GDF15 fusion protein and its use

Abstract

The present invention provides a composition using GDF15 that can be used to treat or prevent metabolic diseases, disorders, or conditions. [Solution] A fusion protein is provided comprising (a) a half-life extension protein, (b) a linker, and (c) a GDF15 protein, arranged in the order (a)-(b)-(c) from the N-terminus to the C-terminus. Also described are nucleic acids encoding the fusion protein, recombinant cells thereof, compositions containing the fusion protein, and methods of using the fusion protein to treat or prevent metabolic diseases, disorders, or conditions.

Description

[Technical Field]

[0001] This invention relates to a GDF15 fusion protein. More specifically, this invention relates to a half-life extension protein. A fusion protein containing protein, linker, and GDF15 protein, A pharmaceutical combination comprising encoding nucleic acids and expression vectors, recombinant cells thereof, and fusion proteins. Regarding the product; a method for producing such a fusion protein, and using such a fusion protein Methods for treating metabolic diseases are also provided. [Background technology]

[0002] GDF15 is a member of the TGFβ family and is a 25kDa homodimer. It is a secreted protein that circulates in the plasma. The plasma concentration of GDF15 is 15 in many individuals. The range is 0-1150 pg / ml (Tsai et al., J Cachexia) Sarcopenia Muscle.2012,3:239~243). GDF15 Plasma concentrations of this substance increase under conditions of injury, cardiovascular disease, and certain types of cancer. This is thought to be a cytoprotective mechanism. High plasma concentrations of GDF15 are associated with cancer. Weight loss due to loss of appetite and cachexia, as well as weight loss in renal failure and heart failure. They are related. In clinical trials, GDF15 levels were found to be associated with the effects of the glycemic index (GDF15) in obese non-diabetic subjects. It was considered an independent predictor of phosphorus resistance (Kempf et al., Eur.J.En). do.2012,167:671~678). A study on twins showed that in a twin pair... It has been shown that the difference in GDF15 levels correlates with the difference in BMI between the pair, This suggests that F15 functions as a long-term regulator of energy homeostasis. (Tsai et al., PLoS One. 2015, 10(7): e013 3362).

[0003] GDF15 has been extensively studied as a biomarker for several cardiovascular diseases and other disease conditions, but the protective role of GDF15 in myocardial hypertrophy and ischemic injury has also been described (Collinson, Curr. Opin. Cardiol. 201 4, 29: 366-371; Kempf et al., Nat. Med. 2011, 1 7: 581-589; Xu et al., Circ Res. 2006, 98: 342 -350). GDF15 has been shown to play an important role in protecting renal tubules and intestine from injury in mouse models of type 1 and type 2 diabetes (Mazago va et al., Am. J. Physiol. Renal Physiol. 201 3; 305: F1249-F1264). GDF15 has been shown to have a protective effect against age-related sensory and motor neuro n loss, and improves recovery after peripheral nerve injury (Strelau et al., J. Neurosci. 2009, 29: 136 40-13648; Mensching et al., Cell Tissue Re s. 2012, 350: 225-238). In fact, GDF15 transgenic mice have been shown to have a longer lifespan than their littermate controls, which suggests that this molecule may function as a long-term survival factor (Wang et al., Ag ing. 2014, 6: 690-700).

[0004] Many reports have shown that treatment with GDF15 protein improves glucose tolerance in mouse models Transgenicity has been shown to improve insulin sensitivity. Transgenicity that overexpresses GDF15 In two independent strains of Nick mice, reductions in body weight and body fat mass, as well as improved glucose tolerance, were observed. (Johnen et al., Nat. Med. 2007, 13:133) 3~1340;Macia et al.,PLoS One.2012,7:e348 68;Chrysovergis et al.,Int.J.Obesity.201 4,38:1555~1564). In GDF15 mice, whole-body energy consumption and Increased oxidative metabolic activity has been reported (Chrysovergis et al.,2 014, ibid.). These are due to increased expression of thermogenic genes in brown adipose tissue, and white This is accompanied by an increase in lipolytic genes in adipose tissue. In mice lacking the GDF15 gene... , increased body weight and body fat mass (Tsai et al., PLoS One. 2013) ,8(2):e55174). The Fc fusion protein of GDF15 is found in obese cynomolgus monkeys. In Dell, when administered once a week for 6 weeks, it reduced body weight and improved glucose tolerance and insulin levels. It has been shown to improve phosphorus sensitivity (International Publication No. 2013 / 113008).

[0005] The effect of GDF15 on body weight is achieved through a reduction in food intake and an increase in energy expenditure. It is thought to be mediated by body weight-dependent and independent mechanisms. This may be improving blood sugar control.

[0006] Considering these observations, increasing GDF15 levels may be associated with metabolic diseases. It is considered a useful treatment method. [Overview of the initiative] [Problems that the invention aims to solve]

[0007] GD may be used to treat or prevent metabolic diseases, disorders, or conditions. Compositions using F15 are needed in this field. [Means for solving the problem]

[0008] This invention exhibits improved solubility / stability and is suitable for the treatment of metabolic diseases, disorders, or conditions. Alternatively, it provides a GDF15 fusion protein that exhibits characteristics indicating it can be used for prevention. This will meet such demands. Examples of such features include, for example, this Subjects administered the fusion protein according to the embodiment of the invention experienced weight loss, improved glucose tolerance, and One benefit is improved nerve sensitivity.

[0009] In a general embodiment, the present invention comprises (a) a half-life extension protein, (b) a linker, and (c) Contains GDF15 protein, with (a)-(b)-(c) in the order from the N-terminus to the C-terminus Regarding fusion proteins arranged in this way.

[0010] In one embodiment of the present invention, the GDF15 protein is human GDF15 protein or It is a functional variant of GDF15. In certain embodiments, the GDF15 protein is a mature GDF15. This includes a protein or a functional variant thereof. In a more detailed embodiment, the GDF15 protein The quality includes amino acid sequences that are at least 90% identical to sequence numbers 6-11. In certain embodiments, the GDF15 protein has the amino acid sequence of SEQ ID NO: 11, for example, It contains an amino acid sequence selected from the group consisting of sequence numbers 6 to 11.

[0011] In one embodiment of the present invention, the half-life extension protein is human serum albumin (HSA) or This is a functional variant of it. In certain embodiments, the half-life extension protein is SEQ ID NO: 1 It includes an amino acid sequence having at least 90% identity with the above. In other specific embodiments, half The protein with extended mitosis contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

[0012] In one embodiment of the present invention, the linker is a flexible linker. In a particular embodiment, The flexible linker is arranged in (GGGGS)n (where n is 2 to 20, preferably 4 to 10). (Includes) In another embodiment of the present invention, the linker of the fusion protein is a structured linker —In certain embodiments, the structured linker is array (AP)n or (EAAAK) Includes n (where n is 2 to 20, preferably 4 to 10).

[0013] In embodiments of the present invention, the fusion protein is sequence numbers 5, 25-30, 36-37, 4 amino acids having at least 90% sequence identity with 0, 48, 55-60, or 64-75 It contains an acid sequence. In a detailed embodiment of the present invention, the fusion protein is SEQ ID NO: 5, 25-3 The present invention comprises an amino acid sequence selected from the group consisting of 0, 40, 55-60, and 70. In a more detailed embodiment, the fusion protein contains the amino acid sequence of SEQ ID NO: 60.

[0014] In another general embodiment, the present invention provides for the isolation nucleic acid encoding the fusion protein of the present invention. Regarding molecules.

[0015] In another general embodiment, the present invention relates to nucleic acid molecules encoding the fusion protein of the present invention. Regarding expression vectors containing this.

[0016] In another general embodiment, the present invention relates to nucleic acid molecules encoding the fusion protein of the present invention. This relates to recombinant host cells, including those containing such cells.

[0017] In another general embodiment, the present invention relates to a method for obtaining the fusion protein of the present invention. The method involves (1) the nucleic acid encoding the fusion protein under conditions in which the fusion protein is produced. (1) Culturing host cells containing the molecule, and (2) the fusion protein produced by the host cell. This includes recovering the product.

[0018] In another general embodiment, the present invention relates to a fusion protein of the present invention and a pharmaceutically acceptable This relates to a pharmaceutical composition containing a carrier.

[0019] In another general embodiment, the present invention relates to nucleic acid molecules encoding the fusion protein of the present invention. The present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0020] In another general embodiment, the present invention relates to a kit comprising the pharmaceutical composition of the present invention.

[0021] In another general embodiment, the present invention relates to a method for treating or preventing metabolic diseases, disorders, or conditions. A law that provides a therapeutic or preventive effective amount of the pharmaceutical compound of the present invention to a subject requiring treatment or prevention. The present invention relates to a method, which includes administering a product. In a particular embodiment, the pharmaceutical composition is used to target It is administered subcutaneously or intravenously.

[0022] According to embodiments of the present invention, in subjects requiring treatment, type 2 diabetes, elevated blood glucose levels, Elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure A method for treating metabolic diseases selected from the group consisting of , or rheumatoid arthritis, is applicable to the target. Ami selected from the group consisting of Sequence IDs 5, 25-30, 40, 55-60, and 70. A therapeutically effective dose of a drug comprising a fusion protein containing an acid sequence and a pharmaceutically acceptable carrier. This includes administering the composition. In one detailed embodiment, such a method involves administering the SEQ ID NO: A therapeutically effective fusion protein comprising a 60-amino acid sequence and a pharmaceutically acceptable carrier. This includes administering a certain amount of the pharmaceutical composition.

[0023] Other aspects, features, and advantages of the present invention are described in the detailed description of the invention and preferred embodiments thereof. This will become clear from the following disclosure, including the form and accompanying claims. [Brief explanation of the drawing]

[0024] The "means for solving the problem" up to now and the "best way to carry out the invention" thereafter The "morphology" will be better understood when read in conjunction with the attached drawings. This invention is illustrated in the drawings. It should be understood that the embodiments shown on the surface are not limited to those embodiments themselves.

[0025] The drawings are as follows: [Figure 1A] The crystal structure of GDF15 is shown, in which a disulfide pairing (C1-C2) between the first and second cysteine ​​residues forms a loop at the N-terminus of the protein. [Figure 1B] The crystal structure of GDF15 is shown, in which a disulfide pairing (C1-C2) between the first and second cysteine ​​residues forms a loop at the N-terminus of the protein. [Figure 2]The effects of subcutaneous administration of fusion proteins based on the embodiments of the present invention (e.g., fusion protein FP1 (SEQ ID NO: 60) and 6xHis-FP1 (SEQ ID NO: 26 with a 6xHis tag attached to the N-terminus)) on food intake in C57Bl / 6 mice are shown, with cumulative food intake 24 hours after administration. The values ​​shown in each bar represent the percentage reduction compared to the solvent (PBS) group ± SEM. All groups consisted of N=8 animals (except for the FP1 16 nmol / kg group, which had N=9). *p<0.05 compared to the solvent; p-values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test. [Figure 3] This study shows the effects of subcutaneous administration of FP1 and 6xHis-FP1 (SEQ ID NO: 26, with a 6xHis tag fused to the N-terminus) on food intake in Sprague-Dolly rats, with cumulative food intake 48 hours after administration shown. The values ​​shown in each bar represent the percentage reduction compared to the solvent (PBS) group ± SEM. Each group consisted of 8 animals. *p<0.05 compared to the solvent; p-values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test. [Figure 4] This chart shows the change in body weight in diet-induced obesity (DIO) during treatment with FP1. The arrows indicate the time (days) of subcutaneous administration after the first dose (day 0). Each group consisted of 8 animals. *p<0.05 in the 1 nmol / kg FP1 group compared to the solvent; #p<0.05 in the 10 nmol / kg FP1 group compared to the solvent; p values ​​were calculated using two-way RM ANOVA and Tukey's multiple comparison test. [Figure 5A] The blood glucose levels of DIO mice during oral glucose tolerance tests (OGTT) after administering FP1 every 3 days (q3d) for 14 days are shown, with each value represented as the area under the curve. Each group consisted of 8 animals. *For the 1 nmol / kg FP1 group compared to the solvent, p<0.05; p-values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test. [Figure 5B]The blood glucose levels of DIO mice during oral glucose tolerance tests (OGTT) after administering FP1 every 3 days (q3d) for 14 days are shown, with each value represented as the area under the curve. Each group consisted of 8 animals. *For the 1 nmol / kg FP1 group compared to the solvent, p<0.05; p-values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test. [Figure 6] This shows the blood glucose levels of DIO mice during treatment with FP1. Each group consisted of 8 animals. *p<0.05 compared to the solvent; p-values ​​were calculated using two-way RM ANOVA and Tukey's multiple comparison test. [Figure 7] This shows the evaluation of a 4-hour fasting homeostasis model (HOMA-IR) for insulin resistance in DIO mice after 14 days of treatment with FP1. Each group consisted of 8 animals. *p<0.05 compared to the solvent; p-values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test. [Figure 8] This graph shows the change in body weight of ob / ob mice between FP1 treatments every 3 days (qd3). The arrows indicate the time (days) of subcutaneous administration after the first dose (day 0). Each group consisted of 9 animals. *P<0.05 in the 10 nmol / kg FP1 group compared to the solvent; #P<0.05 in the 1 nmol / kg FP1 group compared to the solvent; p values ​​were calculated using two-way RM ANOVA and Tukey's multiple comparison test. [Figure 9] This chart shows the blood glucose levels of ob / ob mice during treatment with FP1. The arrows indicate the time (days) of subcutaneous administration after the first dose (day 0). Each group consisted of 9 animals. *p<0.05 in the 10 nmol / kg FP1 group compared to the solvent; #p<0.05 in the 1 nmol / kg FP1 group compared to the solvent; p values ​​were calculated using two-way RM ANOVA and Tukey's multiple comparison test. [Figure 10] The mean (± standard deviation, SD) serum drug concentration-time profiles of FP1 after intravenous (IV) and subcutaneous (SC) administration of 2 mg / kg in C57Bl / 6 mice are shown. [Figure 11]The mean (±SD) serum drug concentration-time profiles of FP1 after 2 mg / kg IV and SC administration in Sprague-Dolly rats are shown. [Figure 12] The mean (±SD) serum drug concentration-time profiles of FP1 after 1 mg / kg IV and SC administration in cynomolgus monkeys, as measured by immunoassay, are shown. [Figure 13] The serum concentrations (ng / mL) of FP1 as complete dimers in cynomolgus monkeys over time, measured by immunoaffinity (IA) capture LC-MS analysis, are shown. [Figure 14] The serum concentrations (ng / mL) of FP1 as a complete dimer over time in cynomolgus monkeys after a single dose of SC, as measured by immunoaffinity capture LC-MS analysis, are shown. [Figure 15] The concentrations of FP1 are shown as percentages (%) of the initial concentrations at 0, 4, 24, and 48 hours after ex vivo incubation in plasma obtained from two human subjects (Sub) as measured by immunoassay. [Figure 16] The mean FP1 concentration is expressed as the percentage of complete dimers at 0, 4, 24, and 48 hours after ex vivo incubation in plasma obtained from two human subjects (Sub) as measured by intact mass immunoaffinity capture LC-MS analysis. [Figure 17] This shows acute food intake in lean C57BL6 male mice before and after administration of different N-terminal deletion mutants of GDF15. (Sequence IDs 92, 111, and 112 compared to the wild-type fusion without deletion (Sequence ID 26 with a 6xHis tag attached to the N-terminus)). N=8 animals per group. *p<0.05 compared to the solvent; p-values ​​were calculated using two-way RM ANOVA and Tukey's multiple comparison test. [Figure 18]This report shows the effect of a single dose of FP2 on food intake in C57Bl / 6 mice. Specifically, it shows the cumulative food intake 24 hours after administration. The values ​​shown in each bar represent the percentage reduction compared to the PBS group (mean ± SEM). All groups consisted of N=8 animals (except for N=6 in 6xHis-FP1). **p<0.01, ***p<0.001,** **p<0.0001; p-values ​​were calculated using two-way ANOVA and Dunnett's multiple comparison test. [Figure 19] This table shows the cumulative food intake measured in Sprague-Dolly rats 24 hours after a single dose of FP2. The values ​​shown in each bar represent the percentage decrease compared to the PBS group (mean ± SEM). Each group consisted of 8 animals. **p<0.01, p-values ​​were calculated using two-way ANOVA and Tukey's multiple comparison test. [Figure 20] This graph shows the percentage change in body weight (%) during q3d treatment with FP2 in DIO mice. Arrows indicate the time of subcutaneous administration of FP2. Each group consisted of 6 animals. *p, 0.05, p-values ​​were compared with the solvent using two-way ANOVA and Tukey's multiple comparison test. [Figure 21A] This shows the area under the curve (AUC) of blood glucose levels during the OGTT after 14 days of FP2 administration in DIO mice. *p<0.05, one-way ANOVA and Tukey's multiple comparison test were used, with N=8 animals in each group. [Figure 21B] This shows the area under the curve (AUC) of blood glucose levels during the OGTT after 14 days of FP2 administration in DIO mice. *p<0.05, one-way ANOVA and Tukey's multiple comparison test were used, with N=8 animals in each group. [Figure 22A] This shows plasma insulin levels during the OGTT after 8 days of FP2 administration (q3d) in DIO mice. *p<0.05, FP2 (0.3 nmol / kg), FP2 (10 nmol / kg), and rosiglitazone relative to the solvent. #p<0.05, compared with rosiglitazone (10 mg / kg) using two-way RM ANOVA and Tukey's multiple comparison test. [Figure 22B]This shows the AUC of plasma insulin levels during the OGTT after 8 days of FP2 q3d administration in DIO mice. *p<0.05, compared to solvent, #p<0.05, compared to rosiglitazone. [Figure 23] This shows the blood glucose levels after 8 days of FP2 q3d administration in DIO mice. *p<0.05, two-way RM ANOVA and Tukey's multiple comparison test were used, with N=8 animals in each group compared to the solvent. [Figure 24] The HOMA-IR of DIO mice after 14 days of q3d treatment with FP2, followed by a 5-hour fast on day 14, is shown. *p<0.05. One-way ANOVA and Tukey's multiple comparison test were used to compare each group (N=8 animals) with the solvent. [Figure 25] This table shows serum concentrations of FP2 in C57Bl / 6 mice after intravenous (IV) and subcutaneous (SC) administration of 2 mg / kg. Each value represents the mean ± SD (n=5 samples per time point). [Figure 26] This table shows serum concentrations of FP2 after intravenous (IV) and subcutaneous (SC) administration of 2 mg / kg in Sprague-Dolly rats. n=5 samples were collected at each time point. [Figure 27] This table shows the plasma concentrations of FP2 in cynomolgus monkeys analyzed by immunoassay. Each value represents the mean ± SD of n=3, except for n=2 of the IV group on day 22 (528 hours). IV = intravenous, SC = subcutaneous. [Figure 28] The plasma concentrations of FP2 as a complete dimer in cynomolgus monkeys, analyzed by LC-MS, are shown. Each value represents the mean ± SD of n=3, excluding n=2 for subcutaneous administration (SC) at 168 hours, n=1 for SC at 120 and 432 hours, and n=1 for intravenous administration (IV) at 168 and 432 hours. [Figure 29] This shows the ex vivo stability (normalized recovery rate (%)) of FP2 in human plasma over 48 hours, as measured by immunoassay. [Figure 30] This shows the ex vivo stability (normalized recovery rate (%)) of FP2 in human plasma over 48 hours, as measured by intact LC / MS. [Figure 31] The concentration-response curves of FP2 and HSA-GDF15:GDF15 heterodimers obtained using the pAKT assay in SK-N-AS cells (N=3) expressing recombinant human GFRAL receptor are shown. [Figure 32] This shows the daily food intake (g) in cynomolgus monkeys before and after a single dose of FP1. *For FP1 at 10 nmol / kg compared to the solvent, p<0.05 was considered. [Figure 33] This shows the percentage change in body weight (%) before and after a single dose of FP1 in cynomolgus monkeys. *For FP1 at 10 nmol / kg compared to the solvent, p<0.05 was assumed. *For FP1 at 3 mg / kg compared to the solvent, p<0.05 was used, and two-way RM ANOVA and Tukey's multiple comparison test were performed on N=8 animals in each group. [Figure 34] This shows the daily food intake (g) in cynomolgus monkeys before and after a single dose of FP2. *p<0.05, compared to the solvent using two-way RM ANOVA and Tukey's multiple comparison test, with N=8 animals per group. [Figure 35] This shows the percentage change in body weight (%) before and after a single dose of FP2 in cynomolgus monkeys. *p<0.05, for 10 nmol / kg FP2 compared to the solvent, #p<0.05, for 3 nmol / kg FP2 compared to the solvent, &p<0.05, for 1 nmol / kg FP2 compared to the solvent (using two-way RM ANOVA and Tukey's multiple comparison test, with N=8 animals in each group). [Modes for carrying out the invention]

