second phosphorus may include 2, 13, 14, or 15 G4S units. Car peptides may be, for example, peptides having 2 to 15 G4S units, 4 to 13 G4S units, 、 6~11 G4S units, 8-9 G4S units, 2-8 G4S units, 2-6 G4S units , 6-8 G4S units, 7-8 G4S units, 7-9 G4S units, 7-10 G4S units, or any value in between. In certain embodiments, AS or GS The unit can be, for example, at the amino terminus of the second linker peptide.

[0076] In certain embodiments, the second linker peptide is glycine-glycine-glycine-glycine It may contain lysine-alanine repeats (i.e., G4A repeats), and the G4A pentapeptide may be G G4A repeats can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, In certain embodiments, the second linker may comprise 12, 13, 14, or 15 G4A units. The anchor peptide may be, for example, 2 to 15 G4A units, 4 to 13 G4A units, 、 6~1 1 G4A unit, 8-9 G4A units, 2-8 G4A units, 2-4 G4A units 2-6 G4A units, 4-6 G4A units, 4-8 G4A units, 6-8 G4A units, 6-9 G4A units, 6-10 G4A units, or anything in between In certain embodiments, the dipeptide glycine-alanine (i.e. , GA unit) unit can be present, for example, at the amino terminus of the second linker peptide.

[0077] In certain embodiments, the second linker peptide can be a polyglycine peptide. Polyglycine peptides are peptides that contain from about 6 to about 50 glycine residues, from about 10 to about 45 glycine residues, about 15 to about 40 glycine residues, about 20 to about 35 glycine residues, about 25 to about 30 glycine residues, about 20 to about 25 glycine residues, or any number therebetween. The second linker peptide of polyglycine may comprise 6, 7, 8, 9, 10, 11, 1 2, 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 It may contain lysine residues.

[0078] The second linker peptide sequence should satisfy the following criteria: (i) expression yield and purity; (ii) enzyme activity; (iii) in vitro potency; (iv) serine xylosylation or serine xylosylation; (v) the lack of potential for rosylation, and the properties of the GLP1-GDF15 fusion protein (e.g. For example, in vivo stability and in vivo efficacy (i.e., GLP1 or GLP1 variant peptides) and GDF15 or GDF15 mutant proteins are capable of binding to GLP1R and GDF Whether or not it can have agonist activity of 15R (GFRAL) receptor The selected region may be selected based on at least one of the following:

[0079] [Table 4]

[0080] GDF15 or GDF15 mutant protein Growth differentiation factor 15 (GDF15) is a transforming growth factor-β (TGF-β) GDF15 is a member of the Gdc2+ superfamily of proteins. It forms a 25-kDa dimer. GDF15 is a secreted protein that circulates in the prostate. Phage inhibitory cytokine-1 (MIC-1), NSAIDs (non-steroidal anti-inflammatory drugs) It is also called placental TGF-β (PTGFβ) and activating gene (NAG-1).

[0081] Although the functions of GDF15 have not yet been fully elucidated, it is thought to play a role in energy homeostasis, body weight regulation, and and cachexia caused by cancer and chronic illness. GDF15 is involved in a number of biological processes. Pharmacologically administered GDF15 reduces energy intake. The potential for regulating GABAergic deficiency and thereby inducing weight loss has been demonstrated in mice, rats, and monkeys. It has been proven that. (Johnen et al., Nat Med 13:1333-40 (2007), Hsu et al., Nature 550:255-9(2017) ,Mullican et al., Nat Med 23:1150-7(2017) ,Tsai et al.,Int J Obesity 42:561-71(201 8) The weight loss mediated by GDF15 administration is due to enhanced glucose homeostasis and hemodynamic regulation. Improves metabolism, including lowering plasma triglycerides and cholesterol (Xiong et al., 2013). et al., Sci Trans Med 9:412(2017)).

[0082] GDF15 binds to the GDNF family receptor alpha-like (GFRAL) and is a transmembrane receptor is present only in neurons of the brainstem (Mullican et al., Hsu et al. al, Yang et al., Emmerson et al.). GDF15 binding At times, GFRAL mediates downstream intracellular phosphorylation, including post-translational modifications of AKT, ERK, and PLCγ. It forms a complex with RET, a tyrosine kinase that stimulates an oxidative cascade. Additional molecular and cellular components of the pathway remain to be elucidated, but they may be involved in the regulation of GDF15 / GFRAL signaling. The net effect of null transmission is reduced food intake and weight loss (Mullican et al., 2003). nd Rangwala, 2018).

[0083] Provided herein is a GLP1-GDF15 fusion protein that includes a fifth component. The fifth component is a GDF15 or GDF15 mutant protein. The protein may comprise a sequence provided in Table 5, and the GDF15 mutant protein may comprise a sequence provided in Table 5. GDF15 or GDF15 variant proteins may include variants of the sequences provided in Table 5. At least 75%, 76%, 77%, 78%, 79%, 80%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% The GDF15 and / or GDF15 variant protein sequences may include the following groups: Criteria: (i) expression yield and purity, (ii) in vitro stability, (iii) in vitro potency, (iv) In Vitro Potency in Combination with GLP1 or GLP1 Variant Proteins (v) lack of serine xylosylation or potential for serine xylosylation, and i) The properties of the GLP1-GDF15 variant protein (e.g., in vivo stability, in vivo Potency (i.e., GLP1 or GLP1 variant peptides and GDF15 or GDF15 variant peptides) The heterologous proteins bind to the GLP1R and GDF15R (GFRAL) receptors, respectively. Based on at least one of the following: can be selected.

[0084] GDF15 or GDF15, which constitutes the fifth component of the GLP1-GDF15 fusion protein The F15 mutant protein is sufficient to activate native GDF15R (GFRAL) It is intended to encompass proteins having homology and functionality. The F15 mutant protein is able to bind to GFRAL in the brainstem, resulting in the The same signaling occurs when GDF15 binds to GFRAL in these neurons. The pathways are designed to produce the same effects on food intake.

[0085] [Table 5]

[0086] GLP1-GDF15 fusion protein As used herein, GL includes first, second, third, fourth, and fifth components as described above. A GLP1 or GDF15 fusion protein is provided. The first component is a mutant peptide, the second component is a linker peptide, and the third component is a The first component is a serum albumin protein, and the fourth component is a second linker peptide. and the fifth component is GDF15 or a GDF15 mutant protein. The DF15 fusion protein may include one of the sequences provided in Table 6. The GDF15 fusion protein has at least 85% identity with one of the sequences provided in Table 6; 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, The GLP1-GDF15 fusion tag may comprise 96%, 97%, 98% or 99% identity. Proteins were assayed based on the following criteria: (i) expression yield and purity, (ii) in vitro potency, (iii) ) in vitro stability, (iv) lack of serine xylosylation, (v) GLP1-GDF15 The physical properties of the fusion protein (e.g., in vivo stability and in vivo efficacy (i.e., G LP1 or GLP1 variant peptide and GDF15 or GDF15 variant protein, Can they have agonistic activity against GLP1R and GDF15R, respectively? (vi) achieving a desirable balance of dual agonism pharmacology in vivo; The selection may be based on at least one of

[0087] For fusion protein therapeutics that deliver dual pharmacology in one molecule, both agonists are intended The pharmacokinetic / pharmacodynamic (PK / P) profiles of each moiety were evaluated to ensure that the doses shown were in the therapeutic range. D) It is important to adjust the properties appropriately. a first linker peptide sequence, a serum albumin protein sequence, a second linker peptide sequence, -peptide sequences and various combinations of GDF15 or GDF15 variant protein sequences. Various glucagon-like peptide-1 (GLP1)-growth differentiation factor 15 (GDF15)-containing Extensive studies of the fusion protein have demonstrated that it is both a GLP1 agonist and a GDF15 agonist. This has led to the discovery of novel molecules with unique properties that deliver optimally balanced doses of The novel GLP1-GDF15 fusion protein by H. SA-GDF15 is effective in associating with GDF15R (GFRAL) in vivo. (See Example 11.) Furthermore, GL used in the same dose range The P1-GDF15 fusion protein (see Example 11) inhibits the exposure of the GLP 1 moiety. Although the amount is 10 to 30 times that of dulaglutide, there are no side effects and it is The HSA-binding domain (GLP1-Fc) was unexpectedly effective in binding to the GLP1R. Delivery of the GLP1 peptide as a fusion with GDF15 increases the in vivo potency of the GLP1 moiety. An unexpected but novel glucagon-like peptide-1 (GLP1)-growth differentiation factor 15 (GDF15) fusion proteins have an optimal balance of both agonists I did.

[0088] [Table 6-1]

[0089] [Table 6-2]

[0090] [Table 6-3]

[0091] [Table 6-4]

[0092]

Table 6-5

[0093]

Table 6-6

[0094]

Table 6-7

[0095]

Table 6-8

[0096]

Table 6-9

[0097]

Table 6-10

[0098]

Table 6-11

[0099]

Table 6-12

[0100]

Table 6-13

[0101]

Table 6-14

[0102] GLP1-GDF15 fusion polynucleotides and vectors In another general aspect, the invention provides a GLP1-GDF15 fusion protein of the invention. The present invention relates to an isolated nucleic acid encoding a protein, which can be synthesized without changing the amino acid sequence of the protein. The ability to alter (e.g., substitute, delete, insert, etc.) the coding sequence of a gene It will be understood by those skilled in the art that the nucleic acid sequence encoding the fusion protein of the present invention may be It will be appreciated by those skilled in the art that the sequence may be varied without changing the amino acid sequence of the protein. It will be.

[0103] In another general aspect, the invention provides an isolated nucleic acid encoding a fusion protein of the invention. The present invention relates to a vector comprising a plasmid, a cosmid, a phage vector, or a viral vector. Any vector known to those of skill in the art in light of the present disclosure can be used, such as vectors that can be used to express the vectors. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include, for example, a promoter, a ribosome binding element, a terminator, an enhancer, To establish the traditional functions of an expression vector: a plasmid p53 gene, a selection marker, and an origin of replication. The promoter may be constitutive, inducible, or reversible. Numerous expression vectors capable of delivering nucleic acids to cells can be used. Transducers are known in the art and can be used to generate fusion proteins in cells. The present invention can be applied to the present invention using conventional cloning techniques or artificial gene synthesis. to generate a recombinant expression vector according to an embodiment of the invention.

[0104] In another general aspect, the invention provides an isolated nucleic acid encoding a fusion protein of the invention. In view of the present disclosure, any host cell known to those of skill in the art may be It can be used for recombinant expression of the fusion proteins of the invention. In some embodiments, The host cell is E. coli TG1 or BL21 cell, CHO-DG44 or CHO-K1 In certain embodiments, the recombinant expression vector is A chemical transfection that stably integrates the recombinant nucleic acid into the host cell genome for efficient expression. By conventional methods such as transfection, heat shock, or electroporation. The vector is transformed into a host cell.

[0105] In another general aspect, the invention provides a method for producing a fusion protein of the invention, comprising the steps of: Culturing cells containing nucleic acid encoding the protein and isolating the protein from the cells or cell culture (e.g., and recovering the fusion protein (e.g., from the supernatant). The expressed fusion protein is produced according to conventional techniques known in the art. The antibodies can be harvested from cells and purified according to the techniques and as described herein. do.

[0106] Pharmaceutical Compositions In another general aspect, the invention provides a GLP1-GDF15 fusion protein of the invention and and / or a GLP1-GDF15 fusion polynucleotide, and a pharma- ceutical acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a pharma- ceutical composition that is The GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion protein of the present invention may be administered together with a suitable carrier. The term "GLP1-GDF15 fusion polynucleotide" refers to a product comprising the GLP1-GDF15 fusion polynucleotide of the present invention. Fusion proteins and / or GLP1-GDF15 fusion polynucleotides, and their The compositions comprising are also useful in the manufacture of a medicament for the therapeutic applications mentioned herein.

[0107] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, or the like. Fa, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome inclusion or other materials well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutical acceptable carrier" refers to a pharmaceutical composition according to the present invention. According to certain embodiments, the present invention refers to a non-toxic material that does not interfere with the biological activity of the composition. In view of the disclosure, any pharma- ceutical acceptable carrier suitable for use in a peptide pharmaceutical composition is contemplated herein. can be used in the invention.