[0026] In the background of the invention, and throughout the specification, various publications, articles, and patents are cited or These references are incorporated herein by reference in their entirety. The documents, procedures, materials, apparatus, articles, etc., included herein are the basis of the present invention. This is to provide context. Such considerations are based on any or all of these things. All of these constitute part of the prior art to any of the disclosed or claimed inventions. This does not condone it.

[0027] Unless otherwise defined, all technical and scientific terms used herein are: This has the same meaning as that generally understood by those skilled in the art to which this invention belongs. If not otherwise specified, certain terms used herein shall have the meanings set forth herein. All patents, published patent applications and publications referenced herein are referenced to Therefore, it is incorporated in the same manner as the entirety described herein. The singular forms "a," "an," and "the" as used in this specification and the appended claims. It should be noted that unless otherwise clearly stated in the context, it refers to multiple objects. stomach.

[0028] The present invention relates to (a) a half-life extension protein, (b) a linker, and (c) GDF15 tannin. A fusion tangent containing protein, arranged in the order (a)-(b)-(c) from the N-terminus to the C-terminus. Regarding protein.

[0029] Embodiments of the present invention comprising a half-life extension protein, a linker, and a GDF15 protein. The fusion protein based on the present invention increases the half-life of the GDF15 protein. Fusion proteins are used as therapeutic agents to treat and prevent metabolic diseases, disorders, or conditions. It was found to exhibit metabolic effects that indicate suitability. Such effects are not limited to these. Although it is not a direct result of the fusion protein, animals administered the fusion protein showed weight loss, improved glucose tolerance, and increased insulin levels. One benefit is improved susceptibility to thrin.

[0030] As used in this specification, the term "fusion protein" refers to proteins whose parts are different. This refers to a protein that is derived from another protein and has two or more parts that are covalently bonded to each other. .

[0031] The fusion protein according to the embodiments of the present invention comprises any GDF15 protein. This is possible. The term "GDF15 protein" as used in this specification refers to a naturally occurring protein. This refers to the wild-type growth and differentiation factor 15 protein, or its functional variant. Proteins are found in humans, or other animals such as mice, rabbits, rats, pigs, dogs, or primates. It can be derived from any mammal, such as a suitable mammal. In terms of application, the GDF15 protein is either human GDF15 protein or a functional variant thereof. In a preferred embodiment, the GDF15 protein is mature GDF15 protein or It is a functional variant of that.

[0032] As used herein, "mature GDF15 protein" refers to the furin of RXXR. GDF15 preprota released from the full-length protein after intracellular cleavage at the cleavage site. This refers to the protein portion. Mature GDF15 protein is linked by disulfide bonds. It is secreted as a homodimer. In one embodiment of the present invention, mature GDF15 protein ( GDF15 (197-308) (abbreviated as Sequence ID No. 6) is a fully-length human GDF15 It contains amino acids 197-308 of the protein.

[0033] As used herein, "functional variant" refers to a variant that is substantially or phasely different from the parent protein. The parent protein possesses sequence identity and retains at least one of the biological activities of the parent protein. This refers to a variant of a protein. Functional variants of the parent protein are considered in this disclosure. Functional mutants can be prepared by means well known in the art. This may include one or more modifications to the amino acid sequence of the product. Such modifications may include, for example, the addition of To improve the thermal stability of lipeptides, alter substrate specificity, and change the optimal pH. The physicochemical properties of polypeptides can be altered by such modifications. If it does not cause the loss or disappearance of all of the biological activity of the protein, then the parent protein It can also alter biological activity.

[0034] According to embodiments of the present invention, functional variants of the parent protein are biological variants of the parent protein. Substitutions of the parent protein that do not significantly affect the activity, preferably conservative amino acid substitutions. Includes. Conservative substitutions are not limited to these, but include basic amino acids (algebraic amino acids). (Ginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), Polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine) (Phaseol and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and within the group of small amino acids (glycine, alanine, serine, threonine, and methionine) Examples of amino acid substitutions include non-standard or non-natural amino acids (e.g., 4-hydroxypropyl amino acids). (Lorine, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, α-methylserine) It is also possible to substitute standard amino acid residues in the parent protein using this method.

[0035] According to another embodiment of the present invention, a functional variant of the parent protein is a variant of the parent protein. This includes the deletion and / or insertion of one or more amino acids. For example, the mature GDF15 protein. Functional variants of mature GDF15 protein include 1, 2, 3, 4, 5, 6, 7, and 8. , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2 Deficiencies of 2, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids Loss and / or insertion, preferably 1 to 30 ami at the N-terminus of the mature GDF15 protein. It may include the absence of an acid.

[0036] According to yet another embodiment of the present invention, a functional variant of the parent protein is the parent protein Substitutions for, preferably conservative amino acid substitutions, and deletions and / or insertions, preferably Includes small deletions and / or insertions of amino acids.

[0037] According to embodiments of the present invention, the fusion protein of the present invention has an amino acid configuration of mature GDF15. GDF15 protein having an amino acid sequence at least 90% identical to the column, for example, GDF 15(197~308)(SEQ ID NO: 6); or the mature GDF15 with a shortened N-terminus. An amino acid sequence that is at least 90% identical to the no-acid sequence, for example, GDF15(200~308 )(Sequence ID 7), GDF15(201~308)(Sequence ID 8), GDF15(202 ~308) (Sequence ID 9), GDF15 (203~308) (Sequence ID 10), or GD Contains F15 (211-308) (Sequence ID 11). GDF15 protein is obtained from food intake. The effects of GDF15 protein on blood glucose levels, insulin resistance, and body weight, etc. As long as at least one of the biological activities of the quality is maintained, SEQ ID NOs: 6, 7, 8, 9, 10 or It may have at least one substitution, insertion, and deletion for 11.

[0038] In certain embodiments, the fusion protein of the present invention is sequence numbers 6, 7, 8, 9, 10, and This includes, but is not limited to, 11 amino acid sequences, including the amino acid sequence of SEQ ID NO: 11. It contains GDF15 protein.

[0039] Any suitable half-life extension protein can be used in the fusion protein according to the embodiments of the present invention. It is possible. The term "half-life prolonged protein" as used in this specification means It is known that half-life-extending proteins extend the half-life of proteins to which they are fused. It may be any protein or a fragment thereof. Examples of such half-life prolonged proteins are However, these are not the only examples, but include human serum albumin (HSA), immunoglobulins. The constant fragment domain (Fc) of robulin (Ig), or transferrin (Tf) Examples include: In embodiments of the present invention, the half-life extension protein is HSA or its functional Includes variants. In certain embodiments of the present invention, the half-life extension protein is less than SEQ ID NO: 1. It contains an amino acid sequence that is at least 90% identical. In a preferred embodiment of the present invention, half-life The extended protein has a cysteine ​​residue at position 34 of the HSA replaced with serine or alanine. Includes HSA or its functional variants.

[0040] In a particular embodiment, the fusion protein of the present invention is selected from the group consisting of SEQ ID NOs: 1 to 3. It contains a half-life-extending protein having the amino acid sequence described above.

[0041] Any suitable linker can be used in the fusion protein according to the embodiments of the present invention. Yes, it is possible. The term "linker" as used herein includes peptide linkers. This refers to the connecting section. The linker ensures proper folding and minimizes dimensional obstruction. It is preferable that the structure of each functional element within the fusion protein is not significantly interfered with. In certain embodiments of the present invention, the peptide linker contains 2 to 120 amino acids. For example, peptide linkers are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 4 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 8 0, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 1 05, 106, 107, 108, 109, 110, 111, 112, 113, 114, 1 It contains 15, 116, 117, 118, 119, or 120 amino acids.

[0042] In embodiments of the present invention, the linker increases the flexibility of the fusion protein element. In a particular embodiment of the present invention, the linker is GS-(GGGGS)n or AS-(GGGGS ) Sequence (GGGGS) n (where n is 2-2) including but not limited to n-GT 0, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 It can be a flexible linker that includes (a certain type of linker).

[0043] In other embodiments of the present invention, the linker is structured. Specific implementation of the present invention Morphologically, the linker includes AS-(AP)n-GT or AS-(EAAAK)n-GT. However, this does not apply to sequences (AP)n or (EAAAK)n (where n is 2 to 2). 0, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 A structured linker can be made which includes (a). In other embodiments of the present invention, the linker is the array (GGGGA) n (PGGGS) n (AGGGS) n Or GGS-(EGK SSGSGSESKST) n -Includes GGS (where n is between 2 and 20).

[0044] In embodiments of the present invention, the fusion protein is sequence numbers 5, 25-30, 36-37, 4 It has at least 90% sequence identity with 0, 48, 55-56, 59-60, or 64-75. It contains the amino acid sequence. In a particular embodiment of the present invention, the fusion protein is SEQ ID NO: 5 , 25-30, 36-37, 40, 48, 55-56, 59-60 and 64-75 It contains an amino acid sequence selected from the group. In a more specific embodiment of the present invention, the fusion protein The chemicals are sequence numbers 5, 25-30, 40, 55-56, 55-56, 59-60, and 7 The amino acid sequence comprises an amino acid sequence selected from the group consisting of 0. In a further, more specific embodiment of the present invention The fusion protein has the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 60, or SEQ ID NO: 26. The fusion protein contains polyhysteric compounds at the amino or carboxyl terminus of the protein. Tags that facilitate purification, such as thidine tags, antigenic epitopes, or binding domains. It may have a small extension.

[0045] The fusion proteins disclosed herein are not limited to those described herein, but include dietary proteins. Effects on intake, oral glucose tolerance test, blood glucose level measurement, insulin resistance analysis, weight change Pharmacokinetic analysis, toxicological analysis, immunoassay for the level and stability of full-length fusion proteins Analysis of GDF15 including saturation and mass spectrometry, as well as ex vivo stability analysis in human plasma. The physical activity can be characterized or evaluated.

[0046] The present invention also provides isolated nucleic acid molecules encoding the fusion protein of the present invention. In the application method, the isolated nucleic acid molecules were SEQ ID NOs: 5, 25-30, 36-37, 40, 48, and 55. Amino acid compounds having at least 90% sequence identity with ~56, 59~60, or 64~75 Encodes a fusion protein containing a sequence. In certain embodiments, the isolated nucleic acid molecule is sequence number From 5, 25-31, 36-37, 40, 48, 55-56, 59-60 and 64-75 Encodes a fusion protein containing an amino acid sequence selected from a group. In terms of morphology, isolated nucleic acid molecules are sequence numbers 5, 25-30, 40, 55-56, 59-60, It encodes a fusion protein containing an amino acid sequence selected from the group consisting of 70. In certain embodiments, the isolated nucleic acid molecule contains the sequence of nucleotides 76-91. nothing.

[0047] According to another embodiment of the present invention, the nucleic acid molecule encoding the fusion protein is an expression vector —It can be contained within. Expression vectors are not limited to these. However, vectors for expressing recombinant proteins, and viral vectors, etc. Examples include vectors used to deliver nucleic acids into the body of an elephant and express them in target tissues. Examples of viral vectors suitable for use with the present invention are not limited to these. Adenovirus vectors, adeno-associated virus vectors, lentiviral vectors These are some examples. The vector may be a non-viral vector. Examples of these, though not limited to them, include plasmids, bacterial artificial chromosomes, and yeast. Examples include maternal artificial chromosomes and bacteriophages. Vectors are, for example, promoters. -, ribosome-binding element, terminator, enhancer, selection marker, or multiple It may include any element to establish the conventional function of the expression vector, such as the origin of expression. can.

[0048] According to another embodiment of the present invention, the nucleic acid molecule encoding the fusion protein is derived from a human fetus. Desired host cells such as Henogenin Kernel Cells (HEK) or Chinese Hamster Ovarian Cells (CHO) To improve expression by recombination from cells, the present disclosure is taken into consideration and is well known in the art. Codon optimization can be performed using technology.

[0049] The present invention also provides a host cell containing a nucleic acid molecule encoding the fusion protein of the present invention. Host cells are not limited to these, but include those that express recombinant proteins. Host cells for this purpose, and hosts for delivering nucleic acids into the target body and expressing them in the target tissue. Examples include cells. Examples of host cells suitable for use with the present invention are not limited to these. While not directly related, HEK or CHO cells are examples.

[0050] In another general embodiment, the present invention relates to a method for obtaining the fusion protein of the present invention. In a typical embodiment, such a method involves (1) under conditions in which a fusion protein is produced, (2) Culturing host cells containing nucleic acid molecules that encode quality, and (3) producing by host cells This includes recovering the fusion protein. The fusion protein is further used in the art. It can be purified using well-known methods.

[0051] In certain embodiments, the fusion protein is expressed in host cells, and is not limited to these. Although it is not something that can be done, affinity chromatography, size exclusion chromatography, ultrachromatography Purification from host cells using a combination of one or more standard purification methods, including filtration and dialysis. Preferably, the fusion protein is purified to be free of proteases.

[0052] The present invention also relates to a pharmaceutical composition comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier. provide.

[0053] The present invention relates to a nucleic acid molecule encoding the fusion protein of the present invention and a pharmaceutically acceptable carrier. The present invention further provides a composition comprising nucleic acid molecules encoding the fusion protein of the present invention. The product is a delivery medium for introducing nucleic acid molecules into cells in order to express fusion proteins. It may include substances. Examples of nucleic acid delivery media include liposomes, natural polymers and compounds. Biocompatible polymers including polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides Artificial viral envelopes, metal particles, and bacteria, baculoviruses, adenoviruses and viruses such as retroviruses, bacteriophages, cosmids, plasmids The technical field describes fungal vectors and their expression in various eukaryotic hosts. Other recombinant media commonly used in this context include:

[0054] A pharmaceutically acceptable carrier is one or more pharmaceutically acceptable excipients, buffers, stabilizers, Alternatively, other materials well known to those skilled in the art may be included. Examples of pharmaceutically acceptable carriers include These are not limited to, but include water, saline solution, buffers, sugars, and other isotonic agents. Polyhydric alcohols, auxiliary substances such as humectants or emulsifiers, preservatives, and combinations thereof. One or more of the following are examples. Such materials must be non-toxic and used in a manner that is appropriate for the purpose of use. The amount and concentration must not interfere with the effect of the active ingredient. The exact properties of the carrier or other materials are important. Routes of administration: for example, intramuscular, subcutaneous, oral, intravenous, cutaneous, mucous membrane (e.g., intestines), nasal cavity Alternatively, it may be determined depending on the intraperitoneal route, etc. For example, liquid pharmaceutical compositions generally consist of water, petroleum, Contains liquid carriers such as animal or vegetable oils, mineral oils or synthetic oils. Physiological saline, dextrose, etc. Alternatively, other sugar solutions, or ethylene glycol, propylene glycol, or polyethylene It may contain glycols such as glycols. The composition for parenteral administration is in lyophilized form. It can be stored as a liquid or in solution, and generally has a stopper through which a subcutaneous injection needle can be inserted. A container with a sterile access port, such as an intravenous solution bag or vial, has a - It can be inserted.

[0055] According to embodiments of the present invention, the pharmaceutical composition comprises one or more additional components, such as another active ingredient. It is possible.

[0056] The present invention also relates to a kit containing the pharmaceutical composition of the present invention. Such a kit contains the dry chemical composition of the present invention. A first container containing a dried fusion protein, and prior to administration to the subject, the dried fusion protein and A second container having an aqueous solution to be mixed, or a container containing the liquid pharmaceutical composition of the present invention. It may include a single container. Such a kit provides a single dose of the pharmaceutical composition of the present invention. It may contain one or more dose units. The kit may include syringes (e.g., liquid syringes and lyosyringes). It may also include instructions for use. Such instructions for use should describe how to use the materials provided in the kit. It is sufficient to describe its use and properties, and that it is suitable for the specific metabolic disease being treated. It is possible.

[0057] This invention relates to type 2 diabetes, elevated blood glucose levels, elevated insulin levels, obesity, dyslipidemia, and diabetes. Metabolic diseases such as diseased nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis. This also relates to the use of the pharmaceutical compositions described herein for the treatment or prevention of harm or condition. According to embodiments of the present invention, metabolic diseases, disorders, or conditions of subjects requiring treatment are treated or The method of prevention involves administering a therapeutically or prophylactically effective amount of the pharmaceutical composition of the present invention to the subject. The invention includes a pharmaceutical composition containing the fusion protein or a nucleus encoding the fusion protein. Any of the pharmaceutical compositions described herein, including those containing an acid, is part of the present invention. It can be used in this way.

[0058] As used herein, "subject" means a treatment by the method according to the embodiments of the present invention. This means any animal that has been treated or given a treatment, specifically mammals, and most specifically humans. As used herein, the term "mammal" encompasses all mammals. Examples of dairy animals are not limited to these, but include cows, horses, sheep, pigs, Non-human primates such as cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, or apes. (NHP), more specifically, humans.

[0059] "Metabolic disease, disorder, or condition" refers to any disorder related to abnormal metabolism. Examples of metabolic diseases, disorders, or conditions that can be treated according to this method include: While not limited to these, other conditions include type 2 diabetes, elevated blood sugar levels, elevated insulin levels, and obesity. , dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or chronic rheumatoid arthritis It can be listed.

[0060] As used herein, the terms “to treat,” “to be treated,” and “treatment” are: This refers to administering a composition to a subject to obtain a desired therapeutic or clinical outcome in that subject. In terms of form, the terms "to treat," "to be treated," and "treatment" refer to the pharmaceutical composition of the present invention. Administering this substance can lead to type 2 diabetes, elevated blood glucose levels, elevated insulin levels, obesity, and lipid levels. This includes conditions such as diabetes, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or chronic rheumatoid arthritis. This refers to reducing, mitigating, or delaying the progression or development of a psychotic disorder.

[0061] The term "therapeutic effective dose" refers to the amount required to produce the desired biological or clinical effect. This refers to the amount of therapeutically active compound. According to embodiments of the present invention, "therapeutic effective amount" is clinical A sufficient amount to produce useful or desired results, including the results described above. The therapeutically effective dose is: It can be administered in one or more doses. In relation to the disease state, the effective dose is... a sufficient amount to improve, stabilize, or delay the progression of the disease. Depending on the administration method, the therapeutically effective dose is for type 2 diabetes, elevated blood glucose levels, elevated insulin levels, and obesity. Diabetic dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis Fusion proteins required to treat or prevent metabolic diseases, disorders, or conditions such as It's about the quantity of quality.

[0062] According to embodiments of the present invention, the pharmaceutical composition of the present invention is administered intramuscularly, subcutaneously, orally, intravenously, orally. Considering this disclosure, the following routes of administration may be considered: skin, mucous membranes (e.g., intestines), nasal cavity, or abdominal cavity. It can be administered to the target by any method well known to those skilled in the art. Specific Embodiments The pharmaceutical composition of the present invention is administered to the subject by intravenous injection or subcutaneous injection.

[0063] The dosage, frequency of administration, and duration of administration of the pharmaceutical composition to the target according to embodiments of the present invention. The parameters are not limited to any particular form. The optimal values ​​for such parameters are Various factors such as the target of treatment, the specific metabolic disease being treated, the severity of the disease, and the route of administration may influence the decision. Therefore, it is possible to determine the desired therapeutic or clinical outcome, and a person skilled in the art can obtain it. The optimal values ​​for such parameters can be determined in this way. For example, a pharmaceutical composition can be used for 1 day It can be administered once, or multiple times a day, for example, two, three, or four times a day. Typical doses range from approximately 0.1 μg / kg to approximately 10 μg / kg, depending on factors such as those mentioned above. The fusion protein concentration can be 0 mg / kg or less, or greater than or equal to that.

[0064] Embodiment Embodiment 1 comprises (a) a half-life extension protein, (b) a linker, and (c) GDF15 A fusion containing a protein, arranged in the order (a)-(b)-(c) from the N-terminus to the C-terminus. Regarding proteins.

[0065] Embodiment 2 describes a GDF15 protein that is a human GDF15 protein or a functional variant thereof. This is a different form of the fusion protein described in Embodiment 1.

[0066] Embodiment 3 is one in which the GDF15 protein is selected from the group consisting of SEQ ID NOs: 6 to 11. The amino acid sequence described in Embodiment 1 includes an amino acid sequence having at least 90% identity with the amino acid sequence. It is a fusion protein.

[0067] Embodiment 4 is an embodiment in which the GDF15 protein contains the amino acid sequence of SEQ ID NO: 11. This is the fusion protein described in Form 1.

[0068] Embodiment 5 is an embodiment in which the GDF15 protein is selected from the group consisting of SEQ ID NOs: 6 to 11. This is the fusion protein described in Embodiment 4, which includes an amino acid sequence.

[0069] Embodiment 6 describes a half-life extension protein that is human serum albumin (HSA) or has a function thereof. The fusion protein is one of the embodiments described in any of Embodiments 1 to 5, including the target mutant.

[0070] Embodiment 7 describes a half-life extension protein having at least 90% identity with SEQ ID NO: 1. The fusion protein described in Embodiment 6 contains the amino acid sequence.

[0071] Embodiment 8 is an embodiment in which the half-life extension protein is selected from the group consisting of SEQ ID NOs: 1 to 3. This is the fusion protein described in Embodiment 7, which includes a mino acid sequence.

[0072] Embodiment 9 is a version of any of Embodiments 1 to 8, wherein the linker is a flexible linker. It is a fusion protein.

[0073] Embodiment 10 is a linker with array (GGGGS)n (where n is 2 to 20) , including, for example, GS-(GGGGS)x8 or AS-(GGGGS)x8-GT, implementation form This is the fusion protein described in Form 9.

[0074] Embodiment 11 is described in any of Embodiments 1 to 9, wherein the linker is a structured linker. It is a fusion protein.

[0075] Embodiment 12 is a linker in array (AP)n or (EAAAK)n (where n is 2 (~20), for example, including AS-(AP)n-GT or AS-(EAAAK)n-GT This is the fusion protein described in Embodiment 11.

[0076] Embodiment 13 is represented by Sequence IDs 5, 25-31, 36-37, 40, 48, 55-60 or A fusion protein containing an amino acid sequence with at least 90% sequence identity between 64 and 75. That is the case.

[0077] Embodiment 14 corresponds to Sequence IDs 5, 25-31, 36-37, 40, 48, 55-60, and The fusion according to Embodiment 13, comprising an amino acid sequence selected from the group consisting of 64 to 75. It is a protein.

[0078] Embodiment 15 is a group consisting of sequence numbers 5, 25-30, 40, 55-60, and 70. The fusion protein described in Embodiment 14 includes the amino acid sequence selected from the above.

[0079] Embodiment 16 encodes a fusion protein described in any one of Embodiments 1 to 15. It is an isolated nucleic acid molecule.

[0080] Embodiment 17 is an isolated nucleic acid molecule containing the nucleotide sequences of SEQ ID NOs. 76-91. .

[0081] Embodiment 18 is an expression vector containing the nucleic acid molecule of Embodiment 16 or 17.

[0082] Embodiment 19 is a host cell containing the nucleic acid molecule of Embodiment 16 or 17.

[0083] Embodiment 20 encodes a fusion protein under conditions in which a fusion protein is generated. (2) Culturing host cells containing nucleic acid molecules, and (3) fusion cells produced by the host cells A fusion tank according to any one of Embodiments 1 to 15, which includes recovering protein. This is a method for producing protein.

[0084] Embodiment 21 involves a recovery step that purifies the fusion protein and removes the protease. This is a method according to Embodiment 20, which includes the following:

[0085] Embodiment 22 is a therapeutically effective amount of the fusion protein described in any one of Embodiments 1 to 15. This is a pharmaceutical composition comprising a substance and a pharmaceutically acceptable carrier.

[0086] Embodiment 23 encodes a fusion protein described in any one of Embodiments 1 to 15. This is a pharmaceutical composition comprising a therapeutically effective amount of nucleic acid molecules and a pharmaceutically acceptable carrier.

[0087] Embodiment 24 is a kit comprising the pharmaceutical composition described in Embodiment 22 or 23.

[0088] Embodiment 25 is a method for treating or preventing metabolic diseases, wherein the treatment or prevention of the disease The subject is administered an effective amount of the pharmaceutical composition described in either embodiment 22 or 23. This is a method that includes the following.

[0089] Embodiment 26 describes metabolic diseases such as type 2 diabetes, elevated blood glucose levels, elevated insulin levels, and obesity. Diabetic dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis This is the method according to Embodiment 25, selected from the group consisting of the following.

[0090] Embodiment 27 is an embodiment in which the pharmaceutical composition is administered subcutaneously or intravenously to the target, as in Embodiment 25 or 2 The method is as described in 6.

[0091] Embodiment 28 describes a patient requiring treatment for type 2 diabetes, elevated blood glucose levels, and insulin Elevated values, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or chronic A method for treating metabolic diseases selected from the group consisting of rheumatoid arthritis, wherein the subject is Amino acids selected from the group consisting of row numbers 5, 25-30, 40, 55-60, and 70. A therapeutically effective pharmaceutical composition comprising a fusion protein containing a sequence and a pharmaceutically acceptable carrier. The method includes administering the substance subcutaneously or intravenously.

[0092] Embodiment 29 describes a patient requiring treatment for type 2 diabetes, elevated blood glucose levels, and insulin Elevated values, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or chronic A method for treating metabolic diseases selected from the group consisting of rheumatoid arthritis, wherein the subject is A therapeutic agent comprising a fusion protein containing the amino acid sequence of sequence number 60 and a pharmaceutically acceptable carrier, The method comprises administering a therapeutically effective amount of a pharmaceutical composition.

[0093] Embodiment 30 is an embodiment of Embodiments 25-29 in which the pharmaceutical composition is administered intravenously or subcutaneously to the target. The method is one of the following.