[0108] Pharmaceutically acceptable acid / anionic salts for use in the present invention include acetate, Salts, benzenesulfonates, benzoates, bicarbonates, bitartrates, bromides, edetate potassium Calcium, camsilate, carbonate, chloride, citrate, dihydrochloride, edetate, eds. Salts, estrates, esylates, fumarates, griseptates, gluconates, gluconates, thamate, glycolyl arsenic acid salt, hexylresorcinate, hydrabamine, bromide Hydrogen salt, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactate Tobionate, malate, maleate, mandelate, mesylate, methyl bromide, Methyl nitrate, methyl sulfate, mucoate, napsylate, nitrate, pamoate, pantothenate Salicylates, phosphates / diphosphates, polygalacturonates, salicylates, stearates, Acetate, succinate, sulfate, tannate, tartrate, tetraoclate, tosylate Also, organic or inorganic salts include, but are not limited to, salts and trithiodides. The acids include hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methyl ethanesulfonic acid, hydroxyethanesulfonic acid, oxalic acid, 2-naphthalenesulfonic acid, p-Toluenesulfonic acid, cyclohexanesulfamic acid, saccharinic acid or trifluoro These include, but are not limited to, acetic acid.

[0109] Pharmaceutically acceptable basic / cationic salts include aluminum, 2-amino- 2-Hydroxymethyl-propane-1,3-diol (tris(hydroxymethyl)amine) (also known as tetrahydrofuran, tetrahydrofuran, or "TRIS"), ammonia, benzathine , t-butylamine, calcium, chloroprocaine, choline, cyclohexylamine, Diethanolamine, Ethylenediamine, Lithium, L-Lysine, Magnesium, Meglumine amine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, These include, but are not limited to, thorium, triethanolamine, or zinc.

[0110] In some embodiments of the present invention, 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 and / or a GLP1-GDF15 fusion protein of the invention in an amount A pharmaceutical formulation comprising the polynucleotide is provided. The pharmaceutical formulation may comprise from about 3.0 to about 10, e.g. , from about 3 to about 7, or from about 5 to about 9. The formulation may further comprise a buffer system, a preservative(s). ), isotonicity agent(s), chelating agent(s), stabilizer(s), and surfactant. The composition may comprise at least one component selected from the group consisting of:

[0111] Formulation of pharma- ceutical active ingredients with pharma- ceutical acceptable carriers is described, for example, in Remington :The Science and Practice of Pharmacy As stated in the 21st edition (2005) and any subsequent revisions, Non-limiting examples of additional components include buffers, diluents, Solvents, tonicity adjusting agents, preservatives, stabilizers, and chelating agents. Acceptable carriers can be used in formulating the pharmaceutical compositions of the present invention.

[0112] In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is a water-soluble Liquid formulations include solutions, suspensions, emulsions, and The aqueous formulation may comprise at least one aqueous emulsion, a gel, or the like. 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, Or contains at least 95% w / w water.

[0113] In one embodiment, the pharmaceutical composition can be administered, for example, via an injection device (e.g., a syringe or an infusion pump). ) can be formulated as an injection that can be injected via the rectum. It may be delivered subcutaneously, intramuscularly, intraperitoneally, or intravenously.

[0114] In another embodiment, the pharmaceutical composition is a solid formulation, e.g., a ready-to-use formulation. or to which the physician or patient adds solvents and / or diluents prior to use, freeze-dried or is a spray-dried composition. Solid dosage forms include compressed tablets and / or coated tablets. and capsules (e.g., hard or soft gelatin capsules). The pharmaceutical compositions may also be, for example, in the form of sachets, dragees, powders, granules, lozenges or tablets for reconstitution. The powder may be in the form of

[0115] The dosage form may be immediate release, in which case the dosage form contains a water soluble or dispersible carrier. or may be delayed-, sustained-, or modified-release, in which case The dosage form includes a water-insoluble polymer that controls the dissolution rate of the dosage form in the gastrointestinal tract. good.

[0116] In other embodiments, the pharmaceutical compositions may be delivered intranasally, bucally, or sublingually.

[0117] The pH of the aqueous formulation may be from pH 3 to pH 10. In one embodiment of the present invention, the pH of the formulation In another embodiment of the present invention, the pH of the formulation is from about 3.0 to about The value is 7.0.

[0118] In another embodiment of the present invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include Arginine, aspartic acid, bicine, citrate, disodium monohydrogen phosphate, Malic acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, vinegar Sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinic acid salts, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, and their derivatives The buffer may be present in a concentration of from about 0.01 mg / mL to about 50 mg / mL, either individually or in aggregate. It may be present at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific buffering agents constitute alternative embodiments of the invention.

[0119] 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, butyl 4-hydroxybutyrate, hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, Ruphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxy Dibenzoate, imidourea, methyl 4-hydroxybenzoate, phenol, 2- Phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, Preservatives include sodium dehydroacetate, thiomerosal, and mixtures thereof. , individually or in aggregates, from about 0.01 mg / mL to about 50 mg / mL, for example about 0.1 mg The preservatives may be present in a concentration of about 100 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising the compound constitute an alternative embodiment of the invention.

[0120] In another embodiment of the invention, the pharmaceutical composition comprises an isotonicity agent. Examples include salts (such as sodium chloride), amino acids (glycine, histidine, arginine, Lysine, isoleucine, aspartic acid, tryptophan, and threonine), Ditols (glycerol, 1,2-propanediol, propylene glycol, etc.), 1, 3-propanediol, and 1,3-butanediol, etc.), polyethylene glycol ( For example, PEG 400, and mixtures thereof. Non-limiting examples of sugars include, for example, fructose, glucose, mannose, and the like. sugar, sorbose, xylose, maltose, lactose, sucrose, trehalose, decanose Cystran, Pullulan, Dextrin, Cyclodextrin, Alpha and Beta-HP CD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose The isotonicity agent may be a monosaccharide, disaccharide, or polysaccharide, or a water-soluble glucan, including An example of such a sugar alcohol is a sugar alcohol, and the term "sugar alcohol" refers to a sugar alcohol having at least one -OH group. Sugar alcohols are defined as C(4-8) hydrocarbons that are Inositol, sorbitol, inositol, galactitol, dulcitol, xylitol Pharmaceutical compositions containing each of the isotonicity agents listed in this paragraph include: The isotonicity agent constitutes an alternative embodiment of the present invention. 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific isotonicity agents constitute alternative embodiments of the invention. Complete.

[0121] In another embodiment of the present invention, the pharmaceutical composition comprises a chelating agent. Examples include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and and mixtures thereof. The chelating agent may be present individually or in aggregates in an amount of about 0.01 mg. / mL to about 50 mg / mL, for example, about 0.1 mg / mL to about 20 mg / mL Pharmaceutical compositions containing each one of these specific chelating agents are alternative embodiments of the invention. It constitutes a state.

[0122] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include The antibacterial agent may contain one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, The therapeutic agent may include one or more protease inhibitors.

[0123] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, the stabilizer being carboxy- / Hydroxycellulose and its derivatives (HPC, HPC-SL, HPC-L, and HPMC, cyclodextrin, 2-methylthioethanol, polyethylene glycol Polyvinyl alcohol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolide salts (such as sodium chloride), sulfur-containing substances, such as monothioglycerol, or The stabilizer is, individually or in aggregates, about 0.01 mg / mL to about 5 mg / mL. It may be present at a concentration of about 0 mg / mL, for example about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific stabilizers constitute alternative embodiments of the invention.

[0124] In a further embodiment of the invention the pharmaceutical composition further comprises one or more surfactants, preferably surfactants The surfactant may include at least one surfactant, or two different surfactants. The term "agent" refers to any compound that is composed of a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. The surfactant refers to a molecule or ion of the surfactant. surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactants are present individually or in aggregates at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific surfactants are alternatives of the present invention. This constitutes an alternative embodiment.

[0125] In a further embodiment of the invention, the pharmaceutical composition further comprises one or more protease inhibitors, e.g. , EDTA, and / or benzamidine hydrochloride (HCl). They may be present individually or in aggregates at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific protease inhibitors are alternative embodiments of the invention. Configure.

[0126] The pharmaceutical compositions of the present invention are useful in reducing the formation of polypeptide aggregates during storage of the compositions. The term "amino acid base" may include one or more Amino acids (methionine, histidine, imidazole, arginine, lysine, isoleucine amine, aspartic acid, tryptophan, threonine, etc.) or their analogs Any amino acid may be present in either its free base form or its salt form. Any stereoisomer of the acid-base may be present (i.e., L, D, or mixtures thereof). Amino acid bases, individually or in combination with other amino acid bases, are present in concentrations of about 0.01 mg / mL to about It may be present at a concentration of about 50 mg / mL, for example about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific amino acid bases constitute alternative embodiments of the invention. do.

[0127] The GLP1-GDF15 fusion protein and / or the GLP1-GDF15 fusion polypeptide of the present invention Pharmaceutically acceptable salts of oligonucleotides include those formed from inorganic or organic acids or bases. Examples of such acid addition salts include the conventional non-toxic salts or quaternary ammonium salts. The following salts are available: acetate, adipate, benzoate, benzenesulfonate, citrate, camphorate Salt, dodecyl sulfate, hydrochloride, hydrobromide, lactate, maleate, methanesulfonate Salts, nitrates, oxalates, pivalates, propionates, succinates, sulfates and tartrates Examples of basic salts include ammonium salts, sodium salts, and potassium salts. Alkali metal salts, alkaline earth metal salts such as calcium salts and magnesium salts of dihydroxybutyric acid Examples of the salts include salts with organic bases such as hexylamino salts, and salts with amino acids such as arginine. Additionally, basic nitrogen-containing groups may be quaternized, for example with alkyl halides. It may be possible.

[0128] The pharmaceutical compositions of the present invention may be administered by any means that achieves their intended purpose. Examples include parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral or intraocular. The administration may be by oral route. Suitable formulations for parenteral administration include: Aqueous solutions of the active conjugate in water-soluble form, e.g., water-soluble salts, acidic solutions, alkaline solutions solutions, aqueous dextrose solutions, isotonic carbohydrate solutions, and cyclodextrin inclusion complexes. Can be obtained.

[0129] The present invention also relates to a method for the preparation of a GLP1-GDF15 fusion protein of the present invention, comprising administering to said patient a pharma- ceutical acceptable carrier. and / or a GLP1-GDF15 fusion polynucleotide. The present invention also includes methods of making the pharmaceutical compositions. The subject invention relates to a GLP1-GDF15 fusion protein and / or a GLP1-GDF15 fusion protein of the invention. The present invention also includes pharmaceutical compositions prepared by mixing any one of the polynucleotides with the nucleic acid.

[0130] Furthermore, the GLP1-GDF15 fusion protein and / or GLP1-GDF15 of the present invention The fusion polynucleotide may have one or more crystalline polymorphs or amorphous crystalline forms. These are also intended to be within the scope of the present invention. The protein and / or GLP1-GDF15 fusion polynucleotide may be dissolved in, for example, water (i.e., As used herein, the compound may form solvates with common organic solvents. The term "solvate" refers to a solvate of the GLP1-GDF15 fusion protein and / or GLP By "solvent" is meant a physical association of a .1-GDF15 fusion polynucleotide with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In the case of, for example, one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, The term "solvate" refers to a solution-phase solvate. The present invention is intended to include both isolatable and isolable solvates. Non-limiting examples of suitable solvates include Examples of the ester include ethanolate and methanolate.

[0131] Within the scope of the present invention are GLP1-GDF15 fusion proteins and / or GLP1 -GDF15 fusion polynucleotides and solvates thereof are included. In addition, the term "administration" in the treatment method of the present invention includes administration of the GLP1-GDF15 fusion tag of the present invention. and / or GLP1-GDF15 fusion polynucleotides, or Polymorphs or solvates thereof that are not expressly intended to be included within the scope of the present invention. to treat, ameliorate, or prevent the syndromes, disorders, or diseases described herein. The provisions of this section include the means to:

[0132] In another embodiment, the present invention provides a method for the preparation of a GLP1-GDF1 5 fusion proteins and / or GLP1-GDF15 fusion polynucleotides.

[0133] The present invention relates within its scope to a GLP1-GDF15 fusion protein and / or G In general, such prodrugs include prodrugs of LP1-GDF15 fusion polynucleotides. The prodrugs include GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion proteins. A functional derivative of a synthetic polynucleotide that is capable of expressing the desired GLP1-GDF15 fusion protein. and / or GLP1-GDF15 fusion polynucleotides in vivo. Thus, the term "administration" in the treatment methods of the present invention includes any of the following: The specifically disclosed GLP1-GDF15 fusion proteins and / or The present invention relates to treatment with a GLP1-GDF15 fusion polynucleotide or to a method specifically disclosed herein. At least, specific GLP1-GDF15 fusion proteins and and / or a GLP1-GDF15 fusion polynucleotide that is converted to a GLP1-GDF15 fusion polynucleotide The present invention also includes treatment with a GLP1-GDF15 fusion protein and / or a GLP1-GDF15 fusion polynucleotide. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in , “Design of Prodrugs” (Ed. H. Bundgaard, Els Evier, 1985).

[0134] The GLP1-GDF15 fusion protein and / or the GLP1-GDF15 fusion polypeptide of the present invention During any of the processes for preparing oligonucleotides, sensitivity in any of the molecules involved It may be necessary and / or desirable to protect groups or reactive groups. This may be achieved by rotective Groups in Organic Chemistry, ed. .JFW Mcommie, Plenum Press, 1973, and TWGr eene & PGMWuts,Protective Groups in O rganic Synthesis,John Wiley & Sons,1991( each of which is incorporated herein by reference in its entirety for all purposes. This can be achieved by means of conventional protecting groups, such as those described in In a subsequent step, if desired, it can be removed using methods known in the art.

[0135] How to use The present invention relates to GDF15 receptor (GDF15F, GFRAL)-mediated syndromes and / or GL Preventing, treating, or ameliorating a P1 receptor-mediated syndrome, disorder, or disease. The method comprises administering to a subject in need thereof an effective amount of the GLP1-GDF1 of the present invention. 5. Fusion Proteins, GLP1-GDF15 Fusion Polynucleotides, and / or Pharmaceutical Compositions to a subject in need thereof.

[0136] The present invention relates to a method for treating a disorder, disease, or condition, or any of said disorders, diseases, or conditions. Preventing, treating, delaying the onset of, or ameliorating one or more symptoms of in a subject in need thereof, comprising administering an effective amount of the GLP1-GD F15 fusion protein, GLP1-GDF15 fusion polynucleotide, and / or pharmaceutical composition Also provided are methods that include administering the composition to a subject in need thereof.

[0137] According to certain embodiments, the disease disorder or condition is obesity, type I or type II diabetes, metastasis, glycemic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance ( For example, glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, congenital hyperinsulinemia, Hypoglycemia, dyslipidemia, atherosclerosis, and diabetic nephropathy due to thrombocytopenia (CHI) and hypertension and hypertension associated with uncontrolled cholesterol and / or lipid levels. Other cardiovascular risk factors, such as osteoporosis, inflammation, and nonalcoholic fatty liver disease disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, eczema, sleep apnea Consisting of rheumatoid arthritis, osteoarthritis, polycystic ovarian syndrome, chronic kidney disease, depression, and / or cancer. is selected from the group.

[0138] According to certain embodiments, a therapeutically effective amount is one, two, three, four, or more of the following effects: (i) the disease, disorder, or condition being treated, or reducing the severity of or ameliorating a disease condition or symptoms associated therewith; i) shortening the duration of the disease, disorder or condition being treated, or the symptoms associated therewith; or (iii) preventing the progression of the disease, disorder or condition being treated, or symptoms associated therewith. (iv) causing regression of the disease, disorder or condition being treated or symptoms associated therewith. (v) causing the progression of the disease, disorder or condition being treated or symptoms associated therewith; (vi) to prevent the onset or progression of the disease, disorder, or condition being treated, or related thereto; (vii) preventing the recurrence of the disease, disorder, or condition being treated or its associated symptoms; (viii) reducing hospitalization of subjects with symptoms associated with the disease being treated; Reducing the length of hospital stay for subjects with a disorder or condition, or symptoms associated therewith; (ix) the survival rate of subjects with the disease, disorder, or condition being treated, or a symptom associated therewith. (iv) enhancing the ability of a subject to treat or inhibit the disease, disorder, or condition being treated, or (xii) preventing or reducing the prophylactic or therapeutic effect of another treatment; (xiii) enhancing or ameliorating the disease, disorder or condition being treated Improving the quality of life of subjects with a condition.

[0139] The therapeutically effective amount or dose depends on the disease, disorder or condition being treated, the means of administration, the target site, and the subject's Physiological condition (including, for example, age, weight, health status), whether the subject is human or animal the type of medication being administered, other medications being administered, and whether the treatment is prophylactic or therapeutic, etc. Treatment doses may vary depending on a variety of factors, including the dosage and dosage of the drug. Optimally titrated up.

[0140] As used herein, "treat," "treating," and and "treatment" all refer to at least one condition related to a disease, disorder, or condition. refers to the improvement or reversal of at least one measurable physical parameter in a subject. The term "treatment" is used to refer to a condition that is not necessarily recognizable in the subject, but may be recognizable in the subject. "Treat," "treating," and "treatment" also refer to the treatment of a disease. to cause regression of, prevent the progression of, or at least slow the progression of, a disease, disorder, or condition; In certain embodiments, the term "treat" or "treat "Treatment" and "treatment" refer to the treatment of a disease, disorder, or condition. It refers to the alleviation, prevention of progression or onset, or shortening of the duration of one or more symptoms. In embodiments, "treat," "treat" and "treatment" refer to the remission of a disease, disorder or condition. In certain embodiments, "treat", "treat" and "treatment" refer to: It refers to improving the survival rate of a subject with a disease, disorder, or condition. "Treat," "treat," and "treatment" refer to the elimination of a disease, disorder, or condition in a subject. vinegar.

[0141] In one embodiment, the present invention provides a method for treating obesity, or any one or more symptoms of obesity. To prevent, delay, or ameliorate the onset of a disease in a subject in need thereof. The present invention provides a method for administering to a patient a therapeutically effective amount of a GLP1-GDF15 fusion protein of the present invention, The GLP1-GDF15 fusion polynucleotide and / or pharmaceutical composition may be administered to a subject in need thereof. In some embodiments, the subject's weight is determined by administering to the subject a dose of 0.1 mg / kg or more of the active ingredient as described herein. The GLP1-GDF15 fusion protein and the GLP1-GDF15 fusion polynucleotide of the present invention the body weight of the subject prior to administration of any of the otes, pharmaceutical compositions, forms, or agents, or The GLP1-GDF15 fusion protein of the present invention as described in the specification, have not received any of the following polynucleotides, pharmaceutical compositions, formulations, drugs, or combinations: For example, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to approx. 1%, approx. 1% to approx. 5%, approx. 2% to approx. 3%, approx. 5% to approx. 10%, approx. 10% to approx. 15%, approx. 15% to approx. 20%, approx. 20% to approx. 25%, approx. 25% to approx. 30%, approx. 30% to approx. Reduced by 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50% .

[0142] In some embodiments, the weight loss is for 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 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 , maintained for about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years .

[0143] The present invention relates to a syndrome, disorder, or disease, or a compound thereof. Prevent, treat, delay the onset of, or ameliorate any one or more symptoms of and a method for administering to a subject in need thereof a compound that inhibits the administration of ... The disease may be obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome ROHM X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia Hyperglycemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia Normal blood vessels, atherosclerosis, diabetic nephropathy, and uncontrolled cholesterol and / or other cardiovascular risk factors, such as hypertension and cardiovascular risk factors related to lipid levels; Osteoporosis, inflammation, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), renal disease, and eczema. The present invention relates to a method for producing a GLP1-GDF15 fusion protein and a GLP1-GDF15 fusion polynucleotide. and / or administering the pharmaceutical composition to a subject in need thereof.

[0144] As used herein, metabolic syndrome refers to hyperglycemia (e.g., high fasting blood sugar), high blood pressure, abnormal cholesterol levels (e.g., low HDL levels), abnormal triglycerides Seride levels (e.g., high triglycerides), a large waistline (i.e., waist circumference), Increased fat in the abdominal area, insulin resistance, impaired glucose tolerance, and high C-reactive protein levels (i.e., proinflammatory state), and plasma plasminogen activator inhibitor-1 and fibrinogen The term "thrombotic status" refers to a subject having any one or more of the following:

[0145] The present invention provides a method for reducing food intake in a subject in need thereof. The method further comprises administering to the subject a therapeutically effective amount of the GLP1-GDF15 fusion protein of the present invention, Administering DF15 fusion polynucleotides and / or pharmaceutical compositions to a subject in need thereof In some embodiments, the subject's food intake includes administering to the subject the food intake according to the present invention as described herein. GLP1-GDF15 fusion proteins and GLP1-GDF15 fusion polynucleotides, with respect to the subject's food intake prior to administration of any of the pharmaceutical compositions, forms, or medicaments, or The GLP1-GDF15 fusion protein of the present invention described in the document Controls not receiving any of the oligonucleotides, pharmaceutical compositions, formulations, agents, or combinations. Compared to the subject, for example, about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0. 5% to approx. 1%, approx. 1% to approx. 5%, approx. 2% to approx. 3%, approx. 5% to approx. 10%, approx. 10% to approx. 15 %, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35 %, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50%.

[0146] In some embodiments, the reduction in food intake is for about 1 week, about 2 weeks, about 3 weeks, about 1 month, Months, 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 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 6 years maintained for about 1 year, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years can be.

[0147] The present invention relates to a method for reducing glycated hemoglobin (A1C) in a subject in need thereof. The present invention provides a method for treating a patient suffering from atopic dermatitis, the method comprising administering an effective amount of a GLP1-GDF15 fusion protein of the present invention to a patient suffering from atopic dermatitis. The present invention relates to a method for the preparation of a pharmaceutical composition, a GLP1-GDF15 fusion polynucleotide, and / or a pharmaceutical composition comprising administering to a subject a subject in need thereof. In some embodiments, the subject's A1C can be determined by administering to the subject a dose of 100 mg / kg of the subject's A1C according to the methods described herein. The GLP1-GDF15 fusion protein of the present invention described in the document of a subject prior to administration of any of the oligonucleotides, compositions, formulations, agents, or combinations. A1C or a GLP1-GDF15 fusion protein of the invention as described herein; GLP1-GDF15 fusion polynucleotides, compositions, forms, agents, or combinations For example, about 0.001% to about 0.01%, About 0.01% to about 0.1%, about 0.1% to about 0.2%, about 0.2% to about 0.3%, about 0. 3% to approx. 0.4%, approx. 0.4% to approx. 0.5%, approx. 0.5% to approx. 1%, approx. 1% to approx. 1.5% , about 1.5% to about 2%, about 2% to about 2.5%, about 2.5% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, or is reduced by about 9% to about 10%.

[0148] In other embodiments, reducing fasting blood glucose levels is achieved in a subject in need thereof. The method further comprises administering to the subject a therapeutically effective amount of a GLP1-GDF15 fusion protein of the invention. The present invention relates to a method for the preparation of a pharmaceutical composition, a GLP1-GDF15 fusion polynucleotide, and / or a pharmaceutical composition comprising administering to a subject a subject in need thereof. The fasting blood glucose level can be measured by administering to a subject in need thereof the G GLP1-GDF15 fusion proteins, GLP1-GDF15 fusion polynucleotides, compositions the fasting blood glucose level of the subject prior to administration of any of the substances, forms, agents, or combinations thereof; or the GLP1-GDF15 fusion protein of the invention, GLP1- Any GDF15 fusion polynucleotide, composition, formulation, agent, or combination Compared to non-treated control subjects, Less than 30 mg / dL, about 130 to less than 120 mg / dL, about 120 to 110 mg / dL less than about 110 to about 100 mg / dL, about 100 to about 90 mg / dL, or about It can be reduced to less than 90 to about 80 mg / dL.

[0149] The present invention relates to a method for regulating GLP1 receptor activity and GDF15 receptor activity. The present invention provides a method for administering to a subject in need thereof an effective amount of a GLP1-GD F15 fusion protein, GLP1-GDF15 fusion polynucleotide, and / or pharmaceutical composition As used herein, "preparation" includes administering a composition to a subject in need thereof. "Inhibit" refers to increasing or decreasing receptor activity.