[0094] Embodiment 31 is for type 2 diabetes, elevated blood glucose levels, elevated insulin levels, obesity, and dyslipidemia. From the group consisting of diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or chronic rheumatoid arthritis Any one of Embodiments 1 to 15 for use in the treatment or prevention of selected metabolic diseases It is a fusion protein as described in [reference]. [Examples]

[0095] The following embodiments of the present invention are intended to further illustrate the essence of the present invention. The examples are not intended to limit the present invention, and the scope of the present invention is defined by the supplementary claims. I hope you understand this.

[0096] Example 1: Design of a fusion molecule containing GDF15 (effect of shortening GDF15) Like other members of the TFGβ family, GDF15 forms dimers within the endoplasmic reticulum. It is cleaved in a furin-like manner to produce the secreted mature GDF15 (amino acids 197-308). It is synthesized as a preproprotein. The secreted mature GDF15 homodimer is approximately 25 kD. a is such that each monomer can form up to four intramolecular disulfide bonds, and one The constituent elements of the homodimer are linked together by intermolecular disulfide bonds.

[0097] In this invention, the crystal structure of GDF15 was determined and is shown in Figures 1A and 1B. The structure is such that the C-terminus of mature GDF15 is embedded in the dimer interface, whereas the N-terminus This indicates that it is exposed. This exposed end is half of the N-terminus of GDF15. This enables the linking of fusion proteins, such as proteins that prolong the mitotic life cycle.

[0098] This crystal structure reveals a novel disulfide pairing pattern of cysteine ​​residues in GDF15. It also shows that TFGβ1 is pairing C1-C3 and C2-C7 (i.e., The pairing between the 1st and 3rd cysteine ​​residues, and the 2nd and 7th cysteine ​​residues While it has pairings between residues, GDF15 has pairings between C1-C2 and C3-C7 It has a pairing (see Figures 1A and 1B). This unique disulfide pairing The link is located at the N-terminus of the protein, away from the cysteine ​​knot which contains other disulfide bonds. This creates a loop formed by the C1-C2 pairings located in that position. The N-terminus of GDF15 is important for dimer formation or the folding of the entire protein. It may not be necessary, and GDF15 and its N-terminal fusion protein are C1 and C2, N-terminal deletions involve the deletion of residues within the C1-C2 loop, or even the C-terminal residue of C2. Therefore, it is predicted that it will not be affected.

[0099] Example 2: Design of a fusion molecule containing GDF15 (influence of linker) Different linkers between HSA molecules and GDF15 molecules were evaluated. (GGG sequence) A flexible linker containing GS)n and a structured linker containing sequence (AP)n or (EAAAK)n We evaluated both the index (where n is between 2 and 20).

[0100] Fusion proteins containing different linkers, their biophysical properties, and dietary intake in lean mice The effects of these factors on the efficacy of the drug, their pharmacokinetic (PK) values ​​in mice, and human blood The ex vivo stability of these mutants was compared. The results for the tested linker mutants are shown in the table. As shown in 1, the molecule containing the (EAAAK)8 linker and SEQ ID NO: 31 is obtained by HPLC. It showed aggregation. The remaining seven linker mutants in Table 1 did not show aggregation.

[0101] [Table 1] * A 6xHis tag was concatenated to the N-terminus for purification purposes.

[0102] These mutants were also identified through in vivo experiments with mice, as well as with human whole blood and blood. The stability of the linker was evaluated by ex vivo stability experiments in plasma samples. The results from these experiments were analyzed using morphology. Anti-GDF15 capture antibody and anti-HSA detection. An immunoassay using antibody pairs is used to measure the presence of both molecules on either side of the linker. This allowed us to assess the degree of completeness of the linker. The overall completeness of the molecule was roughly evaluated. This state was analyzed using liquid chromatography with different surrogate peptide sequences from HSA and GDF15. Analysis was performed by Raffy / mass spectrometry (LC-MS). Immunoassay was performed on all linkers. - The mutants showed a stable PK profile, and all linker mutants No spikes in sample concentration in plasma were observed over 48 hours. (LC-MS) The results were consistent with the immunoassay, and surrogate particles were obtained from different parts of the HSA and GDF15 molecules. The peptide was shown to be complete. Phosphorus was analyzed by LC-MS using a surrogate peptide. The PK profiles of the Kerr variants showed similar trends in different linker variants, and in all cases... The linker mutant also showed detectable levels on day 7. All mutants in Table 1 are: Except for Sequence ID No. 31, the desired biophysical properties and PK values ​​were shown.

[0103] Each linker mutant was subjected to food intake experiments in lean mice to determine their in vivo activity. Sex was evaluated. Table 2 shows the effect of fusion proteins on the reduction of food intake. This shows the effect of linker variants. A clear influence of the linker on the effect was observed. Flexible In the (GGGGS)n linker, the length of the linker can be increased from 2 to 4 to 8 to achieve fusion. The effect of the synthesizing protein was greatly increased. In the stiffer (AP)n linker, this trend was... The effect is not so pronounced, and the degree of freedom of the GDF15 molecule within the fusion protein plays a crucial role. This suggests that they are fulfilling their role.

[0104] [Table 2] * A 6xHis tag was concatenated to the N-terminus for purification purposes.

[0105] Example 3: Design of a fusion molecule containing GDF15 (effect of HSA mutation) Human serum albumin, as a half-life extension protein, is linked to the N-terminus of GDF15. We designed a recombinant protein fused using this method. According to this design, the dimer of GDF15 Because the interface for emulation is not destabilized, natural intermolecular chain disulfide bonds are formed. The fused HSA protein extends from each GDF15 arm into the GDF15 homozygous region. A mer is obtained. According to this approach, HSA-GDF15 homodimers can be produced. Therefore, it only requires one gene.

[0106] Natural human serum albumin protein has 35 disulfide bonds forming 17 disulfide bonds. It contains a cysteine ​​(Cys, C) residue, with Cys-34 being the only free residue in the molecule. is cysteine. This free Cys-34 acts on multiple reactive oxygen species (ROS) and reactive nitrogen species (RNS) by scavenging them, thereby functioning as a free radical scavenger has been reported. Therefore, mutating this free Cys reduces the risk of heterogeneity caused by oxidation to a minimum.

[0107] The free cysteine at position 34 of HSA was mutated to serine or alanine, and these HSA ( C34S) or GDF15 fusion molecules having the HSA (C34A) mutation were analyzed. These molecules were all purified via a three-step purification method consisting of (i) ion-exchange chromatography, (ii) hydrophobic interaction chro matography, and (iii) size-exclusion chromatography When these molecules were initially prepared, HPLC analysis showed that both molecules were pure and free of aggregation (Table 3).

[0108] However, two weeks after preparation, the fusion protein comprising the HSA (C34S) mutation (sequence number 48) showed aggregation as detected by HPLC, whereas the protein comprising the HSA (C34A) mutation (SEQ ID NO: 40) remained free of aggregation even after four weeks.

[0109]

Table 3

[0110] Example 4: Protease cleavage propensity of GDF15 The present inventors observed that the arginine residue at amino acid position 198 (R198) of GDF15 is prone to protease cleavage in HSA -GDF15 fusion molecules. Such cleav age results in a heterogeneous population, which is undesirable for a therapeutic composition. Such cleavage This can be prevented by a protease inhibitor cocktail. The removal of HSA-GDF15 fusion ions was investigated. Table 4 shows the results measured by HPLC. Two types of HSA affinity columns tested for protein purification are shown. In terms of purity, the HSA-GDF15 fusion protein purified by either method was 100%. It was % and was perfect. At low concentrations (2-5 mg / ml), purification was performed by either method. The proteins were also perfectly preserved throughout the entire 4-week test period. However, high concentration At a dose of 40-50 mg / ml, the antibody-based HSA resin (CaptureSele) Only CT Corporation produced a protein that did not contain protease, and this was the case throughout the entire 4-week trial period. It was completely preserved over time. HSA-ligand-based resin (Albupure) The resulting protein was initially complete, but when stored at high concentrations, it deteriorated over time. It was also broken down. Upon addition of a protease inhibitor cocktail (PI) and EDTA, Alb High-concentration HSA-GDF15 fusion protein purified using upure's resin The solution completely stopped. Therefore, purification methods are important for producing stable therapeutic compositions. It plays a role in this. No corresponding degradation was observed in vivo or ex vivo. Once the therapeutic composition is prepared without protease, the fusion protein This indicates that the decomposition of is not a problem in vivo. Therefore, CaptureS Methods like those using elect's resin effectively remove potential proteases during manufacturing. The purification method that can be used effectively produces homogeneous, complete, and stable GDF15 therapeutic agents. It is essential for doing so.

[0111] [Table 4]

[0112] Example 5: N-terminal deletion mutants of GDF15 According to the GDF15 crystal structure shown in Figure 1A and Figure 1B, the GD involved in the deletion mutant F15 N-terminus is predicted not to be critical for dimer formation and overall protein folding. According to this crystal structure, such N-terminal deletions are any potential receptor interactions are also predicted to not be affected. Various deletions at the N-terminus of GDF15 were included HSA-GDF15 fusion proteins were tested for in vivo activity.

[0113] N-terminal deletion variants of GDF15 with the protease cleavage site (R198) of GDF15 removed were designed. Immediately after residue R198 is a potential deamination site composed of residues N199 to G200, and deamination of a substrate is still undesirable in therapeutic compositions. N-terminal deletion of GDF15 can remove both the proteolytic cleavage site and the deamination site. When incorporated into a fusion protein together with HSA, the resulting GDF15 deletion mutants include GDF 15 (201-308, SEQ ID NO: 8), GDF15 (202-308, SEQ ID NO: 9), and GDF15 (211-308, SEQ ID NO: 11) were included. In in vivo experiments in mice, showed that N-terminal deletion mutants of GDF15 still remain active in reducing food intake (Figure 17). The experimental results show that these N-terminal deletion mutants of GDF15 are properly expressed, form correct dimers, and are active in vivo, which has been confirmed .

[0114] Example 6: Inactive mutant of GDF15 Table 5 shows the results of eliminating the in vivo activity of GDF15 and identifying the functional epitope of GDF15. This shows 12 different mutants of GDF15 that were created for this purpose. The body contains five types of single mutants, two types of double mutants, and five types of triple mutants. The body contains these mutations in the HSA-GDF15 fusion protein. Physical properties and activity were evaluated (Table 5). Of the 12 mutants, One mutation was not observed, four formed aggregates over time, and these mutations affected the protein. It was shown that folding and biophysical properties were impaired. The remaining seven types of mutants Of these, four have a single mutation in GDF15, and these mutants were used in mice. The study examined the decrease in food intake compared to the wild type. Three single mutants (I89) were tested. R, I89W, and W32A) lost in vivo activity, but the remaining mutant (Q60W) was wild It showed activity similar to that of the viable form. These results indicate that the I89R, I89W, and W32A mutations are... This demonstrates that it inhibits the interaction of GDF15 receptors / co-receptors, and the functional effects of GDF15. This suggested that the pitope was located around residues I89 and W32. The numbering is based on the mature GDF15 present in the fusion protein. For example, "1 " refers to the first amino acid of mature GDF15 (SEQ ID NO: 6), and "89" refers to mature GDF1 This refers to the 89th amino acid in a protein.

[0115] [Table 5] * A 6xHis tag was concatenated to the N-terminus for purification purposes.

[0116] Example 7: Expression and purification method Expression To achieve expression levels exceeding 20 ml, the cells were grown in Expi293® expression medium. Expression was performed using HEK Expi293® cells. The cells were subjected to 12°C under 8% CO2 conditions. The cells were grown at 37°C with shaking at 5 RPM. An Expi293(trademark) expression kit was used. Then, the cells are divided into 2.5 × 10⁶ cells per 1 ml. 6 They were transfected individually. For every liter of cells, 1 mg of total DNA was diluted in 25 ml of Opti-MEM and mixed with 2.6 ml of water. Dilute 1 ml of Expi293 (trademark) reagent in 25 ml of Opti-MEM and leave at room temperature for 5 minutes. Incubated. Diluted DNA and diluted Expi293 reagent were added together and incubated at room temperature. The cells were incubated for 20 minutes. Next, this DNA complex was added to the cells. The cells were shaken. I placed it in an incubator overnight. The day after transfection, I used a 5ml kit of Enha Dilute ncer1 in 50 ml of the kit's Enhancer2, and use the two Enhancers The entire amount was added to the cells. The transfected cells were returned to the incubator for another 4 days. They were then harvested. The cells were concentrated by centrifuging at 6000g for 30 minutes, and then proceeded to the purification process. The sample was filtered using a 0.2 μm filter while standing.

[0117] Expression was also performed in CHO cells. Plasmids were purified and characterized. Prior to the action, plasmid DNA20 containing the coding region of HSA-GDF15 One aliquot of 0 μg was linearized by restriction enzyme digestion with ACL I. Digestion with limiting endonucleases ensures the reliable removal of ampicillin resistance genes. Two 15 μg aliquots of linearized DNA were transferred to a BTX ECM 830 Electro Cell Manipulator (Harvard Apparatus, Massachusetts Holliston) to transfect two 1×10 7 cells (designated as transfection pools A and B ). The cells were electroporated three times at 250 V in a cuvette with a 4 mm electrode gap, with a 15 mill second pulse length and 5 second interval between pulses. The transfected cells were transferred to MACH-1 + L-glutamine in a shake flask and incubated for 1 day . Transfection pool A and transfection pool B were centrifuged, resuspended in MA CH-1 + MSX, transferred to a shake flask and incubated for 6 days. Transfected HSA fusion protein-producing cells from transfection pool A and transfection pool B were pooled and seeded in methylcellulose on day 8 after electroporation .

[0118] Purification Two-step purification using CaptureSelect resin and size exclusion chromatography was used. Cell supernatant from transiently transfected Expi293™ cells was loaded at an appropriate volume of 10 mg protein per ml of resin onto a pre-equilibrated (PBS, p H 7.2) HSA CaptureSelect column (CaptureSelect Human Albumin Affi nity Matrix sold by ThermoFisher S cientific). After loading, unbound proteins were removed by washing the column with 10 column volumes ( CV) of PBS (pH 7.2) HSA-GDF15 bound to ram in 10 CV of 20 mM Tris (pH 7.0) 2M MgCl 2 Elution occurred. The peak fraction was pooled, filtered (0.2 μm), and aged at 4°C in PB. Dialysis was performed against S (pH 7.2). After dialysis, the protein was filtered again (0.2 μL), and appropriate After concentrating to the appropriate volume, use a 26 / 60 Superdex 200 column (GE Hea Loaded into Ithcare. High-purity size exclusion chromatography (SEC) The protein fractions eluted from the sample (measured by SDS-PAGE) were pooled. Protein concentration was measured at 280 nm using a BioTek Synergy HTTM spectrophotometer. Absorbance was used for measurement. The quality of the purified protein was assessed by SDS-PAGE and analytical size extraction. Evaluated by HPLC (SE-HPLC, Dionex HPLC Systems). The endotoxin levels were measured using LAL assay (Pyrotell(registered trademark)-T, Assoc Measurements were taken using a device from Cape Cod Inc.

[0119] A two-step purification process using Albupure resin and SEC was employed. HSA-GDF15 fusion Synthetic proteins are immobilized with synthetic triazine ligands that selectively bind to HSA. Using AlbuPure resin (ProMetic BioSciences) Purified at room temperature. The supernatant for expression was poured into AlbuPure resin. Then, the resin was first 4 First, 15 CV PBS (pH 7.2), then 4CV 50mM Tris (pH 8.0), Washed with 0 mM NaCl buffer. The HSA-GDF15 bound to the column was subjected to 100m³ of water. The protein was eluted with 4CV of PBS buffer (pH 7.2) containing sodium octanoate (M). The fraction containing the quality is placed in a spin concentrator with a cutoff value of 30,000 kDa molecular weight. Concentrate to a volume of 10 mL using Amicon, then use PBS buffer (pH 7.2) The solution is then passed through a 26 / 60 Superdex S200pg column (GE) that has been equilibrated in step 7.2. The SEC fraction containing the HSA-GDF15 dimer was identified and analyzed by SDS-PAGE. The samples were pooled for use. Protein purity was evaluated by SDS-PAGE and SE-HPLC. Ta.

[0120] Examples 8-14 and 19 illustrate the present invention having the amino acid sequence of SEQ ID NO: 60. We are characterizing a fusion protein. This fusion protein contains glycine and serine. HSA maturation via the 42-amino acid linker GS-(GGGGS)8, which consists of residues. It is a fully recombinant protein that exists as a homodimer of a fusion with human GDF15. The predicted molecular weight of this fusion protein is 162,696 Da, and it is located at position 34 of the HSA. One of the naturally occurring free cysteine ​​molecules has been mutated into serine. This particular HSA-GDF15 fusion molecule is involved. For simplicity, the combined protein will be simply referred to as "FP1" in the following examples. . FP1 6xHis tagged variant containing AS-(GGGGS)x8-GT linker ( 6xHis-FP1 (SEQ ID NO: 26) was used for comparison in some of the following examples.

[0121] Example 8: Effect of FP1 on food intake in C57B1 / 6 mice The purpose of this experiment is to assess the dose-response of FP1 to inhibiting food intake in C57B1 / 6 mice. The objective was to demonstrate the effects of sex.

[0122] Male C57Bl / 6 mice were acclimatized in BioDAQ cages for a minimum of 72 hours. Subsequently, the mice were divided into six groups of eight based on their food intake, and the groups were formed 24 hours prior to the grouping. They were divided. Between 4:00 and 5:00 p.m., the animals' weight was measured and the solvent or FP1 was included. The composition was administered by subcutaneous injection. For each cage, the change in food intake was measured using BioDAQ. The system continuously recorded data for 48 hours after injection. In this experiment, 6xHi s-FP1 was used for comparison.

[0123] The results (Figure 2 and Table 6) are expressed as the average cumulative food intake over a predetermined time interval. The results showed that subcutaneous administration of FP1 to C57Bl / 6 mice was effective at all doses and time points tested. It was shown that food intake was significantly inhibited in animals treated with the solvent. 6xHis- FP1 reduced food intake at a dose of 8 nmol / kg.

[0124] [Table 6] The data is expressed as mean ± SEM. * p ≤ 0.05 for PBS; ** p ≤ 0.01 for PBS; *** Against PBS p ≤ 0.001; **** p ≤ 0.0001 for PBS One-way ANOVA: Tukey's multiple comparison test; n=8 / group

[0125] Example 9: FP relative to food intake in Sprague Dawley rats Effect 1 The purpose of this experiment is to use FP1 to inhibit food intake in Sprague-Dolly rats. The objective was to demonstrate the effects of dose responsiveness.

[0126] Male Sprague Dolly rats were allowed to acclimate in a BioDAQ cage for at least 72 hours. Then, the rats were divided into 6 groups of 8 rats each based on their food intake, and the 24 hours prior to the experiment were measured. The animals were divided into loops. Between 4:00 PM and 5:00 PM, their weight was measured, and they were given a solvent or fusion treatment. A protein-containing composition was administered by subcutaneous injection. Changes in food intake were observed for each cage. This was continuously recorded using the BioDAQ system for 48 hours after injection. For comparison, we used the 6xHis-FP1.

[0127] The results are shown in Figures 3 and 7. Subcutaneous administration of FP1 resulted in a 2.5 nm reduction compared to solvent-treated animals. Food intake was inhibited at doses of ol / kg and 10 nmol / kg. The inhibition rate was 24 and After 48 hours, a statistically significant difference was reached only at the highest dose tested (10 nmol / kg). FP Drug 1 reduced food intake at a dose of 8 nmol / kg, and its effect was significant at 24 and 48 hours. That was the case.

[0128] [Table 7] The data is expressed as mean ± SEM. * p ≤ 0.05 for PBS; ** p ≤ 0.01 for PBS One-way ANOVA: Tukey's multiple comparison test; n=8 / group

[0129] Example 10: Glucose homeostasis and The effect of FP1 on body weight The purpose of this experiment was to ensure that the DIOC57Bl / 6 mice were fed throughout the entire two-week treatment period. By evaluating the effects of FP1 on food intake, body weight, and glucose homeostasis... there were.

[0130] The body weight of male DIO mice was measured, and FP1 was administered every three days on days 0, 3, 6, 9, and 12. (q3d) 2 mL / kg was administered subcutaneously. The same procedure was followed for the solvent-treated group and the rosiglitazone-treated group. PBS was administered according to the regimen. Rosiglitazone was administered as a control at 0.015% in the diet. They were given free access inside. The weight of the mice and their diet was recorded daily. A blood glucose meter (One Tou) was used. ch (registered trademark) Ultra (registered trademark), Lifescan, Milpey, California Blood glucose levels were measured using a Bruker Mini-GX. Body fat mass and lean body mass were measured using a Bruker Mini-GX. Using Spec LF110, time-domain NMR (TD-NMR) was used to determine the state of the awakened Mau Quantitative analysis was performed using S. In the oral glucose tolerance test (OGTT), mice were fasted for 4 hours. 2 g / kg of glucose was administered orally at a dose of 10 mL / kg to 0, 30, 60, and 90. The tails were cut off and blood glucose levels were measured 120 minutes after glucose administration. Insulin was measured a few minutes later.

[0131] At the end of the experiment, the mice were euthanized by CO2 inhalation, and final blood samples were collected. Serum After placing it in a 96-cell plate filled with wet ice, it was stored at -80°C. The liver was removed, and the liver The fat content relative to the total mass of organ slices is measured according to the manufacturer's instructions, using Bruker Min Evaluation was performed using TD-NMR with iSpec mq60.

[0132] An insulin-resistant fasting homeostasis model (HOMA-IR) was developed using fasting blood glucose levels (m It is calculated by dividing the product of (g / dL) and insulin level (mU / L) by the factor of 405. Ta.

[0133] DIO mice were treated with 1 nmol / kg and 10 nmol / kg of FP1 in q3d. As a result, body weight (Table 8) and food intake (Table 9) decreased. The amount of decrease is described below. The value reached a statistically significant level only at a specific point in time.

[0134] FP1 was found in DIO mice at levels of 1 (days 2-14) and 10 nmol / kg (days 1-14). Weight loss was observed at doses of ) (Table 8 and Figure 4). A significant decrease in food intake was observed at 1 nmol. The dose was measured at / kg on days 1 and 2 of the experiment, and at a dose of 10 nmol / kg on days 1, 8, and 9. This was recognized (Table 9).

[0135] [Table 8]

[0136] [Table 9]

[0137] In the OGTT conducted on day 14 of the experiment, FP1 was effective at all three doses tested. Blood glucose levels were significantly lower compared to solvent-treated animals at all time points from time point 0 onward. Table 10). When this was further quantified as the total area under the curve (AUC) and ΔAUC, the results were obtained. All three doses tested showed significantly lower levels compared to the solvent (Table 10 and Figures 5A and 5B). ).

[0138] [Table 10]

[0139] Blood glucose levels were measured at the start of the experiment (day 0), day 7, and day 13 (Table 11, and Figure 6). FP1 was administered at doses of 1 nmol / kg and 10 nmol / kg on day 13 of the experiment. It statistically significantly reduced blood glucose levels.

[0140] [Table 11]

[0141] During the OGTT, the plasma insulin level in FP1 was 0.1 nmol / kJ at 30 minutes. The dose was significantly higher than that of the corresponding solvent group at g doses of 1 and 10 nmol / kg at the same time point. The amount was low (Table 12). The insulin fluctuation during the OGTT, as measured by total AUC, At a dose of 0.1 nmol / kg of FP1, the levels were higher than in the solvent group (Table 12), and at 1 and 10 nm. The levels were lower at ol / kg doses. In all cases, statistical significance was only found at the lowest dose. The difference was reached. At 90 minutes, mice treated with 1 and 10 nmol / kg of FP1 were more Although low insulin levels were observed, this effect did not show a statistically significant difference. Insulin sensitivity The HOMA-IR, used as an indicator of sex, was measured on the 14th day of the experiment. At this point, FP 1 reduced HOMA-IR at 10 nmol / kg and improved insulin sensitivity. (Table 13 and Figure 7)

[0142] [Table 12] Data is expressed as mean ± SEM. n = 8 / group * p<0.05 compared to the solvent-treated group.

[0143] [Table 13] The data is expressed as mean ± SEM. n=8 / group * p<0.05 compared to the solvent-treated group.

[0144] The magnitude of weight loss achieved by day 13 is measured in absolute body fat or body mass, regardless of the dosage. No measurable change in fat percentage (%) occurred (Table 14). At a dose of 10 nmol / kg A significant decrease in absolute lean body mass was observed. This decrease was expressed as lean body mass percentage (%). In this case, it was not observed. A final autopsy was performed on the 15th day of the experiment to measure the liver weight. Table 15). FP1 is calculated as absolute liver weight and percentage of body weight (%) at a dose of 10 nmol / kg. It reduced liver weight. A reduction was observed at a dose of 1 nmol / kg, but which one was the cause? The parameters did not reach statistically significant values. Liver fat was measured by NMR in a biopsy. (Table 16). FP1 fusion protein was administered at doses of 1 and 10 nmol / kg in liver biopsies. It reduced the liver fat content, expressed as a percentage of weight (%). This reduction was observed at higher doses. It was significant.

[0145] [Table 14] Data is expressed as mean ± SEM. n = 8 / group * p<0.05 compared to the solvent-treated group.

[0146] [Table 15] Data is expressed as mean ± SEM. n = 8 / group * p<0.05 compared to the solvent-treated group.

[0147] [Table 16] Data is expressed as mean ± SEM. n = 8 / group * p<0.05 compared to the solvent-treated group.

[0148] Example 11: Effects of FP1 on blood glucose levels and body weight in ob / ob mice The purpose of this experiment was to study the effects of an 8-day study in obese, hyperglycemic leptin-deficient ob / ob mice. The objective was to evaluate the effects of FP1 on body weight and blood glucose levels throughout the treatment process.

[0149] The body weight of male ob / ob mice was measured, and FP1 was administered every 3 days on days 0, 3, and 6 (q3 d) 2 mL / kg was administered subcutaneously. The weight of the mice and their diet was recorded daily. Blood glucose meter Blood glucose levels were measured daily using [a specific method]. At the end of the experiment, the mice were euthanized, and the final blood samples were taken. It was collected.

[0150] FP1, at a dose of 1 nmol / kg, was used in ob / ob mice compared to solvent-treated mice. Then, a significant reduction in weight was observed from day 2 to day 8 (expressed as a percentage of starting weight). (It can be used). FP1 was administered at a dose of 10 nmol / kg to ob / ob mice, which showed solvent-treated results. The weight loss from day 1 to day 8 for the subject is expressed as a percentage of the starting weight. (Table 17 and Figure 8).

[0151] [Table 17]

[0152] FP1 was administered at a dose of 10 nmol / kg to ob / ob mice on day 1 and during the experiment. On day 2, and from day 4 to day 8, the dietary blood glucose levels of solvent-treated mice were reduced. A decrease in blood glucose levels was observed at 1 nmol / kg, but this effect did not reach a statistically significant value. (Table 18 and Figure 9).

[0153] [Table 18]

[0154] Example 12: Pharmacokinetics of Multiple Species Pharmacokinetics in mice FP1 was administered to female C57Bl / 6 mice at a dose of 2 mg / kg in PBS (pH 7). The drug was administered intravenously and subcutaneously. After both administration routes, blood samples were collected, and the serum was processed to obtain the drug. The drug concentration was measured over a 7-day period. The concentration of FP1 was measured by immunoassay. The serum drug concentration / time profiles are summarized in Tables 19 and 20 and shown in Figure 10.

[0155] [Table 19]

[0156] [Table 20]

[0157] Pharmacokinetic analysis revealed that in C57Bl / 6 mice after subcutaneous and intravenous administration, The final half-lives of FP1 were shown to be 1.67 and 1.57 days (Table 21). FP1 is a type of phosphate. The drug showed an average bioavailability of approximately 71% after administration.

[0158] [Table 21] Note: * Tmax (Median)

[0159] Pharmacokinetics in rats FP1 was administered to female Sprague Dolly rats at a dose of 2 mg / kg in PBS (pH 7). The drug was administered intravenously and subcutaneously according to the prescribed dosage. After both administration routes, blood samples were collected and serum was processed. The drug concentration was measured over a 7-day period. The concentration of FP1 was measured by immunoassay. The drug concentration / time profiles in serum are summarized in Tables 22 and 23, and shown in Figure 11. show.

[0160] [Table 22]

[0161] [Table 23] * The results were confirmed through repeated analysis.

[0162] Pharmacokinetic analysis showed that in Sprague-Dolly rats after subcutaneous and intravenous administration... The final half-lives of FP1 were shown as 1.34 and 1.51 days, respectively (Table 24). FP1 It showed an average bioavailability of approximately 23% after subcutaneous administration.

[0163] [Table 24] Note: * Tmax (Median)

[0164] Pharmacokinetics in monkeys FP1 is a naive male crab-eating macaque (Macaca fascicularis). The drug was administered intravenously and subcutaneously at a dose of 1 mg / kg in PBS (pH 7). Afterward, blood samples were collected, serum was processed, and immunoassay bioanalysis was performed. Drug concentrations were measured over 21 days. The serum drug concentration / time profile is shown in Table 25. These are shown together in section 26 and in Figure 12.

[0165] [Table 25]

[0166] [Table 26]

[0167] Pharmacokinetic analysis revealed that in cynomolgus monkeys after subcutaneous and intravenous administration, the levels were 8. The final half-lives of FP1, 5 and 9.2 days, were shown, and the mean bioavailability after subcutaneous administration was also shown. The percentage was approximately 88% (Table 27).

[0168] [Table 27] Note: * Tmax (Median)

[0169] Immunoaffinity capture LC-MS analysis was used to analyze the serum of cynomolgus monkeys after intravenous and subcutaneous administration. The concentration of the complete dimer present was quantified (Tables 28 and 29, and Figures 13 and 14). The concentrations measured by this method are similar to those measured by immunoassay (IA). Therefore, FP1 circulates as a complete dimer, and in cynomolgus monkeys, undetectable metabolic anemia This indicates a lack of stability.

[0170] [Table 28]

[0171] [Table 29]

[0172] The concentrations of the test substance in cynomolgus monkey serum after intravenous and subcutaneous administration were also measured by immunoaffinity capture trypsin. The HSA region within FP1 was measured by digestion LC-MS / MS analysis (Tables 30 and 31). It is located near the N-terminus of the region, near the N-terminus of GDF15, and near the C-terminus of GDF15, respectively. The trypsin-digested peptide that does this is ALV(ALVLIAFAQYLQQSPFE DHVK), ASL (ASLEDLGWADWVLSPR), and TDT (TDTGVS LQTYDDLLAK was selected. These peptides were viewed as surrogate peptides for FP1. I understand. All concentrations of the surrogate peptides were measured against each other and also by immunoassay. The concentration is comparable, the GDF15 sequence within FP1 is completely preserved, and complete HS is observed in vivo. It was shown that it is concatenated to the A sequence.