[0150] In some embodiments, an effective amount of a GLP1-GDF15 fusion protein of the invention and and / or GLP1-GDF15 fusion polynucleotide, or a form, composition, or The drug is administered once daily, twice daily, three times daily, four times daily, or five times daily to subjects who need it. In another embodiment, an effective amount of the compound of the present invention is administered once a day, six times a day, seven times a day, or eight times a day. GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide The nucleotide, or a form, composition, or medicament thereof, is administered to a subject in need thereof within 2 days. 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, It is given three times a month or four times a month.

[0151] Another embodiment of the invention is a method for treating a disease, disorder, or syndrome, or a method for treating said disease, disorder, or syndrome. to prevent, treat, delay the onset of, or otherwise affect one or more symptoms of the syndrome; The present invention includes a method of inducing remission in a subject in need thereof, the method comprising administering In the therapy, an effective amount of the GLP1-GDF15 fusion protein of the present invention, GLP1-GDF15 Administering the fusion polynucleotide and / or pharmaceutical composition to a subject in need thereof. In certain embodiments, the combination therapeutic agent is a second therapeutic agent. , the combined therapy is surgical therapy.

[0152] As used herein, the term "combined" refers to the administration of two or more therapeutic agents to a subject. In this context, it refers to the use of multiple therapeutic agents.

[0153] As used herein, combination therapy refers to the administration of an effective amount of a GLP1-GDF15 fusion of the invention. Proteins and / or GLP1-GDF15 fusion polynucleotides or forms thereof, The composition or drug may be administered simultaneously with one or more additional therapeutic agents or one or more surgical therapies. In some embodiments, one or more additional therapies are administered to a subject in need thereof. The drug or surgical treatment may comprise an effective amount of the GLP1-GDF15 fusion protein of the invention and / or G In other embodiments, one or more The above additional therapeutic or surgical therapy may be administered in the form of an effective amount of a GLP1-GDF15 fusion protein of the invention. The subject may be administered the same week or month as the GLP1-GDF15 fusion polynucleotide and / or the do.

[0154] The present invention also relates to the treatment of any of the diseases, disorders, syndromes, or conditions described herein using combination therapy. The term "preventing, treating, delaying the onset, or ameliorating a disease using a method" refers to The combination therapy is also contemplated to be carried out in a subject in need thereof, and the combination therapy comprises an effective amount of GLP1 of the present invention. -GDF15 fusion proteins, GLP1-GDF15 fusion polynucleotides, and / or The pharmaceutical composition may be used in combination with any one or more of the following therapeutic agents: administering to the subject: a dipeptidyl peptidase-4 (DPP-4) inhibitor (e.g., (e.g., sitagliptin, saxagliptin, linagliptin, alogliptin, etc.);GL P1 receptor agonists (e.g., short-acting, such as exenatide and lixisenatide) GLP1 receptor agonists; intermediate-acting GLP1 receptor agonists such as liraglutide; Long-acting GLP-1 drugs, such as extended-release exenatide, albiglutide, and dulaglutide receptor agonists; sodium-glucose cotransporter-2 (SGLT-2) inhibitors (e.g. (e.g., canaglifozin, dapaglifozin, empaglifozin, etc.); bile acid sequestrants (e.g., colesevelam, etc.); dopamine receptor agonists (e.g., bromocriptine, fast release insulin; biguanides (e.g. metformin); insulin; oxytomod sulfonylureas (e.g., chlorpropamide, glimepiride, glipizide, glimepiride, Brid, glibenclamide, glibornuride, glisoxepide, glyclopyramide, trametinib azamide, tolbutamide, acetohexamide, cabutamide, etc.); and thiazolidinediones (e.g., pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, dalglitazone, (e.g., ritazone, englitazone, netoglitazone, rivoglitazone, troglitazone, etc.) In some embodiments, the dose of the additional therapeutic agent(s) is 5 fusion protein and / or GLP1-GDF15 fusion polynucleotide In some embodiments, the GLP1-GDF15 of the present invention is reduced when administered. In combination with fusion proteins and / or GLP1-GDF15 fusion polynucleotides When used together, the additional therapeutic agent(s) have a lower It may be used in doses.

[0155] The present invention relates to a method for treating any of the diseases, disorders, syndromes, or conditions described herein using combination therapy. to prevent, treat, delay the onset of, or ameliorate a disease or condition that requires The combination therapy is intended to be carried out in a subject for whom the ... GDF15 fusion proteins, GLP1-GDF15 fusion polynucleotides, and / or pharmaceuticals The composition is administered to a subject in need thereof in combination with a surgical treatment. In certain embodiments, the surgical therapy is bariatric surgery (e.g., Roux-en-Y gastric bypass surgery, etc. Which gastric bypass surgery; sleeve gastrectomy; adjustable gastric band surgery; duodenal switch gastric diversion; intragastric balloon; gastric plication, and combinations of these) .

[0156] One or more additional therapeutic or surgical therapies may be administered in the form of an effective amount of a GLP1-GDF15 fusion of the invention. The protein and / or GLP1-GDF15 fusion polynucleotide are administered on the same day. In an embodiment, the GLP1-GDF15 fusion protein and / or GLP1-GDF 15 The fusion polynucleotide may be administered before, after, or simultaneously with additional therapeutic or surgical therapy. The use of the term "in combination" does not restrict the order in which therapeutic agents are administered to a subject. For example, a first therapeutic agent (e.g., a composition described herein) can be administered to a subject in combination with a second therapeutic agent. Before administration of the drug (e.g., 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h) 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, at the same time as, or after (e.g. For example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 The administration can be made after 1 week, 6 weeks, 8 weeks, or 12 weeks.

[0157] Embodiment The present invention also provides the following non-limiting embodiments. Embodiment 1 is a glucagon-like peptide-1 (GLP1) / growth factor 15 (GDF1 5) A fusion protein, the GLP1-GDF15 fusion protein is a GLP1 peptide. , a first linker peptide, a serum albumin protein, a second linker peptide, and Contains GDF15 protein.

[0158] In a second embodiment, the GLP1 peptide is an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4. The GLP1-GDF15 fusion protein of embodiment 1 comprises the amino acid sequence

[0159] In a third embodiment, the first linker peptide is selected from the group consisting of SEQ ID NOs: 5 to 25. 3. The GLP1-GDF15 fusion protein according to embodiment 1 or 2, comprising an amino acid sequence It is.

[0160] In a fourth embodiment, the serum albumin protein is selected from SEQ ID NO: 26 or SEQ ID NO: 27. GLP1-GDF1 according to any one of embodiments 1 to 3, comprising an amino acid sequence as defined above. 5 fusion protein.

[0161] In a fifth embodiment, the second linker peptide is selected from the group consisting of SEQ ID NOs: 28 to 30. GLP1-GDF15 according to any one of embodiments 1 to 4, comprising an amino acid sequence It is a fusion protein.

[0162] In a sixth embodiment, the GDF15 protein is selected from SEQ ID NO: 31 or SEQ ID NO: 32. 6. The GLP1-GDF15 fusion protein according to any one of embodiments 1 to 5, comprising an amino acid sequence It is a synthetic protein.

[0163] In embodiment 7, the GLP1-GDF15 fusion protein is selected from the group consisting of SEQ ID NOs: 33-74 and 84. A GLP1-GDF15 fusion protein comprising an amino acid sequence selected from the group consisting of: be.

[0164] Embodiment 8 is a method for producing a GLP1-GDF15 fusion protein according to any one of embodiments 1 to 7. An isolated nucleic acid encoding a protein.

[0165] Embodiment 9 is a vector comprising the isolated nucleic acid of embodiment 8.

[0166] Claim 10 relates to a host cell comprising the isolated nucleic acid of embodiment 8 or the vector of claim 9. It is a cell.

[0167] Embodiment 11 is a method for producing a GLP1-GDF15 fusion protein according to any one of embodiments 1 to 7. The pharmaceutical composition comprises a protein and a pharma- ceutical acceptable carrier.

[0168] Embodiment 12 relates to treating or preventing obesity in a subject in need thereof. A method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of embodiment 11. The method includes providing a

[0169] Embodiment 13 is a method for treating a patient suffering from atopic dermatitis by administering an effective amount of a pharmaceutical composition to a subject in need thereof. and about 5% to about 10%, about 10% to about 15%, or about 20% to about 25%, or about 5% to about 10%, or about 20% to about 25%, or about 5% to about 10%, or about 5% to about 15 ... %, about 15% to about 20%, or about 20% to about 25% body weight, This is the method described above.

[0170] Example 14 is directed to treating or preventing a disease or disorder in a subject in need thereof. The method is carried out by administering a therapeutic agent to a subject having a disease or disorder, the disease or disorder being obesity, type I or type II diabetes, metabolic syndrome, Syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia Lysineemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atheroma arteriosclerosis, diabetic nephropathy, and uncontrolled cholesterol and / or lipid Other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with quality levels, osteoporosis, inflammation Disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) , renal disease, and eczema, and the method further comprises administering to the subject an effective amount of any one of claims 1 to 11. The method comprises administering the pharmaceutical composition described above to a subject in need thereof.

[0171] Embodiment 15 is the method of embodiment 14, wherein the disease or disorder is obesity.

[0172] Embodiment 16 relates to the method of embodiment 14, wherein the disease or disorder is type I diabetes. be.

[0173] Embodiment 17 is the method of embodiment 14, wherein the disease or disorder is type II diabetes. It is.

[0174] Embodiment 18 is a method according to embodiment 14, wherein the disease or disorder is metabolic syndrome. This is a method according to the present invention.

[0175] Embodiment 19 is the method of embodiment 14, wherein the disease or disorder is a renal disease. .

[0176] In embodiment 20, the disease or disorder is nonalcoholic steatohepatitis (NASH). 15. The method according to embodiment 14.

[0177] In embodiment 21, the disease or disorder is non-alcoholic fatty liver disease (NAFLD). 15. The method according to embodiment 14,

[0178] Embodiment 22 relates to a method of reducing food intake in a subject in need thereof. The method comprises administering an effective amount of the pharmaceutical composition of embodiment 11 to a patient in need thereof. administering to the subject.

[0179] Embodiment 23 is a method for treating a patient suffering from atopic dermatitis by administering an effective amount of a pharmaceutical composition to a subject in need thereof. and about 5% to about 10%, about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 5% to about 50%, or about 6% to about 60%, or about 7% to about 80%, or about 9% to about 90%, or about 10% to about 10 ...20 15%, approx. 15% to approx. 20%, approx. 20% to approx. 25%, approx. 25% to approx. 30%, approx. 30% to approx. 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50% food intake 23. The method of embodiment 22, wherein the

[0180] Embodiment 24 relates to a method for modulating GLP1 receptor activity in a subject in need thereof. The method comprises administering an effective amount of the pharmaceutical composition of embodiment 11 to a patient in need thereof. administering to a subject in need thereof.

[0181] Embodiment 25 relates to a method for modulating GDF15 receptor activity in a subject in need thereof. The method comprises administering an effective amount of the pharmaceutical composition of embodiment 11 to a patient having a pulmonary circulation, to a subject in need thereof.

[0182] Embodiment 26 is any one of embodiments 11 to 25, wherein the pharmaceutical composition is administered by injection. This is one method.

[0183] Embodiment 27 is an embodiment in which the injection is delivered subcutaneously, intramuscularly, intraperitoneally, or intravenously. 26.

[0184] Embodiment 28 is a variant of embodiment 1, in which the pharmaceutical composition is administered in combination with a second therapeutic agent. The method according to any one of 1 to 27.

[0185] Embodiment 29 is a method for administering the pharmaceutical composition daily, weekly, or monthly to a subject in need thereof. The method according to any one of embodiments 11 to 28, wherein

[0186] Embodiment 30 is a pharmaceutical composition administered once, twice, three times, four times, five times, or six times per day. 30. The method of embodiment 29, wherein

[0187] Embodiment 31 is a pharmaceutical composition administered once, twice, three times, four times, five times, or six times per week. 30. The method of embodiment 29, wherein

[0188] Embodiment 32 is a method for administering the pharmaceutical composition once, twice, three times, or four times a month. 30. The method according to embodiment 29.

[0189] Embodiment 33 is a method for producing a GLP1-GDF15 fusion protein according to any one of embodiments 1 to 7. 9. A kit comprising the protein of embodiment 8, the isolated nucleic acid of embodiment 8, and / or the vector of embodiment 9. .

[0190] Embodiment 34 is a kit according to embodiment 33, wherein the kit further comprises an injection device. be.

[0191] Embodiment 35 is a method for producing a GLP1-GDF15 fusion protein according to any one of embodiments 1 to 7. A method for producing a pharmaceutical composition comprising a GLP1-GDF15 fusion protein, with a pharma- ceutical acceptable carrier to obtain a pharmaceutical composition.

[0192] Embodiment 36 is a method for producing a GLP1-GDF15 fusion protein according to any one of embodiments 1 to 7. A method for producing a protein comprising: Cultivating the acid-containing cells under conditions that produce the GLP1-GDF15 fusion protein and recovering the GLP1-GDF15 fusion protein from the cell or cell culture. The present invention relates to a method for EXAMPLES

[0193] Example 1: Combination of GLP1 and GDF15 agonists The potential additive weight loss effects of combining GLP1 and GDF15 agonists were examined in a diet-based setting. The study was conducted in diet-induced obese (DIO) mice. Liraglutide, a GLP1 agonist alone, , or in combination with the HSA-GDF15 (SEQ ID NO: 81) molecule for 8 days. -GDF15 was given every 2 days, while liraglutide was administered subcutaneously daily. Any single agent that demonstrates the potential for additivity when these independent mechanisms are combined was greater in animals receiving both agonists compared with those receiving either agonist (Fig. 1 ).

[0194] Example 2: Design of a GLP1-GDF15 fusion protein GLP1 peptide or GLP1 variant peptide, serum albumin protein, and GD The recombinant fusion protein consisting of the F15 protein or the GDF15 mutant protein is The GLP1 peptide or the GLP1 variant peptide was designed as follows: or via a first linker peptide at the C-terminus of the GLP1 variant peptide, Human serum albumin was linked to the N-terminus of albumin via a second linker peptide at the C-terminus. The present invention binds to the N-terminus of GDF15 protein or a GDF15 protein mutant via a tide. This design avoids perturbing the GDF15 dimerization interface and avoids the native interchain disulfide. The aim is to enable the formation of a homodimer of approximately 170 kDa. The organism was designed to be produced recombinantly and only required one gene ( Figure 3).

[0195] The GLP1 peptide or GLP1 mutant peptide may be a human GLP1 peptide having a mutation ( 7-36) Peptides from Gila monster that agonize human GLP1 receptors and exendin 4(9-39). (A8S, A30E) mutations (numbering of mutation positions is from the start of the fusion protein refers to the GLP1 peptide variants in Table 1, and SEQ ID NO: 2 refers to the human GLP1 peptide variants (A8G, G22E, R3 SEQ ID NO: 3 contains the exendin 4 peptide (UniProtKB -P26349 48-86).

[0196] Native human serum albumin (UniProtKB-P02768 25-609) is Contains 35 cysteine ​​(Cys, C) residues that form 7 intramolecular disulfide bonds, making it unique This free Cys-34 is capable of reacting with multiple reactive oxygen species. Free radical scavenging by capturing reactive oxygen species (ROS) and reactive nitrogen species (RNS) It has been shown that the jar functions as a Intensive Care, 3:4 (2013)). Therefore, this free Cys was converted to Se. r to generate SEQ ID NO:26, minimizing the risk of heterogeneity due to chemical reactivity and oxidation. It has been miniaturized.

[0197] The N-terminus of mature GDF15 (UniProtKB-Q99988 197-308) is It contains the site responsible for proteolysis (R198) and the site responsible for deamidation (N199). Therefore, the fusion proteins were designed to express GDF15(201-308) with their responsible sites deleted. ) (SEQ ID NO:31).

[0198] Example 3: Expression and purification methods GLP1-GDF15 fusion protein, GLP1-first linker-serum albumin ( Serum albumin (e.g., HSA, GSA)- 2 linker-GDF15 protein, and / or the GDF15 protein used in the above examples. Proteins were cultured in HEK Expi293F™ (ThermoFisher Science). ntific, Catalog No. A14527) or ExpiCHO-S™ (Ther moFisher Scientific, Catalog No. A29127; Waltham For expression in HEK Expi293F™, The plasmid encoding the GLP1-GDF15 fusion protein was prepared according to the manufacturer's recommendations. Therefore, cells were transfected by transient transfection. Expi293F cells were incubated in a shaking incubator set at 37°C, 8% CO2 and 125 RPM. In a incubator, Expi293™ Expression Medium (ThermoFisher Science On the day of transfection, the cells were maintained in suspension in 1 mL of entificant. 2.5×10 6 Subculture the cells so that they can be diluted into cells and ensure cell viability of 95% or more. Transient transfections were performed using ExpiFectamine™ 2 93 Transfection Kit (ThermoFisher Scientific) One microgram of plasmid was used for every 1 mL of diluted cells to be transfected. The smid DNA was diluted in OptiMEM™ SFM complex medium. Tamine™ 293 reagent was used in a ratio of 1:2.6 (v / v, DNA:reagent). This was also diluted in OptiMEM™ and allowed to incubate at room temperature for 5 minutes. The DNA and transfection reagent were combined for 20 minutes to form DNA / lipid complexes. After overnight incubation, Expi293™ filters were added to the cells. The cells were then treated with 500 mM erythrocyte sequestration medium and ExpiFectamine™ 293 enhancer. The cells were cultured at 37° C. for 4 days with shaking, and the culture supernatant was then harvested.

[0199] ExpiCHO-S™ cells were incubated at 37° C., 8% CO2 and 125 RPM. In a shaking incubator, culture the cells in ExpiCHO™ Expression Medium (ThermoFisher On the day of transfection, the cells were maintained in suspension in 1 ml 6.0 x 10 per L 6 Passaging cells so that cells can be diluted and cell viability The transfection was performed using ExpiFectamine ( CHO Transfection Kit (ThermoFisher Scientific For every 1 mL of diluted cells to be transfected, 1 microliter of gram of plasmid DNA was used and diluted in OptiPRO™ SFM complex formation medium. Add ExpiFectamine™ CHO Reagent in a 1:3 ratio (v / v, DNA The diluted DNA and transfection are used in the same manner as above. The transfection reagent is combined for 1 min to allow the DNA / lipid complexes to form, and then the cells are After overnight incubation, ExpiCHO™ feed and Expi Fectamine™ CHO enhancer was added to the cells. The cells were incubated at 32° C. for 5 days. After culturing with shaking for 1 h, the culture supernatant was collected.

[0200] GLP1-GDF15 fusion proteins were prepared by single-step affinity capture or affinity capture followed by Either of the two-step processes using a size exclusion chromatography (SEC) polishing step for preparation Transiently transfected ExpiCH was purified from the culture supernatant by Cell supernatant from O™ cells was added at an appropriate volume of 10 mg protein per mL of resin. , pre-equilibrated (dPBS, pH 7.2) HSA CaptureSelect column (CaptureSelect provided by ThermoFisher Scientific The results were loaded onto the Human Albumin Affinity Matrix (Human Albumin Affinity Matrix). After loading, the column was washed with up to 12 column volumes (CV) of dPBS pH 7.2, followed by and washing with 3 CV of 1 M NaCl in 50 mM sodium phosphate, pH 7.4. The unbound proteins and impurities were removed by elution. The fusion protein was diluted with 10% 0.1 M sodium acetate, pH 3.5 for up to 10 CV. % (by volume) of 1 M Tris (untitrated). Peak fractions were pooled. After filtering through a 0.2 μm membrane, the buffer was exchanged for dPBS (pH 7.2) or Either the mixture was cooled to 30°C or proceeded to the SEC step.

[0201] In the SEC step, the protein obtained in the capture step is concentrated to an appropriate volume and then separated by 26 / 60 Superdex200 column (GE Healthcare; Little Chal The SEC column was loaded with high purity. The eluted protein fractions (measured by SDS-PAGE) were pooled. Protein concentrations (obtained by either method) were analyzed using a BioTek Synergy HT™ spectrophotometer. The absorbance at 280 nm was measured in a spectrophotometer. The quality of the purified protein was confirmed by SDS-PAH chromatography. GE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system Endotoxin levels were assessed by LAL assay (Pyrotell Registered trademark)-T, Associates of Cape Cod;East Falm Measurements were performed using a 30-μm CT scanner (outh, MA).

[0202] Example 4: Dual targeting by delivery of both GLP1 and GDF15 agonists on one molecule Combined and additive effects Additive effects achieved by delivering both GLP1 and GDF15 agonists on a single molecule The possibility of obtaining this potential was investigated by administering the agonist fusion with gorilla serum albumin (GSA) for 8 days. The study was carried out by assessing the weight loss of DIO mice. Mice were treated with 8 nmol / kg GLP1-GSA as a surrogate for HSA in offspring. -GDF15 (SEQ ID NO: 72), GLP1-GSA-GDF15 (I89R) (SEQ ID NO: 73, I89R mutation abolishes GDF15 activity), GLP1(9-36)-GSA- Received subcutaneous administration of GDF15 (SEQ ID NO: 74) or GSA-GDF15 (SEQ ID NO: 75) The latter two were both designed to recreate degraded GLP1. Groups of animals served as controls and benchmarks, one group receiving only the vehicle and the other receiving the GLP1 antagonist. The patient was administered dulaglutide, a stimulant for the treatment of rheumatoid arthritis. Mice were administered GLP1(9-36)-GSA-GDF15 (SEQ ID NO: 74), G LP1-GSA-GDF15(I89R) (SEQ ID NO: 73), and GSA-GDF15( Mice administered SEQ ID NO: 75) lost more weight and showed increased expression of GLP1R and GFRAL The concept that both can be agonized by a single molecule, resulting in additive effects. This provided evidence (Figure 2).

[0203] Example 5: In vitro human GLP1R potency, ex vivo human, and in vivo mouse plasma GLP1 peptide or GLP1 peptide variant and first linker peptide for stability The effect of Different GLP1 peptides or GLP1 peptide variants and different first linker peptides The data show that hGLP1-overexpressing HEK cells are involved in the regulation of cyclic adenosine monophosphate (cAMP) levels. had different effects on in vitro hGLP1 receptor potency in assays measuring The Lance competitive cAMP immunoassay (Perkin Cell-based assays to measure intracellular cAMP production using a 100-μg / mL ELISA kit (Elmer, Waltham, MA) Fusions were screened for in vitro GLP1R potency in a 3-drug assay. Clonal HEK293 cells stably expressing mouse or human GLP1R were used in the assay. The data obtained were used to analyze the data using Prism statistical software (GraphPad Compound EC 50 The value was calculated In general, the GLP1 variant (SEQ ID NO: 1) exhibits a potent inhibitory effect in an in vitro hGLP1R assay. , is less potent than the other two GLP1 variants (SEQ ID NO:2 and SEQ ID NO:3).

[0204] Ex vivo stability in human plasma was evaluated. Briefly, fresh, non-frozen human blood Plasma was prepared from heparinized blood by centrifugation. The fusion protein was added without gentle mixing. Then, the cells were incubated in this substrate for 0, 4, 24, 48, 72, and 96 hours at 37°C. The stability of the GLP1 domain of the fusion molecule over time in human plasma was examined using immunoaffinity capture–transformation assays. Trypsin digestion was monitored by LC-MS / MS analysis. Selected trypsin The peptide, HSE (HSEGTFTSDVSSYLEGQAAK) (SEQ ID NO: 7 6), HGE-1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77), and and HGE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78) is a GLP1 peptide or These are located at the N-terminus of the GLP1 mutant peptides (SEQ ID NOs: 1, 2, and 3), respectively. Selected tryptic peptides from these were subjected to anti-GDF15 immunoaffinity capture and tryptic digestion. The results were then monitored by LC-MS / MS to determine whether the GLP1 had an intact N-terminus. By this method, all molecules tested were found to be significantly higher in human plasma than The results showed moderate stability of GLP1 over time.