[0173] [Table 30]

[0174] [Table 31]

[0175] Human plasma stability assay The objective of this study was to analyze the ex vivo stability of FP1 in human plasma. Fresh, unfrozen human plasma was centrifuged to separate heparin from the heparin of two subjects (one male, one female). Prepared from blood with added ions. FP1 was gently mixed in this substrate with 0, 4, 24 and The samples were incubated at 37°C for 48 hours. The concentration of FP1 was measured by immunoassay. The mean difference (%) from the starting concentration (0 hours) was in the range of -4.1 to -12.9. No increase over time was observed, and FP1 remained stable in ex vivo human plasma for up to 48 hours. This was shown (Table 32 and Figure 15).

[0176] [Table 32]

[0177] Using immunoaffinity capture LCMS, complete ionization of the The total dimer concentration was quantified. The concentrations measured by this method were analyzed over time (0, 4, 24, and It is stable for 48 hours, and FP1 remains stable in ex vivo human plasma for up to 48 hours. It was shown that the molecules are maintained in a mer (Table 33 and Figure 16).

[0178] [Table 33]

[0179] Example 13: IR method To detect anti-drug antibodies (ADAs) in animal and clinical samples, an immune response (IR) assay is used. I is developed. The IR assay assesses the state of ADA in terms of pharmacokinetics / toxicokinetics (PK / TK). This identifies ADA-positive samples for comparison with the results, and involves exposure to FP1 and drug activity. This enables the evaluation of the state. Clinical IR assays screen serum samples, Used to confirm the specificity of ADA-positive samples and to measure the ADA titer of confirmed positive samples. The development of a neutralizing antibody (NAb) assay is being used in Phase 1 of the program for ADA-positive subjects. This is done for use with confirmed positive samples from the individuals. Furthermore, it is used in Phase 2 of the program. Therefore, the cross-reactivity of ADA to endogenous GDF15 is determined. Prior to the In-Human (FIH) trial, immunogenicity risk was assessed, and further immunogenicity risk assessment was conducted. If an assessment of the characteristics of the epidemic response is deemed appropriate, it may be carried out.

[0180] Example 14: Toxicity Plan Since the endogenous target receptor for GDF15 has not been identified, in vitro results in FP1 There is no data on combination or functionality. However, data on rats, mice, and cynomolgus monkeys are available. Drug and efficacy studies of single and multiple doses in these species have shown that FP1 in these species Its activity has been demonstrated to reduce food intake, decrease body weight, and regulate oral glucose tolerance. It has been shown that, based on the results of efficacy tests, the receptors in rats and monkeys in these species The endogenous effect of FP1 on (in humans) has not been fully characterized. With this understanding, they will likely be designated as rodent and non-rodent toxicology species, respectively.

[0181] In Examples 15-19, the amino acid sequence of SEQ ID NO: 92 (codon optimized 1 The actual nucleotide sequence has ) and 110 (codon-optimized 2) encoded by the nucleotide sequence. We will characterize another exemplary fusion protein of the present invention as described in Example 5. The molecule consists of 42 amino acid linkers made up of glycine and serine residues, GS-(GGGGS )8 via mature human GDF15 deletion mutants of HSA(C34S) (201-308 It is a completely recombinant protein that exists as a homodimer of a fusion with (Sequence No. 8). One naturally occurring free cysteine ​​at position 34 of the HSA has been mutated into serine. For simplicity, the A-GDF15 fusion protein will be referred to as "FP2" in the following examples. It will be used as a weapon.

[0182] Example 15: Effect of FP2 on food intake in C57Bl / 6 mice FP2 was developed to reduce food intake in male C57Bl / 6 mice after a single administration. We evaluated the following: Taconic Biosciences (Hudson, New York) Male C57Bl / 6N mice (10-12 weeks old) obtained from ) were used in the experiment. The animals were kept individually in temperature-controlled rooms with a 12-hour light-dark cycle (6 AM / 6 PM). They were allowed to freely access water and food. Male C57Bl / 6 mice were kept in BioDAQ cages. The mice were allowed to acclimate to the environment for at least 72 hours. After that, the mice were divided into groups of 8 based on their food intake. The animals were divided into 6 groups during the last 24 hours. Between 4:00 PM and 5:00 PM, the animals' weight was measured. The amount of food intake was measured and administered by subcutaneous injection. Changes were continuously recorded using the BioDAQ system for 48 hours after compound administration. In this experiment, 6xHis-FP1 was used for comparison.

[0183] FP2 was found to be effective at 12, 24, and 48 hours after administration at all dose levels tested. It showed a significant effect in reducing the amount of food intake (Table 34). In mice, at all time points and all A decrease in the percentage change in food intake compared to PBS was observed at the dose level (Table 3). 5).

[0184] [Table 34] The data is expressed as mean ± SEM. each, * p ≤ 0.05 for PBS ** p ≤ 0.01 for PBS *** p ≤ 0.001 for PBS **** p ≤ 0.0001 for PBS The statistical analysis methods used were ANOVA and Dunnett's multiple comparison test. Excluding the group administered 6xHis-FP1 at 8 nmol / kg (n=6), n=8 / group.

[0185] [Table 35]

[0186] The appetite-suppressing effect of FP2 compared to the relative amount of food intake of each PBS control. It is expressed as a percentage decrease.

[0187] The data is expressed as mean ± SEM. each, * p ≤ 0.05 for PBS ** p ≤ 0.01 for PBS *** p ≤ 0.001 for PBS **** p ≤ 0.0001 for PBS The statistical analysis methods used were ANOVA and Dunnett's multiple comparison test. Excluding the group receiving 6xHis-FP1 at 8 nmol / kg (n=6), the total number of participants was 8 / group.

[0188] Example 16: Effect of FP2 on food intake in Sprague-Dolly rats Food intake and weight increase in male Sprague Dolly rats after a single administration of FP2. The ability to reduce was evaluated. Animals weighing 200-225g were given Charl Obtained and delivered from es River Labs (Wilmington, Massachusetts). Used within one week. The animals were kept in a temperature-controlled room with a 12-hour light-dark cycle, using Alp Each animal was placed in a cage containing dry bedding and a concentrated plastic tube. The animals are allowed to drink water freely, and are fed Irradiated Certi, which is food for experimental rodents. fied PicoLab® Rodent Diet 20, 5K75 * (A Through SAP (Quakertown, Pennsylvania), Purina Mills ( The rats were given (supplied from St. Louis, Missouri). Prior to administration, each rat was given The animals' weights were measured and recorded.

[0189] The animals were allowed to acclimate to the environment in BioDAQ cages for a minimum of 72 hours. After that, rats were fed. Based on their intake, the animals were divided into six groups of eight each during the last 24 hours. 4:00 PM Between 0:00 and 5:00, the animals' body weight was measured, and the solvent or compound was administered by subcutaneous injection. For each cage, changes in food intake were measured using the BioDAQ system after compound administration. The data was continuously recorded for 48 hours. In this experiment, 6XHis-FP1 was used for comparison. Ta.

[0190] The dose-dependent decrease in food intake after a single dose of FP2 was examined. 0.3 nmo At a dose of l / kg, no significant difference in food intake was observed. At 1 nmol / kg, A significant effect of reducing food intake was observed after 12 hours, but not after 24 or 48 hours. It was not observed. At dose levels of 3 and 10 nmol / kg, there was a significant decrease in food intake. This was observed at all time points and at all dose levels (Table 36, Figure 19). A decrease in the percentage change in food intake was observed compared to PBS (Table 37).

[0191] [Table 36] The data is expressed as mean ± SEM. each, * p ≤ 0.05 for PBS ** p ≤ 0.01 for PBS ****p ≤ 0.001 for PBS The statistical analysis methods used were ANOVA and Dunnett's multiple comparison test. n=8 / group

[0192] [Table 37] The appetite-suppressing effect of FP2 compared to the relative amount of food intake of each PBS control. It is expressed as a percentage decrease. The data is expressed as mean ± SEM. each, * p ≤ 0.05 for PBS ** p ≤ 0.01 for PBS *** p ≤ 0.001 for PBS The statistical analysis methods used were ANOVA and Dunnett's multiple comparison test. n=8 / group

[0193] Example 17: Food intake, body weight, and The effect of FP2 on glucose homeostasis FP2 was repeatedly administered to male DIO C57Bl / 6 mice over an 8-day period, and food intake was also monitored. We evaluated its ability to reduce intake and body weight, and to improve glucose homeostasis. Male obtained from Taconic Biosciences (Hudson, New York) In the experiment, sex DIO C57Bl / 6 mice (21 weeks old, fed a high-fat diet for 15 weeks) were used. The mice were used in a temperature-controlled room with a 12-hour light-dark cycle (6 AM / 6 PM). They were housed individually, given free access to water, and given research food D12492 (Research D iets (New Brunswick, New Jersey) administered the substance to the mice for one week. The mice were allowed to acclimate to the environment in the upper mouse rearing room before being used in the experiment. The evaluation items for the experiment were food intake and body weight. Weight, body composition, and blood glucose levels (OGTT, blood glucose) were measured. Movement was performed one day prior to administration. The weight of the objects was measured and they were grouped according to their body weight (BW). They were administered to mice by subcutaneous injection. The animals administered FP2 received this compound on days 0, 3, 6, 9, and It was administered on day 12. The solvent group and the rosiglitazone group received sterile PBS on these days. It was administered as follows. Rosiglitazone was given freely in the diet at a rate of 0.015% by weight. Weight and food intake were recorded daily for 15 days. Blood glucose levels were recorded on days 0, 7, and 13. Measurements were taken. An oral glucose tolerance test (OGTT) was performed on day 14. During the OGTT, Insulin levels were measured at the selected time. On day 15, the mice were euthanized with CO2 and exposed to The final blood sample for dew was collected by cardiac puncture. A total of 15 mice were collected, with 3 mice from each treatment group. We performed separate PK arm operations at the mortar.

[0194] Exposure-response (ER) analysis of FP2 in DIO mice Most animals in the pharmacodynamics (PD) (efficacy) arm are likely immunogenic, The drug concentration was undetectable on the last experimental day when the PK sample was obtained. Therefore, Instead of using individual PKs from the PD arm, use the average PK profile from the PK arm. Then, the percentage change in body weight from baseline in the PD arm at the corresponding dose level. Exposure-response tests were conducted (on days 3, 6, and 9, respectively). In this method, the PK arm was used to test for the drug. It is assumed that the drug exposure will exhibit similar behavior to that of the PD arm.

[0195] E max Model (GraphPad Prism 6, logarithm of agonist for reaction) The exposure was correlated with the response data using the value (logarithmic values ​​were converted to drug concentrations). l Slope was set to 1. EC 50 EC for the value 10 The model to which this was applied is E max The estimated values ​​were different (E max = -4.26%, -8.18%, and -9. Despite 85%, the increase was less than double on days 3, 6, and 9. On day 9, some animals However, the data from day 9 still showed the disappearance of drug exposure due to the formation of potential ADAs. ER parameters based on this require careful interpretation.

[0196] Two weeks of F2-week The effects of P2 exposure were evaluated in diet-induced obese male C57Bl / 6 mice. 0.3nm FP2 of 1.7-3.3 nM in the ol / kg treatment group, and 7.1 in the 1.0 nmol / kg treatment group. ~14nM, 20.8~41.6nM in the 3.0nmol / kg treatment group, and 10nmol The trough exposure values ​​of FP2 from 28.5 to 112.9 in the / kg treatment group were measured using the experimental PK arm. It was maintained until day 9 (n=2 or 3, Table 49). Even though q3d administration was continued from day 9 onwards... Nevertheless, a decrease in circulating values ​​was observed in most animals (Table 49). This accelerated clearance Consistent with the experiment, the majority of animals in the PD arm showed undetectable circulating FP2 levels on day 15. They possessed (Table 50).

[0197] Treatment of DIO mice with q3d FP2 compared to solvent treatment (Table 43 and Figure 23) This reduces food intake (Table 38), body weight (Tables 39, 40, and Figure 20), and dietary blood glucose levels. A significant decrease in food intake was observed at 0.3 nmol / kg on days 2, 5, and 8. Visually, at 1.0 nmol / kg, the effect lasted from day 1 to day 7, while at 3.0 nmol / kg, the effect lasted from day 1. On day 1, day 2, day 4-6, and day 8, at 10.0 nmol / kg, on day 1 and day 3 This was observed from day 1 to day 6, day 8, and day 9. The percentage change in body weight was 0.3 nmol / For kg, the period is from day 5 to day 13, and for 1.0 nmol / kg and 10.0 nmol / kg, it is 3 From day 1 to day 13, the 3.0 nmol / kg level was statistically significant from day 4 to day 13. Body weight The change (g) was observed from day 8 for 0.3 nmol / kg and from day 6 for 1.0 nmol / kg. From the eyes, at 3.0 nmol / kg, symptoms appear from day 7, and at 10.0 nmol / kg, from day 5. The effect was significant. The reduction in dietary blood glucose levels lasted 7 days in animals at a dose level of 3.0 nmol / kg. The effect was significant visually, and in animals at dose levels of 3.0 and 10.0 nmol / kg, on day 13... It was significant.

[0198] DIO mice treated with q3d FP2 showed improved solvent performance in oral glucose challenge. Compared to the science group, improved glucose tolerance was observed on day 14 (Table 41, Figures 21A and 12B). The glucose levels were measured at 30 minutes in the 0.3 nmol / kg group and at 60 minutes in the 1.0 nmol / kg group. In 120 minutes, the 3.0 nmol / kg group took 120 minutes, while the 10.0 nmol / kg group took 3 The levels were significantly lower at 0, 90, and 120 minutes. The area under the curve was significant in all dose groups. The insulin levels during the glucose challenge were 0.3 and 10.0 nmol / kJ. The g group showed significantly lower levels at 30 minutes (Table 42, Figures 22A and 22B). Furthermore, solvent-treated animals and In comparison, DIO Mau after 14 days of q3d treatment with FP2 at 10.0 nmol / kg A significant decrease in the fasting HOMA-IR calculation was observed, indicating improved insulin sensitivity. (Table 44 and Figure 24).

[0199] Body composition was measured by MRI on day 1 and day 13 prior to the start of the experiment (Tables 47 and 4). 8) DIO-Mauve treated with 1.0 nmol / kg and 10.0 nmol / kg FP2 While S showed a significant decrease in body fat mass on day 13, lean body mass decreased in all treatment groups. No changes were observed. On the 13th day, the 10.0 nmol / kg treatment group and the solvent treatment group were different. Compared to the previous period, a significant increase in lean body mass (%) and a significant decrease in body fat percentage (%) were observed. -1 day The changes in lean body mass from day 1 to day 13 were 0.3 nmol / kg and 1.0 nmol / kg. The results were significant in the 10.0 nmol / kg treatment group, with lean body mass (%) being 1.0 and 3. Significant changes were observed in the 0 and 10.0 nmol / kg treatment groups. The changes from day 1 to day 13 were as follows: Compared to the solvent, all treatment groups showed significant improvements in body fat mass and lean body mass (%).

[0200] Endogenous mouse GD was obtained between solvent-treated animals and mice treated with q3d FP2 for 15 days. No significant difference was observed in F15 serum levels (Table 46).

[0201] Conclusion: These results suggest that higher drug exposure generally occurs in the dose groups experimented on days 3, 6, and 9. In all cases, a larger percentage change in weight from baseline at the population level is observed. This indicates that.

[0202] Two weeks of exposure to FP2 resulted in decreased food intake, weight loss, and elevated blood glucose levels in DIO mice. This led to a decrease in q3d, and improvements in glucose tolerance and insulin sensitivity. q3d values ​​of 1.0, 3.0, and A significant reduction in food intake was observed over multiple days at a concentration of 10.0 nmol / kg. Weight significantly decreased from 3 to 5 days after the start of the experiment. On day 13, the blood glucose level after eating was 3.0. A significant decrease was observed after q3d administration of 10.0 nmol / kg FP2. (Fasting-induced HOMA-IR) Insulin sensitivity, as indicated by a significant decrease in q FP2 at 10.0 nmol / kg, is indicated by q Obtained 14 days after 3d administration. On day 13, q3d with 10.0 nmol / kg FP2. In treated DIO mice, there was a significant increase in lean body mass (%) and body fat percentage (%). A significant decrease was observed.

[0203] [Table 38] Each value is based on data from 8 animals, except for the case where n=7 (indicated by ^). This represents the average ± SEM value per unit time per group. * p<0.05 relative to the solvent The statistical analysis methods used were two-way ANOVA RM and Tukey's multiple comparison test. .

[0204] [Table 39] Each value represents the mean ± SEM per unit time and per group for data from 8 animals. . * p<0.05 relative to the solvent The statistical analysis methods used were two-way ANOVA RM and Tukey's multiple comparison test. .

[0205] [Table 40] Each value represents the mean ± SEM per unit time and per group for data from 8 animals. . * p<0.05 relative to the solvent The statistical analysis methods used were two-way ANOVA RM and Tukey's multiple comparison test. .

[0206] [Table 41] Each value represents the mean ± SEM per unit time and per group for data from 8 animals. . * p<0.05 relative to the solvent The statistical analysis methods used for blood glucose levels were two-way ANOVA RM and Tukey's method. Multiple comparison tests, For AUC, we used one-way ANOVA and Tukey's multiple comparison test.

[0207] [Table 42] Each value represents the mean ± SEM per unit time and per group for data from 8 animals. . * p<0.05 relative to the solvent The statistical analysis methods used for insulin levels were two-way ANOVA RM and Twitch. Key multiple comparison tests, For AUC, we used one-way ANOVA and Tukey's multiple comparison test.

[0208] [Table 43] Each value represents the mean ± SEM per unit time and per group for data from 8 animals. . * p<0.05 relative to the solvent The statistical analysis methods used were two-way ANOVA RM and Tukey's multiple comparison test. .

[0209] [Table 44] Each value represents the mean ± SEM per unit time and per group for data from 8 animals. . * p<0.05 relative to the solvent The statistical analysis methods used were one-way ANOVA and Tukey's multiple comparison test.

[0210] [Table 45] Each value represents the mean ± SEM per unit time and per group for data from 8 animals. . * p<0.05 relative to the solvent The statistical analysis methods used were one-way ANOVA and Tukey's multiple comparison test.

[0211] [Table 46] Each value represents the mean ± SEM per unit time and per group for data from 8 animals. . * p<0.05 relative to the solvent The statistical analysis methods used were one-way ANOVA and Tukey's multiple comparison test.

[0212] [Table 47] Each value represents the mean ± SEM per unit time and per group for data from 8 animals. . * p<0.05 relative to the solvent The statistical analysis methods used were one-way ANOVA and Tukey's multiple comparison test.

[0213] [Table 48] Each value represents the mean ± SEM per hour and per group for data from 8 animals . * p < 0.05 vs. vehicle The statistical analysis methods used are one-way ANOVA and Tukey's multiple comparison test.

[0214]

Table 49

[0215]

Table 50

[0216] Example 18: Pharmacokinetics and Immune Response of FP2 in Multiple Species Pharmacokinetics in Mice The pharmacokinetic properties of FP2 after subcutaneous administration to female C57Bl / 6 mice were evaluated. F P2 was administered to female C57Bl / 6 mice (Sage Laboratories, Missouri St. Louis) in PBS (pH 7.3 to 7.5) at a dose level of 2.0 mg / kg, via subcutaneous administration (n = 5 samples per time point) and intravenous administration (n = 5 samples per time point). Collection of the sample at the final time point was performed by terminal exsanguination. Blood samples were collected, serum was processed, and drug concentration was measured up to 168 hours. The concentration of FP2 was measured by immunoassay . Plasma drug concentration profiles are summarized in Tables 51 and 52, and shown in Figure 25.

[0217] Pharmacokinetic analysis of FP2 in C57Bl / 6 mice showed that after intravenous and subcutaneous administration... The final half-lives were approximately 1.51 days and 1.76 days, respectively, and the average bio-life after subcutaneous administration was approximately 1.51 days and 1.76 days, respectively. The availability was shown to be approximately 61%.

[0218] [Table 51]

[0219] [Table 52]

[0220] [Table 53]

[0221] Pharmacokinetics in rats FP2 is Sprague Dolly Rat (Sage Laboratories, Ms. In St. Louis, California, a dose level of 2.0 mg / kg in PBS (pH 7.3-7.5) was observed. The samples were administered subcutaneously (n=5 samples per time point) and intravenously (n=5 samples per time point). The final sample was collected by the final bloodletting. Blood samples were collected and serum was treated. The drug concentration was measured for up to 168 hours. The concentration of FP2 was measured by immunoassay. The drug concentration profiles in plasma are summarized in Tables 54 and 55, and shown in Figure 26. The pharmacokinetic parameters calculated from this data are summarized in Table 56.

[0222] Pharmacokinetic analysis of FP2 in Sprague-Dolly rats showed that intravenous and subcutaneous administration... The final half-lives after administration were approximately 1.46 days and 1.37 days, respectively, and the average after subcutaneous administration was The bioavailability was shown to be approximately 28%.

[0223] [Table 54]

[0224] [Table 55]

[0225] [Table 56]

[0226] Pharmacokinetics in monkeys FP2 was administered to three male crab-eating macaques in PBS (pH 7.0-7.6) for 1 m³ each. The drug was administered subcutaneously at g / kg and intravenously at 1 mg / kg. Blood samples were collected and the plasma was processed. Drug concentrations were measured until day 21.

[0227] The pharmacokinetics (PK) of FP2 were determined by a single intravenous (IV) dose (1.0 mg / kg) administered to cynomolgus monkeys. The plasma was characterized after administration of ) and subcutaneously (SC) (1.0 mg / kg). The drug concentration-time profiles were obtained from immunoassay and LCMS analysis, as shown in Table 57. The drug concentration-time profile in plasma after IV administration is shown in 58, respectively, and immunoassay The results of the CE and LCMS analyses are shown in Tables 59 and 60, respectively. Immunoassay data The results are shown graphically in Figure 27, and the LCMS data is shown in Figure 28.

[0228] Using the results of immunoassay analysis, the end half-life of FP2 (t) based on the mean NCA 1 / 2) The time intervals after IV and SC administration were approximately 7.05 days and approximately 8.51 days, respectively. Table 61 shows the mean PK parameters after administration of V and SC. Results of immunoassay bioanalysis Using this, the final half-life of FP2 estimated with the average non-compartment model (t1 / 2 The time intervals after IV and SC administration were approximately 7.05 days and 8.51 days, respectively. The mean bioavailability (F%) is AUC in cynomolgus monkeys after SC administration. 0~最終 Based on this, it is estimated to be approximately 98.5%, AUC 0~無限 Based on this, it is estimated to be approximately 109.2%. It was done.

[0229] [Table 57] N / A = Not applicable

[0230] [Table 58] -=The initial measurement failed, and there was not enough sample to perform a repeat analysis. #=The tube was excluded from the analysis due to an error in labeling. N / A = Not applicable

[0231] [Table 59] N / A = Not applicable

[0232] [Table 60] -=The initial measurement failed, and there was not enough sample to perform a repeat analysis. N / A = Not applicable #=The tube was excluded from the analysis due to an error in labeling.

[0233] [Table 61] The PK parameter is the mean value based on NCA of the PK data from the immunoassay. * Tmax (Median)

[0234] Human plasma stability assay The ex vivo stability of FP2 was investigated in fresh heparinized plasma at 37°C for up to 48 hours. Fresh, unfrozen human plasma was centrifuged into two subjects (one male, one female). Prepared from valine-enriched blood. FP2 was gently mixed in this substrate at concentrations of 0, 4, and 24. The samples were incubated at 37°C for 48 hours. The concentration of FP2 was determined by an immunoassay. The substrate was measured under assay conditions by performing LCMS after independent immunoaffinity capture. The concentration of the complete dimer present was quantified.

[0235] In immunoassay methods, the recovery rate from the starting concentration ranged from 104.8 to 94.1. No decrease over time was observed, and FP2 remained stable in ex vivo human plasma for up to 48 hours. This was shown (Figure 29 and Table 62). In the LCMS method, the concentration was stable over time, and J NJ-64739090 was maintained as a complete dimer in ex vivo human plasma for up to 48 hours. This was shown to be the case (Figure 30 and Table 63).

[0236] [Table 62]

[0237] [Table 63]

[0238] Example 19: Effects of FP1 and FP2 in cynomolgus monkeys FP1 and FP1 in relation to food intake and body weight after a single dose in naive cynomolgus monkeys The impact of point 2 was evaluated.

[0239] FP1 was administered to a cohort of naive cynomolgus monkeys at three doses: 1, 3, and 10 nmol / kg. The drug was administered subcutaneously at the specified dose level. A solvent-treated group was also included. Animals were blinded during the treatment. The experiment consisted of two parts. Weekly baseline food intake measurement and data collection, 4 weeks after single dose of compound. Data collection was performed over a total of 6 weeks. Plasma drug exposure was measured on days 1, 7, 14, 21, and 28 of administration. It was measured in the eye.

[0240] Treatment of cynomolgus monkeys with a single dose of FP1 showed improved food intake and overall health compared to solvent treatment. Weight was reduced (Figures 32-33). A significant decrease in daily food intake was 10 nmol / kg. This was observed at dose levels on days 4, 5, 6, and 8-12 (Figure 32). Daily food intake The weekly mean values ​​significantly decreased during the second week after administration at a dose level of 10 nmol / kg. 3 At nmol / kg dose levels, the average weekly food intake was obtained before the second week of administration. It showed a significant decrease rate (%), and at a dose level of 10 nmol / kg, the levels at 1 and 2 weeks after administration were A significant percentage reduction (%) was observed from the average weekly food intake before administration. Weight change from day 0. A significant decrease in the rate (%) was observed at a dose level of 3 nmol / kg on day 28, reaching 10 nmol / At the kg dose level, this was observed on days 14, 21, and 28 (Figure 33).

[0241] FP2 was administered to a cohort of naive cynomolgus monkeys at three doses: 1, 3, and 10 nmol / kg. The drug was administered subcutaneously at the specified dose level. A solvent-treated group was also included. Animals were blinded during the treatment. The experiment consisted of 5 Weekly baseline food intake measurement and data collection, 1 week of processing, and 5 weeks of drug-free interval. Data collection was performed over a total of 11 weeks. Plasma drug exposure was measured at doses 1, 7, 14, 21, and 28. Measurements were taken on days 35 and 42.

[0242] Treatment of cynomolgus monkeys with a single dose of FP2 showed improved food intake and overall health compared to solvent treatment. Weight was reduced (Figures 34-35). A significant decrease in daily food intake was due to a reduction of 3 nmol / kg. At dose levels 3, 5-8, 10 and 12, at dose levels 10 nmol / kg, 3- This was observed on days 38 and 40 (Figure 34). The weekly average daily food intake was 3nmo. At a dose level of l / kg, the levels decreased significantly in the first week after administration, and at a dose level of 10 nmol / kg... It decreased significantly between weeks 1 and 6. At a dose level of 3 nmol / kg, at 2 weeks after administration, A significant percentage decrease (%) was observed in the average weekly daily food intake compared to the week prior to administration, with 10 nmol. At dose levels of / kg, at weeks 1 and 6 after administration, the average weekly daily food intake was compared to the pre-administration amount. A significant decrease (%) was observed from week 1. A significant decrease (%) in the percentage change in weight from day 0 was observed. At a dose level of 1 nmol / kg, the effect occurred on days 21-42, and at a dose level of 3 nmol / kg. This was observed from day 14 to day 42, and from day 7 to day 42 at a dose level of 10 nmol / kg (Figure). 34).

[0243] Example 20: HSA-GDF15: GDF15 heterodimer The biological activity of the HSA-GDF15:GDF15 heterodimer was investigated.

[0244] To produce HSA-GDF15:GDF15 heterodimers, two types of constructions are used. The first construct is mature GDF via a glycine / serine linker. This was defined as containing HSA fused to the N-terminus (AA203~308) of 15 molecules (SEQ ID NO: 9). 3) The second construct is N of mature GDF15 via a glycine / serine linker. 6×histidine-tagged HSA and HRV3C fused at the terminals (AA197~308) The plasmids included a rotease cleavage site (SEQ ID NO: 94). These plasmids were manufactured by the manufacturer. According to the protocol, the Expi293(trademark) expression system (Thermo Fishe Simultaneous transfection was performed in a 1:1 ratio using (r Scientific). Tides are heterodimers and homodimers in which monomers are linked by disulfide bonds. It was secreted as the HSA-GDF15 protein, which contains both forms of the body.

[0245] The cell supernatant from transiently transfected Expi293(trademark) cells was transfected. The sample was collected 5 days after septic culture, centrifuged for clarification, and then sterile filtered (0.2 μm PES membrane). Corning (the company) performed the procedure. The clarified supernatant was treated with 20 mM sodium phosphate and 500 mM N. Histrap HP column (GE Healthca) equilibrated with aCl, pH 7.4 Loaded into the column (re-company). After loading, unbound proteins were balanced in equilibration buffer. They were removed by washing. Both heterodimers and homodimers bound to the column. HSA-GDF15 protein containing the form of 20 mM sodium phosphate, 150 mM imitation Dazole was eluted at pH 7.4. Each fraction of the eluent was pooled, and 6× histidine-tagged H2O was extracted. Incubate overnight at 4°C in the presence of RV3C enzyme (Janssen) to obtain HSA-G A DF15:GDF15 heterodimer was generated. After incubation, the protein dissolved... After removing imidazole by dialyzing the solution into equilibration buffer, HisTrap HP color I tested it again. HSA-GDF15:GDF15 heterodimer is 20 mM sodium phosphate Histidine was eluted during the washing process with 50 mM imidazole at pH 7.4, but The glycated protein was retained. The heterodimer was equilibrated in 1×DPBS, pH 7.2. HiLoad26 / 60 Superdex 200 pg column (GE Health Further purification by size exclusion chromatography (SEC) using hcare (a company). HSA-GDF15: GDF15 heterodimer with high purity (measured by SDS-PAGE) The fraction of the eluent from SEC containing (determined) was pooled and filtered. The protein concentration was measured by Bio Absorbance was measured at 280 nm using a Tek SynergyHTTM spectrophotometer. The sample was purified. The quality of the protein was assessed using SDS-PAGE and size exclusion HPLC (Ultimate). Endotoxin levels were evaluated using the ate3000 HPLC system. (Pyrotell(registered trademark)-T, Associates of Cape Cod Measurements were taken using (company name). The purified protein was stored at 4°C.

[0246] SK-N-AS cells (ATCC) that stably express the GDF15 receptor (GFRAL) The cells were then seeded into growth medium (10% FBS) in a 96-well plate 24 hours before the assay. After 24 hours, the culture medium was replaced with 200 μl of DMEM medium supplemented with 1% HI horse serum. Furthermore, the cells are incubated in a 37°C incubator for 3 hours to induce a starvation state. Next, the medium supplemented with 1% HI horse serum was replaced with 200 μl of AB1, and the temperature was set to 37°C. The sample was incubated for another 2 hours in the incubator. To perform the assay, AB1 was used. Aspirate from all wells, add 100 μl of different concentrations of the test compound to AB2, and play. The sample was incubated in a 37°C incubator for 15 minutes. After 15 minutes, the test solution was removed. Remove the contents, add 30 μl of lysis buffer (as provided in the detection kit), and place the plate in a cool, dry place. The sample was shaken on a plate shaker at warm temperature for 30 minutes. 16 μl of the dissolved sample was then used for detection. Transfer the mixture to a 384-well assay plate and add 4 μl of antibody for HTRF pAKT detection. After incubating the plates at room temperature overnight, the HTRF signal was applied to the Envision (According to Perkin Elmer.)