[0205] The in vivo stability in mice was evaluated. The fusion protein was diluted with 2 mM The drug was administered subcutaneously to male C57Bl / 6 mice at a dose of 1000 mg / kg. Blood samples were collected at 0, 4 and 6 h after administration. K3E Sarstedt blood collection tubes with protease inhibitors at 24 and 48 hours Plasma was prepared by centrifugation. The stability of the GLP1 domain of the protein over time was assessed using immunoaffinity capture-trypsin digestion-LC-MS / MS analysis. Selected tryptic peptides, namely HSE( HSEGTFTSDVSSYLEGQAAK) (SEQ ID NO: 76), HGE-1 (HGEG TFTSDVSSYLEEQAAK) (SEQ ID NO: 77), and HGE-2 (HGEGTF TSDLSK) (SEQ ID NO: 78) is a fusion protein containing SEQ ID NOs: 1, 2, and 3 These selected tryptic peptides were analyzed using anti-GDF15 After immunoaffinity capture and trypsin digestion, the N-terminal This method was used as a surrogate measure of the concentration of the fusion protein containing intact GLP1. Therefore, all molecules tested induced moderate GLP1 stabilization over time in mice in vivo. It showed its sexuality.

[0206] Table 7 shows the GLP1R overexpression profiles for the indicated GLP1-GDF15 fusion proteins. in vitro GLP1 receptor potency measured by cAMP levels in live cells, both using mass spectrometry The present invention provides the results of ex vivo human plasma stability and in vivo mouse plasma stability evaluated by do.

[0207] [Table 7]

[0208] Example 6: O-linker peptide synthesis by removing serine residues in the first and second linker peptides Removal of linked xylosylation Xylose glycans have been reported to be attached to glycine-serine linkers. (Spahr et.al, mAbs 6(4):904-914), glycine Molecules containing a -serine linker were tested for O-linked xylose levels. The samples were then diluted into guanidine-HCl buffered at pH 8.0 for denaturation, followed by The solution was prepared by adding DTT for reduction and incubating at 37°C for 1 hour. After reduction, the samples were diluted with freshly prepared iodoacetamide for 6 h at room temperature in the dark. DTT was then added to the sample to remove any unreacted iodoacetamide. The samples were then diluted in 50 mM Tris, 1 mM CaCl2, pH 8.0. , using Zeba Spin Desalting columns according to the manufacturer's protocol. The digestion reaction was desalted and digested with trypsin (Promega) at 37°C for 4 hours. To quench the reaction, TFA was added to each sample. The digested samples were kept at 4°C. and injected into LC / MS within 24 hours.

[0209] The digested samples were analyzed using an Agilent Infinity 1290 UHPLC (Agilent Infinity Agile Lent Technologies at a flow rate of 0.1 mL / min nt AdvanceBio Peptide Map Micro Bore Rap The column temperature was maintained at 65°C. Mass spectrometry grade HPLC solvents (A: 0.1% formic acid and B: 100% ACN in 0.1% formic acid) ) was purchased from VWR. Proteolytic peptides were diluted in 0.1% FA at 2% to 40%. The column was eluted with a 50 min gradient of ACN at 300 rpm. The spray voltage was 3.5 kV, 20 Using a sheath gas, 7 auxiliary gas, an ion transfer tube at 299°C, and an evaporator at 100°C, The column effluent was thermally injected through a heated electrospray ionization probe (HESI). o Implemented into an Orbitrap Q-Exactive mass spectrometer.

[0210] The top two data-dependent experiments were run on a 150 nm NMR spectrometry system with Orbitrap detection, 70,000 resolution, and a mass range of 15 0-2000m / z, AGC target 1.0e6, max injection time 50ms, 1 micro The scan was performed with the precursor scan set to positive polarity. Precursor Determination The criteria were monoisotopic precursor selection, peptide, charge state: 2–7, dimer Dynamic Exclusion: 6.0 seconds, and Precursor Strength Threshold: 5e4. The cursapeptides were isolated by a quadrupole with an isolation width of 1.6 m / z and sent to the collision cell. A collision energy of 28 resulted in higher-energy collision dissociation (HCD) of the peptide. These fragments were then transferred to the Orbitrap for mass measurement. The lap settings were 17,500 resolution, 200-2000 m / z range, and AGC target 5. e5, maximum injection time 100 ms, 1 microscan, and the space acquired in centroid mode. It was Kutlu.

[0211] Peptide mapping data was collected from Byonic (Protein Metrics Inst. c) Processed using a search algorithm. The custom libraries and their monoisotopic masses were used as search parameters for Ser-specific modifications. The extracted ion chromatogram (XIC) areas of modified and unmodified peptide species were To quantify the protein-based results, Biologic search results were analyzed using Biologic (Protein Immunoassay). Imported into n Metrics Inc.

[0212] GLP1-GDF15 fusion protein containing the first and second linker peptides of G4S The protein harbors various levels of O-linked cleavage on the serine residues of the first and second linker peptides. Table 8 shows the xylosylation of three such proteins transiently expressed from ExpiCHO cells. Molecular results: Xylose levels are protein context dependent, and GLP1 peptide or a first linker peptide that links the GLP1 peptide variant to the HSA protein. The results ranged from "not detected" to 1% for HSA protein and GDF15 protein or was 11.61% in the second linker peptide bound to the GDF15 protein mutant. ~56.2%. Potential O-linked xylosyl to the linker peptide To avoid the risk of polymerization, the first and / or second linker peptides may contain a serine residue. We designed and generated GLP1-GDF15 fusion proteins that lack the GLP1- The GDF15 fusion protein may be a fusion protein comprising a linker containing AP repeats, G4A repeats, or polyglycine repeats. It contained.

[0213] [Table 8]

[0214] Example 7: In vitro potency against GLP1R and GDF15R The in vitro activity of the GLP1-GDF15 fusion protein was determined using GLP1-GDF15 expression in a cell-based assay. Both LP1R and GDF15R potency were tested. ) activity, stably overexpressing either human or cynomolgus GFRAL Phosphorylated AKT (Ser4) in transfected SK-N-AS human neuroblastoma cells 73) levels were determined by measuring GFRA levels with various concentrations of fusion molecules. Phosphorylation of AKT after treatment of L-expressing cells was measured using Phospho- AKT (Ser473) Assay kit (Cisbio, Beford, MA) was used. The data obtained were used to analyze the data using Prism statistical software (GraphP Ad Software San Diego) was used for compound EC 50 The values ​​were calculated. The GLP1-GDF15 fusion protein tested in 50 is 4.6 to 6.9 nM It has a similar potency to GDF15R in range.

[0215] The efficacy of the fusion GLP1R was examined by overexpressing human, mouse, or cynomolgus GLP1R. cAMP levels in stably transfected clonal HEK293 cells for The intracellular levels of fusion molecules were determined by measuring the intracellular levels of fusion molecules after treatment of cells with various concentrations of fusion molecules. cAMP production was measured using the Lance competitive cAMP immunoassay (P The measurements were performed using a 300-millimeter meter (Erkin Elmer, Waltham, Massachusetts). The data obtained were used to calculate the variance of the variance in the Prism statistical software (GraphPad Compound EC was performed using the software San Diego 50 values ​​were calculated.

[0216] A first linker that binds a GLP1 peptide or a GLP1 peptide variant to an HSA protein. The serine residue in the anchor peptide was replaced with alanine or removed (G4S to G4 When the linker is switched to a poly-G linker of similar length, the GLP1 activity is increased in the human or was not affected when tested in the mouse GLP1R assay. A second linker that connects the protein to the GDF15 protein or the GDF15 protein variant. - Peptides were changed from GS-8x (G4S) to GA-8x (G4A) or 10x (AP). Changing the first linker (5X(G4 A), 8X(G4A), 5X(AP), 10X(AP), 20X(AP), 25X(AP) Fusion proteins with increasing numbers of repeats of The results showed that these fusion proteins activated GLP1R signaling with varying potency. It has been demonstrated that

[0217] [Table 9]

[0218] [Table 10]

[0219] Example 8: Effect of the first and second linker peptides on protein purity and stability Size-exclusion high performance liquid chromatography (SE-HPLC) was used to identify the major species, Both high molecular weight (HMW) species representing aggregates and low molecular weight (LMW) species representing fragments were observed. The purity and stability of the molecule were examined by quantifying the proportion of To determine protein purity, 20 μg of protein was dissolved in 1x DPBS, pH 7.2 ( Gibco, Cat. No. 14190-136) mobile phase with Tosoh TSK gel l Injected onto a BioAssist G3SWXL (cat. no. 20026) column. Protein species were eluted at a flow rate of 1 mL / min at room temperature, and UV-280 nm absorbance values ​​were recorded. A Dionex Ultimate3000 HPLC system equipped with a variable wavelength detector was used. To determine protein stability, 100 μg of protein was Columns were loaded onto a Tosoh TSKgel with 0.2 M sodium phosphate, pH 7.0 mobile phase. The mixture was injected onto a BioAssist G3SWXL (catalog number 20026) column. Protein species were eluted at room temperature at a flow rate of 0.7 mL / min, and UV-280 nm absorbance values ​​were recorded as A Dionex Ultimate3000 HPLC system equipped with a variable wavelength detector was used. Data were analyzed using Chromeleon software. Ta.

[0220] GLP1-GDF15 fusion protein was derived from transient expression in ExpiCHO cells. When prepared and purified by affinity capture according to the method described in Example 2, 10x (AP) A second linker peptide (SEQ ID NO: 28) (connects the HSA protein to the GDF15 protein or GLP1-GDF15 fusion protein (which binds to GDF15 protein mutants) The material is a molecule containing the GA-8x(G4A) second linker peptide (SEQ ID NO:29). In comparison, the proportion of low molecular weight (LMW) species was lower (Table 10). The final GLP1-GDF15 fusion protein was obtained by removing LMW species using The purity was >97% by SE-HPLC.

[0221] The stability of these GLP1-GDF15 fusion proteins was tested under defined stress conditions. To enforce chemically induced oxidative stress, samples were treated with 0.1% final concentration of hydrogen peroxide. After exposure to 100 mM NaCl and incubation in the dark for 6 hours, the reaction was stopped by adding catalase. In the metal-induced oxidation condition, ferrous iron was added to the sample at a final concentration of 30 μM. After incubation for 1 h, the reaction was stopped by adding EDTA. To test this, samples were dialyzed into 50 mM acetate, pH 3.5 buffer, held for 6 hours, and then The samples were dialyzed against 1 M sodium phosphate, pH 7.4 to test the stability of the samples under thermal stress. To obtain a concentration of approximately 10 mg / mL, an ultracentrifugal filter with a molecular weight cutoff of 30 KDal was used. The mixture was concentrated to 100 ml and kept in PBS at 40° C. for 2 weeks. These samples subjected to low pH or thermal stress conditions were incubated at room temperature for 20 min with 1 mL / The samples were analyzed under analytical size exclusion chromatography (TOSOH column) at a flow rate of 1 min. The data were collected at UV-280 nm (Agilent 1100 LC system). Data analysis was performed on a Chemstation (Agilent).

[0222] Table 11 shows the stability of the GLP1-GDF15 fusion protein under thermal stress (40°C for 2 weeks). The effect of the second linker on the quality of the peptide is shown. The GLP1-GDF15 fusion protein containing GA-8x(G4A) GLP1-GDF15 fusion protein containing a second linker peptide (SEQ ID NO: 29) Compared to the quality, lower levels of LMW species were formed, and the AP second linker peptide , which was shown to be more thermally stable compared to the G4A second linker peptide. Under the stress conditions tested (low pH, metal or chemically induced oxidation), the levels of LMW species in the samples The signal remains at a minimal level similar to the unstressed sample.

[0223] Differential scanning calorimetry (DSC) was performed to determine the GLP1-HSA-GDF15 protein Thermal stability was determined using an automated MicroCal VP-Capillary DSC instrument. Samples were evaluated at approximately 0.5-1 mg / mL in PBS pH 7.4 buffer using a heat sink. The scan ranged from 25°C to 95°C at a heating rate of 1°C / min. A filtering period of 10 seconds and 100 ms were used for each run. The data were processed using the non-two-state fitting function in the table. These GLP1-GDF15 fusion proteins are GLP1 peptides or Similar to the unmutated HSA-GDF15 fusion protein, the fusion protein was incubated at 63–72°C. Melting temperature (T m ) (Table 12).

[0224] [Table 11]

[0225] [Table 12]

[0226] [Table 13]

[0227] Example 9: Ex vivo human plasma testing The stability of the GLP1-GDF15 fusion protein was evaluated ex vivo in human plasma. Briefly, fresh, non-frozen human plasma was generated from heparinized blood by centrifugation. The fusion protein was incubated in this substrate for 0, 4, 24, 48, 72, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4 The fusion molecule was incubated for 1 h at 37° C. for 96 h. The concentration of the fusion molecule in human plasma over time was Immunoaffinity capture with anti-GDF15 antibody and anti-HSA antibody ("whole format") or anti-GL P1 N-terminal specific antibody ("GLP1 activity format") Tables 13 and 14 show the results of these two immunoassays.

[0228] [Table 14]

[0229] [Table 15]

[0230] Example 10: Pharmacokinetics in multiple species Pharmacokinetics in mice GLP1-GDF15 fusion proteins derived from SEQ ID NOs: 50, 45, 46, and 44 was administered to female C57Bl / 6 mice at IV and SC doses of 5 mg / kg in PBS, pH 7. After both routes of administration, blood samples were taken and plasma was processed to determine drug concentrations over a 4-day period. The concentrations of analytes in plasma after IV and SC administration were also measured using immunoaffinity capture assays. Selected tryptic peptides were measured by tryptic digestion-LC-MS / MS analysis. , i.e., ALV (ALVLIAFAQYLQQSPFEDHVK) (SEQ ID NO: 79) , HGE-1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77), and H GE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78), and TDT (TDTGVS LQTYDDLLAK) (SEQ ID NO: 80) is the N-terminus of the HSA protein, the GLP1 peptide and the N-terminus of a GLP1 peptide variant, and a GDF15 protein or GDF15 tRNA. These surrogate peptides are located near the C-terminus of the protein variants. By sequencing, the respective regions of the GLP1-GDF15 fusion protein (GLP1, This enables the evaluation of the pharmacokinetics of HSA and GDF15. are summarized in Tables 15 to 22.

[0231] [Table 16]

[0232] [Table 17]

[0233] [Table 18]

[0234] [Table 19]

[0235] [Table 20]

[0236] [Table 21]

[0237] [Table 22]

[0238] [Table 23]

[0239] Noncompartmental pharmacokinetic analysis (NCA) using data from Tables 15 to 22 , SC of four GLP1-GDF15 fusion proteins tested in C57BL / 6 mice. and 14 to 23 hours after IV administration for GLP1 peptide or GLP1 peptide variants. , 22 to 49 hours for GDF15 protein or GDF15 protein variants, and A terminal half-life of 22-47 hours was found for the HSA protein (Table 23).

[0240] [Table 24]

[0241] Pharmacokinetics in monkeys GLP1-GDF15 fusion proteins derived from SEQ ID NOs: 33, 50, 45, 46, and 44 Protein was administered to naive male crabmoths at a dose of 0.5 mg / kg SC in PBS, pH 7. The mice were administered the same dose of the antibody to monkeys (Macaca fascicularis). Blood samples were taken, plasma was processed, and immunoreactive samples were analyzed. Drug concentrations were measured up to 21 days using immunoassay bioanalysis. The antibody was designed to capture GDF15 and to recognize intact GLP1 (active) or HS Detection by either antibodies recognizing A ("total") or A ("total") was included. Plasma drug concentration vs. time The profiles are summarized in Tables 24 and 25.

[0242] [Table 25]

[0243] [Table 26]

[0244] Pharmacokinetic analysis of the data in Tables 24 and 25 revealed that the active ingredient in cynomolgus monkeys after SC administration Terminal half-lives of 2-3 days for the offspring and 5-8 days for the whole molecule were found. (Table 26).

[0245] [Table 27]

[0246] Example 11: In vivo efficacy of GLP1-GDF15 fusion proteins Assessment of food intake in C57BL / 6 male mice: The purpose of this study was to 6 SEQ ID NOs: 50, 33, 49, 45, 46, and 50A on the inhibition of food intake in mice The objective of this study was to demonstrate the in vivo pharmacokinetic effects of the molecule corresponding to 44. The subjects were administered GLP1-GDF15 fusion protein (test article) or The food weight per cage was measured before and after subcutaneous administration of the control (PBS vehicle or dulaglutide). The change in food intake was recorded every 24 hours. The results were the percentage change in food intake compared to 24 hours before administration. is expressed as and depends in part on the circulating concentration of the respective test article used in the test (Figure 4) Circulating concentrations of test articles were assessed using immunoaffinity capture-trypsin digestion-LC-MS / MS analysis. The levels of selected tryptic peptides, i.e., H the N-terminus of the SA protein, the N-terminus of the GLP1 peptide or GLP1 peptide variant, and Located near the C-terminus of the GDF15 protein or GDF15 protein mutant, respectively ALV (ALVLIAFAQYLQQSPFEDHVK) (SEQ ID NO: 79), HGE- 1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77), and HGE-2 ( HGEGTFTSDLSK) (SEQ ID NO: 78), and TDT (TDTGVSLQTYD The VVS peptide (VVSVLTVL) (SEQ ID NO: 80) was monitored. HQDWLNGK) (SEQ ID NO: 82) are located in the Fc portion of dulaglutide. By monitoring the surrogate peptides of GLP1-GDF15 fusion protein, This allows for pharmacokinetic evaluation of each area (GLP1, HSA, GDF15). Drug concentrations are shown in Figure 5. Results show that the C Subcutaneous administration to 57BL / 6 mice significantly inhibited food intake compared with vehicle-treated animals. It was shown that.

[0247] Evaluation of GLP1R-mediated food intake effects in GFRAL knockout mice:C57 Inhibition of food intake in BL / 6 mice is mediated by GLP1R and GDF15R (GFRAL) The association of SEQ ID NOs: 50, 33, 45, 46, and 4 on food intake. To determine the GLP1R-specific effects of 4, mice deficient in GFRAL were used. The goal of these experiments was to determine whether the fusion protein effectively inhibited the HSA-GDF15 fusion in mice. The fusion protein inhibits GLP1R association when administered at a dose predicted to be The objective of the present invention was to determine the extent to which the present invention is applicable (see Figures 1 and 2 and U.S. Patent Application Publication No. 20170327 Food intake for 24 hours was compared with the GLP1-GD diet from 4:00 to 5:00 pm. F15 fusion protein (test article: 3 and 10 nmol / kg) or control (PBS vehicle or The change in food weight per cage was measured over 24 hours. Fusion molecules containing intact GLP1 peptide or GLP1 peptide variants were recorded every 1 h. Circulating concentrations of GLP1 were analyzed by immunoaffinity capture-trypsin digestion-LC-MS / MS analysis and A tryptic peptide located near the N-terminus of the peptide or GLP1 peptide variant, HGE -1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77) or HGE-2 ( Selective monitoring of HGEGTFTSDLSK) (SEQ ID NO: 78) and administration of 2 The results were determined after 4 hours. The results are graphed as the percentage change in food intake versus plasma concentration of α-peptide mutants (Figure 6, 7 and 8). The results show that fusion proteins with SEQ ID NOs: 50, 33, 45, 46, and 44 The protein was delivered at doses previously demonstrated to be effective against HSA-GDF15. Surprisingly, GLP1R association was Although the exposure of the GLP1 moiety was 10-30 times that of dulaglutide, Fc-G The GLP1 structure of the fusion protein induced the desired pharmacokinetic response similar to that of the GLP1 agonist. If the compound was as potent as dulaglutide in vivo, it would inhibit GLP1R assembly. The doses are much higher than desired, resulting in side effects not anticipated in humans, such as nausea and vomiting. Therefore, the fusion with HSA-GDF15 could cause adverse events. Delivery of GLP1 peptides is used to achieve the desired balance of the two agonists. Affects the in vivo potency of P1R.

[0248] Assessment of GLP1R-mediated glucose tolerance in fasted DIO mice: GLP1R in the pancreas Agonism results in enhanced insulin secretion and increased glucose uptake, and is associated with diet-induced This can be measured using an intraperitoneal glucose tolerance test in obese mice. The purpose of this study was to determine whether the fusion protein is effective in the treatment of HSA-GDF15 fusion in mice. The extent of GLP1R association by the fusion protein when administered at a dose predicted to be The objective of the present invention was to determine whether the present invention is a method for manufacturing a semiconductor device (see FIGS. 1 and 2 and U.S. Patent Application Publication No. 20170327560 (No. A1). Male C57BL / 6 mice were cultured for 6 to 20 weeks of age to induce obesity. They were maintained on Research Diet D12492 until feeding began at age 10 years. Using recorded blood glucose and body weight measurements, mice were randomized into treatment groups. Mice were then transferred to clean cages with access to water and food for the duration of the test. Controls (PBS vehicle and dulaglutide) and test articles (SEQ ID NOs: 33, 50, 49, 4 5, 46, 44, and 36) (1, 3, and 10 nmol / kg) subcutaneously at the time of food removal. Seventeen hours after dosing, baseline glucose was collected at 1 g / kg body weight. After the glucose bolus, the rats were given 10, 30, 45, 60, and 100 mg of glucose each. Blood glucose was measured at 120 min. Circulating plasma concentrations of fusion molecules containing the fusion complex were determined using immunoaffinity capture-trypsin digestion-LC-MS S / MS analysis and the GLP1 peptide or GLP1 peptide variant near the N-terminus Tryptic peptide, HGE-1 (HGEGTFTSDVSSYLEEQAAK) (sequence Selective monitoring of HGE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78) Glucose was determined immediately after the 120 min time point by averaging and quantification. The area under the curve after roughening and normalization to baseline glucose concentration (delta AUC) was calculated. The results were calculated for each animal. The results are graphed as the percentage change in delta AUC (compared to vehicle treatment) versus the dose (Figure 9, Figure 1 0, FIG. 11, and FIG. 12). The results were as follows: The delivery of fusion proteins carrying 36 and 37 was shown to be effective for HSA-GDF15. The study showed that the previously demonstrated doses of ribavirin resulted in GLP1R association. GLP1R association was observed even though the GLP1 moiety was 10-30 times more exposed than dulaglutide. Regardless of the mechanism of action, the fusion proteins induced the desired pharmacokinetic responses similar to those of Fc-GLP1 agonists. If the GLP1 component of protein was as potent as dulaglutide in vivo, For example, GLP1R association may be much higher than desired, resulting in human side effects such as nausea and vomiting. Therefore, HSA-GDF could cause adverse events as expected. Delivery of the GLP1 peptide as a fusion with 15 provides the desired balance of the two agonists. To achieve this, the in vivo efficacy of GLP1R is affected.

[0249] Assessment of GLP1R association in primates by graded glucose infusion: In non-human primates Insulin secretion during intravenous graded glucose infusion (GGI) in mice was measured using the sequence The GLP1R association of 45 and 44 was evaluated. The purpose of these experiments was to determine whether the fusion proteins Administered at doses predicted to be effective against HSA-GDF15 fusion in human primates The aim of this study was to determine the extent of GLP1R association by the fusion protein (Mul Lican et al., 2017 and U.S. Patent Application Publication No. 20170327560 ( A1). The GGI procedures were performed using baseline and dose-response data for 14 days between the two GGI procedures. In sedated cynomolgus monkeys after a 16-hour overnight fast, the recovery period was compared with that of On day 1, animals were administered the vehicle immediately after food removal. On day 2, animals were anesthetized. A baseline was established with two blood samples taken 10 min apart. GGI1 was measured at 0 min. Glucose infusion was started at 8 mg / kg / min, followed by 12, 16 and 24 mg / kg / min. Each infusion was administered over a 40-minute period. Blood samples for the determination of urinary and C-peptide were taken at 20-minute intervals and the results were compared with those for the time series. The values ​​were plotted as number of samples and the area under the curve (AUC) was determined for each analyte measured in each animal. Animals were then graded based on baseline insulin secretion (ISR) from GGI1. On day 15, animals were randomized to receive the test article at various dose concentrations (0.1 to 1. Test article at 1 mg / kg fusion protein and 0.016-0.1 mg dulaglutide After administration, the subjects were fasted overnight. The second GGI (GGI2) was administered in the same manner as described on the second day. The study was performed on day 16. Test articles bearing intact GLP1 peptide or peptide variants were administered to the mice. The immunoassay was specifically designed to monitor the Compound exposure was assessed in plasma samples taken at time point 0. Data in Figure 13 show that intact G Glucose AU compared to baseline for plasma concentrations of test article with LP1 Reported as dose-induced fold change in ISR AUC normalized to C. The results are shown in SEQ ID NOs: 45 and 44. It has previously been shown that delivery of a fusion protein having The results showed that the doses demonstrated resulted in GLP1R association. The 1R interaction was observed despite the exposure of the GLP1 moiety being 10-30 times that of dulaglutide. The fusion protein induced the desired pharmacokinetic response similar to that of an Fc-GLP1 agonist. If the GLP1 component of this compound was as potent as dulaglutide in vivo, LP1R association is much higher than desired, resulting in symptoms in humans such as nausea and vomiting. Therefore, HSA-GDF15 and Delivery of the GLP1 peptide as a fusion of the agonist and agonist may achieve the desired balance of the two agonists. Thus, it affects the in vivo efficacy of GLP1R.

[0250] The results shown in Figures 6-13 demonstrate the in vivo activity or potency of GLP1 in preclinical species. , compared with the Fc-fused GLP1 peptide of dulaglutide, homodimeric GLP1-GDF 15 They have demonstrated that the dual agonist has a reduced effect on the expression of agonists. agonist (GLP1 peptide or GLP1 peptide variant and GDF15 protein or This allows for a balance between GDF15 protein variants and, therefore, the expected therapeutic range. Effective concentration to achieve GDF15 protein or GDF15 protein variant exposure within the This enables delivery of GLP1 peptide or GLP1 peptide variants in vivo.

[0251] Weight loss efficacy of GLP1-GDF15 fusion protein after repeated administration in mice Evaluate the ability of the GLP1-GDF15 fusion protein to induce weight loss in diet-induced obesity Mice were treated with various concentrations of the test articles (SEQ ID NOs: 45 and 44), duplexes, and Raglutide or vehicle control was administered subcutaneously daily for 7 days. Body weight was monitored throughout the study. Figure 14 shows the percent change in body weight over time relative to day 0 (just before the first dose). Circulating concentrations of test articles were determined by immunoaffinity capture-trypsin digestion-LC-MS / MS analysis. Selected tryptic peptides, i.e., HSA protein, were measured after 7 days of treatment. the N-terminus of a protein, the N-terminus of a GLP1 peptide or a GLP1 peptide variant, and GDF15 ALV( ALVLIAFAQYLQQSPFEDHVK) (SEQ ID NO: 79), HGE-1 (HGE GTFTSDVSSYLEEQAAK) (SEQ ID NO: 77), and HGE-2 (HGEGT FTSDLSK) (SEQ ID NO: 78), and TDT (TDTGVSLQTYDDLLAK ) (SEQ ID NO: 80) was monitored. VVS peptide (VVSVLTVLHQDWL NGK) (SEQ ID NO: 82) are located in the Fc portion of dulaglutide. By monitoring the tides, This enables pharmacokinetic evaluation of the following areas: GLP1, HSA, and GDF15. The results are shown in Figure 15. The results show that repeated administration of SEQ ID NOs: 45 and 44 reduced the risk of developing obese mice with diet-induced obesity. It was shown that it caused weight loss in a concentration-dependent manner.

[0252] Assessment of the feeding effects of a GLP1-GDF15 fusion protein in non-human primates.

[0253] The ability of the GLP1-GDF15 fusion protein to reduce food intake was evaluated in spontaneously obese mice. The study was conducted in monkeys. Subjects were administered various concentrations of the drug with and without the active GLP1 moiety. Test articles (SEQ ID NOs: 44 and 83, respectively, AP10 phosphorylation fused to GDF15 peptide) SEQ ID NO:83, which contains the HSA peptide fused to a carcinoma, is described in U.S. Pat. No. 10,336,812. The subjects were administered either 100 mg of 100% ethanol (previously disclosed in US Pat. No. 6,333,625) or vehicle subcutaneously QW over a period of 4 weeks. Food intake was monitored prior to and throughout the study. The 3-day percentage change in food intake after each dose is shown in Figure 16. Concentrations of SEQ ID NOs: 44 and 83 in plasma were determined for the "total" molecule (HSA detection) and for the "active" GL Two separate immunoassays to detect the P1 portion (detection of the N-terminal GLP1 peptide) The plasma drug concentrations were determined using the method shown in Figures 17 and 18. The results were as follows: and 83 administration reduced food intake in spontaneously obese non-human primates. .

[0254] of GLP1-GDF15 fusion protein after 21 days of dose escalation in non-human primates Assessment of food intake and body weight effects.

[0255] Ability of GLP1-GDF15 fusion protein to reduce food intake and cause weight loss The efficacy of the drug was tested in spontaneously obese cynomolgus monkeys. On the other hand, test articles containing only the active GDF15 or only the active GLP1 moiety (sequences 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, 2 Nos. 44, 83, and 84; SEQ ID NO: 84 is a fusion peptide of GDF15 having the I89R mutation. GLP1 fused to AP5 linker peptide fused to AP10 linker peptide peptides; GDF15 peptides having the I89R mutation are disclosed in U.S. Pat. No. 10,336 (previously disclosed in US Pat. No. 6,812) was administered subcutaneously every 3 days for a period of 21 days. The initial dose given on day 0 was 0.7 nmol / kg, with a maximum dose of 20 nmol / kg. The subjects were titrated up to 1000 mg at each dose. Daily food intake during the extraction is shown in Figure 19. Body weight was monitored throughout the study. Figure 20 shows the percentage change in body weight over time relative to baseline. Cynomolgus monkeys on study The concentration of the test article in plasma was determined using the "whole" molecule (HSA detection; SEQ ID NOs: 44, 83, 84) and and the "active" GLP1 portion (N-terminal GLP1 peptide detection; SEQ ID NOs: 44 and 84 only) Plasma drug concentrations were measured using two separate immunoassay methods to detect The results are shown in Figures 21 and 22. The results show that repeated administration of SEQ ID NOs: 44, 83, and 84 significantly reduced the incidence of spontaneous has been shown to reduce food intake and subsequent weight loss in obese non-human primates. Ta.

[0256] Those skilled in the art may make changes to the above-described embodiments without departing from the broad inventive concept. It will be understood that the invention is therefore not limited to the particular embodiments disclosed. without limitation, and is intended to encompass modifications within the spirit and scope of the invention as defined by this description. is understood to be intended.

[0257] All documents cited are incorporated herein by reference. [1] A glucagon-like peptide-1 (GLP1) / growth differentiation factor 15 (GDF15) fusion protein, the GLP1-GDF15 fusion protein comprising a GLP1 peptide, a first linker peptide, a serum albumin protein, a second linker peptide, and a GDF15 protein. [2] The GLP1-GDF15 fusion protein according to [1] above, wherein the GLP1 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4. [3] The GLP1-GDF15 fusion protein according to [1] above, wherein the first linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 25. [4] The GLP1-GDF15 fusion protein described in [1] above, wherein the serum albumin protein comprises an amino acid sequence selected from SEQ ID NO: 26 or SEQ ID NO: 27. [5] The GLP1-GDF15 fusion protein according to [1] above, wherein the second linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 28 to 30. [6] The GLP1-GDF15 fusion protein described in [1] above, wherein the GDF15 protein comprises an amino acid sequence selected from SEQ ID NO: 31 or SEQ ID NO: 32. [7] The GLP1-GDF15 fusion protein according to [1] above, wherein the GLP1-GDF15 fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33 to 74 and 84. [8] An isolated nucleic acid encoding the GLP1-GDF15 fusion protein described in [1] above. [9] A vector comprising the isolated nucleic acid described in [8] above.

[10] A host cell comprising the isolated nucleic acid described in [8] above or the vector described in [9] above.

[11] A pharmaceutical composition comprising the GLP1-GDF15 fusion protein described in [1] above and a pharma- ceutical acceptable carrier.

[12] A method of treating or preventing a disease or disorder in a subject in need thereof, the disease or disorder being selected from the group consisting of obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (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), renal disease, and eczema, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to

[11] above.

[13] A method for reducing food intake in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described in

[11] above.

[14] A method for regulating GLP1 receptor activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described in

[11] above.

[15] A method for regulating GDF15 receptor (GFRAL) activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described in

[11] above.

[16] The method according to

[12] above, wherein the pharmaceutical composition is administered by injection.

[17] A kit comprising the GLP1-GDF15 fusion protein described in [1] above, the isolated nucleic acid described in [8] above, and / or the vector described in [9] above.

[18] The kit described in

[17] above, further comprising an injection device.

[19] A method for producing a pharmaceutical composition comprising the GLP1-GDF15 fusion protein described in [1] above, comprising combining the GLP1-GDF15 fusion protein with a pharma- ceutical acceptable carrier to obtain the pharmaceutical composition.

[20] A method for producing the GLP1-GDF15 fusion protein described in [1] above, comprising culturing a cell containing a nucleic acid encoding the GLP1-GDF15 fusion protein under conditions for producing the GLP1-GDF15 fusion protein, and recovering the GLP1-GDF15 fusion protein from the cell or culture.

Claims

1. Glucagon-like peptide-1 (GLP1) / growth differentiation factor 15 (GDF15) fusion protein The GLP1-GDF15 fusion protein comprises a GLP1 peptide and a first a linker peptide of the formula (I), a serum albumin protein, a second linker peptide, and a GD and a GLP1-GDF15 protein.

2. The GLP1 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4; The GLP1-GDF15 fusion protein of claim 1.

3. The first linker peptide is selected from the group consisting of amino acids 5 to 25.

2. The GLP1-GDF15 fusion protein of claim 1, comprising the sequence:

4. The serum albumin protein is an amino acid sequence selected from SEQ ID NO: 26 or SEQ ID NO:

27. The GLP1-GDF15 fusion protein of claim 1, comprising the amino acid sequence

5. The second linker peptide is an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-30.

2. The GLP1-GDF15 fusion protein of claim 1, comprising the amino acid sequence

6. The GDF15 protein comprises an amino acid sequence selected from SEQ ID NO:31 or SEQ ID NO:

32.

2. The GLP1-GDF15 fusion protein of claim 1, comprising the sequence:

7. The GLP1-GDF15 fusion protein is selected from the group consisting of SEQ ID NOs: 33-74 and 84. The GLP1-GDF15 fusion protein of claim 1, comprising an amino acid sequence selected from Quality.

8. 2. An isolated nucleic acid encoding the GLP1-GDF15 fusion protein of claim 1.

9. A vector comprising the isolated nucleic acid of claim 8.

10. 10. A host cell comprising the isolated nucleic acid of claim 8 or the vector of claim 9.

11. A GLP1-GDF15 fusion protein according to claim 1 and a pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising:

12. A method of treating or preventing a disease or disorder in a subject in need thereof. The disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, Insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, Congenital hyperinsulinism (CHI) causing hypoglycemia, dyslipidemia, and atherosclerosis, Associated with diabetic nephropathy and uncontrolled cholesterol and / or lipid levels Other cardiovascular risk factors such as hypertension and cardiovascular risk factors, osteoporosis, inflammation, non-alcoholic Fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), kidney disease, and eczema The method comprises administering an effective amount of the pharmaceutical composition of claim 11 to the administering to said subject in need thereof.

13. 1. A method of reducing food intake in a subject in need thereof, comprising: The method comprises administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 11. The method includes:

14. A method of modulating GLP1 receptor activity in a subject in need thereof. The method comprises administering an effective amount of the pharmaceutical composition of claim 11 to the subject in need thereof. The method comprises administering to

15. The present invention relates to a method for modulating GDF15 receptor (GFRAL) activity in a subject in need thereof. The method comprises administering an effective amount of the pharmaceutical composition according to claim 11 to a patient requiring the same. administering to said subject in need thereof.

16. The method of claim 12, wherein the pharmaceutical composition is administered by injection.

17. 10. The GLP1-GDF15 fusion protein of claim 1, the isolated nucleic acid of claim 8, and / or a kit comprising the vector according to claim 9.

18. The kit of claim 17 , further comprising an injection device.

19. A method for producing a pharmaceutical composition comprising the GLP1-GDF15 fusion protein of claim 1. The method comprises combining the GLP1-GDF15 fusion protein with a pharma- ceutical acceptable carrier. to obtain the pharmaceutical composition.

20. 2. A method for producing the GLP1-GDF15 fusion protein of claim 1, comprising: The GLP1-GDF15 fusion protein is produced under conditions for producing the GLP1-GDF15 fusion protein.

5. Culturing cells containing a nucleic acid encoding the fusion protein, and and recovering the GLP1-GDF15 fusion protein from the

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