[0247] EC 50 Values ​​are applied to GraphPad Prism® nonlinear regression (curve fitting). The calculation was performed using [a specific method / tool]. The data was divided into three separate experiments, each giving three overlaps per data point. Expressed as mean ± standard error (SE). HSA-GDF15: GDF15 heterodimer The molecule was identified by mass spectrometry. The leftward shift of the heterodimer curve indicates HSA- GDF15:GDF15 heterodimers are related homodimers that have additional albumin. This suggests that it is more potent in inducing pAKT compared to molecules.

[0248] Example 21: Thermal stability of linker The thermal stability of different linkers connecting HSA and GDF15 was investigated. And to evaluate the properties of aggregation, HSA-GDF15 fusion tanks with different linkers were used. The protein was diluted to 10 mg / ml. After adding EDTA and methionine, the sample was heated at 40°C. The sample was incubated for 14 days as follows. Then the sample was diluted to a concentration of 1 mg / ml and size was measured. Evaluation was performed using exclusion high-performance liquid chromatography (SE-HPLC). Complete protein sequence The percentage of aggregates and fragments (%) was quantified for these proteins. Table 64 The HSA-GDF15 protein, which has a linker consisting of AP repeats, is affected by heat stress. This demonstrates that it is most stable against fragmentation under certain conditions.

[0249] We will evaluate whether these linkers affect the interaction between GDF15 and its receptor. Therefore, GFRAL-FC fusion proteins are coated onto plates to create anti-GDF15 or anti-HS proteins. Immunoassays that use A for detection are performed using GDF15 (Janssen) and HSA (K erafast, Inc. (Boston, Massachusetts) is a monoclonal antimicrobial agent. This was performed using human bodies. This assay showed that all of these linkers shown in Table 66 It was also shown that it has similar binding affinity to the receptor.

[0250] [Table 64]

[0251] The present invention has been described in detail above, with reference to specific embodiments thereof, but those skilled in the art will understand. The present invention may be modified and altered in various ways without departing from the spirit and scope of the invention. This should be obvious. The present invention includes the following embodiments. [1] a. Half-life extension proteins, b. Linker and, c. Contains GDF15 protein, A fusion protein with (a)-(b)-(c) sequences arranged from the N-terminus to the C-terminus. [2] The GDF15 protein is a mature GDF15 protein or a functional variant thereof. , the fusion protein described in [1] above. [3] The GDF15 protein is associated with at least 9 SEQ ID NOs. 6, 7, 8, 9, 10, or 11. The fusion protein described in [1] above, comprising an amino acid sequence having 0% identity. [4] The GDF15 protein is selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, 10, and 11. The fusion protein described in [3] above, comprising the selected amino acid sequence. [5] The aforementioned half-life-extending protein is human serum albumin (HSA) or a functional variant thereof. A fusion protein described in any of the above [1] to [4]. [6] The aforementioned half-life extension protein has at least 90% identity with SEQ ID NO: 1. A fusion protein as described in [5] above, comprising an acid sequence. [7] The aforementioned half-life extension protein is an amino acid selected from the group consisting of SEQ ID NOs: 1, 2, and 3. A fusion protein as described in [6] above, comprising an acid sequence. [8] The fusion according to any one of [1] to [7] above, wherein the linker is a flexible linker. protein. [9] The linker includes the sequence (GGGGS)n (where n is 2 to 20), [8] The fusion protein described above.

[10] The fusion described in any of [1] to [7] above, wherein the linker is a structured linker. protein.

[11] The linker is in the sequence (AP)n or (EAAAK)n (where n is between 2 and 20). The fusion protein described above

[10] , including )

[12] The linker is (GGGGA)n, (PGGGS)n, (AGGGS)n, or GG From S-(EGKSSGSGSESKST)n-GGS (where n is between 2 and 20) A fusion according to any of the above [1] to [7], comprising an amino acid sequence selected from the group. protein.

[13] Sequence numbers 5, 25-30, 36-37, 40, 48, 55-56, 59-60, 64- 75, 92, 113, 115, 117, 119, 121, 123, 125, or 127 A fusion protein containing an amino acid sequence with at least 90% sequence identity.

[14] Sequence IDs 5, 25-30, 40, 55-56, 59-60, 70, 92, 113, 11 Selected from the group consisting of 5, 117, 119, 121, 123, 125, and 127. A fusion protein as described above

[13] , comprising a mino acid sequence.

[15] A nucleotide compound encoding a fusion protein as described in any of the above [1] to

[14] Isolated nucleic acid molecules containing columns.

[16] Sequence numbers 76-91, 95, 109, 110, 114, 116, 118, 120, 12 The isolated nucleic acid molecule described in

[0015] above, having at least 90% sequence identity with 2, 124, 126, and 128.

[17] Sequence numbers 76-91, 95, 109, 110, 114, 116, 118, 120, 12 The above includes a nucleotide sequence selected from the group consisting of 2, 124, 126, and 128. The isolated nucleic acid molecule described in

[16] .

[18] Includes a nucleic acid molecule encoding a fusion protein as described in any of the above [1] to

[14] vector.

[19] Sequence numbers 76-91, 95, 109, 110, 114, 116, 118, 120, 12 The above includes a nucleotide sequence selected from the group consisting of 2, 124, 126, and 128. The vector described in

[18] .

[20] Includes a nucleic acid molecule encoding a fusion protein as described in any of the above [1] to

[14] host cell. [twenty one] A method for producing a fusion protein as described in any of the above [1] to

[14] , (1) Contains nucleic acid molecules encoding the fusion protein under conditions in which the fusion protein is generated. Culturing host cells, (2) Recovering the fusion protein produced by the host cell, method. [twenty two] A fusion protein described in any of the above [1] to

[14] and a pharmaceutically acceptable carrier A pharmaceutical composition containing the following. [twenty three] A nucleic acid molecule encoding a fusion protein as described in any of the above [1] to

[14] , and a drug A pharmaceutical composition comprising a scientifically acceptable carrier. [twenty four] A method for treating or preventing metabolic diseases, wherein the subject requiring treatment or prevention of the disease, A method comprising administering an effective amount of the pharmaceutical composition described in

[22] or

[23] above. [twenty five] In patients requiring treatment for a disease, type 2 diabetes, elevated blood glucose levels, elevated insulin levels, Obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, and rheumatoid arthritis A method for treating diseases selected from a group consisting of metabolic diseases selected from a group consisting of ci The subject includes a fusion protein containing the amino acid sequence of SEQ ID NO: 60, and a pharmaceutically acceptable substance. A method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a carrier to be contained.

[26] In patients requiring treatment for a disease, type 2 diabetes, elevated blood glucose levels, elevated insulin levels, Obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, and rheumatoid arthritis A method for treating diseases selected from a group consisting of metabolic diseases selected from a group consisting of ci The subject includes a fusion protein containing the amino acid sequence of SEQ ID NO: 92, and a pharmaceutically acceptable substance. A method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a carrier to be contained.

[27] A dimer containing two polypeptide chains, where each chain has a HS from the N-terminus to the C-terminus. The A region includes a linker and a GDF15 region, and the HSA region includes sequence numbers 1, 2, or It contains an amino acid sequence having at least 90% identity with 3, and the GDF15 region is Amino acids having at least 90% identity with SEQ ID NOs: 6, 7, 8, 9, 10, or 11 A dimer containing an array.

[28] The HSA region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3. Including the dimer described in

[27] above.

[29] The GDF15 region is selected from the group consisting of sequence numbers 6, 7, 8, 9, 10, and 11. The dimer described in

[27] above, comprising the amino acid sequence.

[30] The dimer according to

[0027] above, wherein the two polypeptide chains are linked by a disulfide bond.

[31] The HSA region and the GDF15 region are linked by a polypeptide linker. The dimer described in

[27] above.

[32] The HSA region and the GDF15 region are connected by a flexible linker. The dimer described in

[31] .

[33] The polypeptide linker is sequence (GGGGS)n (where n is 2 to 20) The dimer described above

[32] , including the above.

[34] The HSA region and the GDF15 region are connected by a structured linker. The dimer described in

[31] .

[35] The polypeptide linker is sequence (AP)n or (EAAAK)n (where n is 2 The dimer described above

[34] , which includes (~20).

[36] The linker is (GGGGA)n, (PGGGS)n, (AGGGS)n, or GG From S-(EGKSSGSGSESKST)n-GGS (where n is between 2 and 20) The dimer described in

[31] above, comprising an amino acid sequence selected from the group.

[37] (i) Includes the first HSA region at the N-terminus, the linker, and the first GDF15 region at the C-terminus. (ii) a first polypeptide chain and a second polypeptide chain containing a second GDF15 region A dimer comprising and, wherein the HSA region comprises at least 9 with SEQ ID NOs: 1, 2, or 3. It contains an amino acid sequence having 0% identity, and the first GDF15 region is SEQ ID NO: 6, It contains an amino acid sequence having at least 90% identity with 7, 8, 9, 10, or 11, The second GDF15 region is at least 9 of sequence numbers 6, 7, 8, 9, 10, or 11. A dimer containing an amino acid sequence with 0% identity.

[38] The HSA region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3. Including the dimer described in

[37] above.

[39] The GDF15 region is selected from the group consisting of sequence numbers 6, 7, 8, 9, 10, and 11. The dimer described in

[37] above, comprising the amino acid sequence.

[40] The aforementioned linker is (GGGGS)n, (AP)n, (EAAAK)n, (GGGGA) n, (PGGGS)n, (AGGGS)n, and GGS-(EGKSSGSGSESKST An amino acid sequence selected from the group consisting of n-GGS (where n is 2 to 20) Including the dimer described in

[37] above.

[41] The dimer according to

[0037] above, wherein the two polypeptide chains are linked by a disulfide bond.

[42] A poly(P) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11. Lipeptide.

[43] Isolated nucleic acid molecules containing nucleotide sequences selected from the group consisting of SEQ ID NOs: 96-108 .

[44] This includes nucleic acid molecules containing nucleotide sequences selected from the group consisting of SEQ ID NOs: 96-108. Vector.

[45] This includes nucleic acid molecules containing nucleotide sequences selected from the group consisting of SEQ ID NOs: 96-108. host cells. [A1] a. The amino acid sequence of Sequence ID No. 2, b. Amino acid sequence (GGGGS) n (where n is 7 or 8) (Sequence ID 42 or It is a linker having a length of 35-48 amino acids, including 43) c. A shortened GDF15 consisting of the amino acid sequence of SEQ ID NO: 8, They are arranged in the order (a)-(b)-(c) from the N-terminus to the C-terminus, The C-terminus of the amino acid sequence of Sequence ID No. 2 is, via the linker, the shortened G A fusion protein fused to the N-terminus of DF15. [A2] The linker then processes the sequence (GGGGS)n (where n is 8) (sequence number 43) Including the fusion protein described in [A1] above. [A3] The linker is composed of the amino acid sequence of Sequence ID No. 12, as described in [A1] above. A fusion protein. [A4] A fusion protein containing the amino acid sequence of SEQ ID NO: 92. [A5] Isolated nucleic acid containing the nucleotide sequence encoding the fusion protein described in [A1] above child. [A6] The above [A5] includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 97-99. Isolated nucleic acid molecules as described. [A7] A vector containing a nucleic acid molecule encoding the fusion protein described in [A1] above. [A8] The above [A7] includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 97-99. The vector shown. [A9] A host cell containing a nucleic acid molecule encoding the fusion protein described in [A1] above. [A10] A method for producing the fusion protein described in [A1] above, (1) Contains nucleic acid molecules encoding the fusion protein under conditions in which the fusion protein is generated. Culturing host cells, (2) Recovering the fusion protein produced by the host cell, method. [A11] A pharmaceutical composition comprising the fusion protein described in [A1] above and a pharmaceutically acceptable carrier. thing. [A12] A pharmaceutical composition comprising the fusion protein described in [A4] above and a pharmaceutically acceptable carrier. thing. [A13] A pharmaceutical composition according to [A11 or 12] above for treating or preventing metabolic diseases. [A14] Type 2 diabetes, elevated blood glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, Metabolic disorders selected from the group consisting of myocardial ischemic injury, congestive heart failure, and rheumatoid arthritis. A pharmaceutical composition for treating a disease selected from the group consisting of diseases, wherein Sequence ID No. 60 A pharmaceutical composition comprising a fusion protein containing the amino acid sequence and a pharmaceutically acceptable carrier. . [A15] Type 2 diabetes, elevated blood glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, Metabolic disorders selected from the group consisting of myocardial ischemic injury, congestive heart failure, and rheumatoid arthritis. A pharmaceutical composition for treating a disease selected from the group consisting of diseases, wherein Sequence ID No. 92 A pharmaceutical composition comprising a fusion protein containing the amino acid sequence and a pharmaceutically acceptable carrier. . [A16] Isolated nucleic acid containing the nucleotide sequence encoding the fusion protein described in [A4] above child. [A17] A vector containing a nucleic acid molecule encoding the fusion protein described in [A4] above. [A18] A host cell containing a nucleic acid molecule encoding the fusion protein described in [A4] above. [A19] A method for producing the fusion protein described in [A4] above, (1) Contains nucleic acid molecules encoding the fusion protein under conditions in which the fusion protein is generated. Culturing host cells, (2) Recovering the fusion protein produced by the host cell, method. [A20] The isolated nucleic acid molecule described in [A16] above, comprising the nucleotide sequence of SEQ ID NO: 95. [A21] The isolated nucleic acid molecule described in [A16] above, comprising the nucleotide sequence of SEQ ID NO: 110. [B1] a. Half-life extension protein containing the amino acid sequence of Sequence ID No. 2, b. Amino acid sequences (GGGGS)n, (AP)n, (GGGGA)n, (PGGGS) A linker containing n or (AGGGS)n (where n is between 4 and 20), c. An amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, 10, and 11. Contains GDF15 protein, The proteins are arranged in the order (a)-(b)-(c) from the N-terminus to the C-terminus, and the half-life extension protein The C-terminus of the amino acid sequence of the substance is linked via the linker to the N-terminus of the shortened GDF15. A fusion protein formed by fusing with it. [B2] The linker is an amino acid sequence (GGGGS) n (where n is 7 or 8 (SEQ ID NO: 4) The GDF15 protein has a length of 35 to 48 amino acids, including 2 or 43) The fusion protein described in [B1] above, wherein the quality contains the amino acid sequence of SEQ ID NO: 6 or 7. [B3] The fusion protein described in [B1] above, comprising the amino acid sequence of SEQ ID NO: 60. [B4] Sequence numbers 5, 25-29, 36-37, 40, 48, 55-56, 59, 64-75, Includes an amino acid sequence selected from 113, 115, 117, 119, 123, or 125 , fusion protein. [B5] A dimer containing two polypeptide chains, where each chain has a HS from the N-terminus to the C-terminus. Region A, amino acid sequences (GGGGS)n, (AP)n, (GGGGA)n, (PGGGS) Linkers including )n or (AGGGS)n (where n is 4 to 20), and GDF It includes 15 regions, the HSA region includes the amino acid sequence of SEQ ID NO: 2, and the GDF15 A dimer in which the region contains the amino acid sequence of SEQ ID NOs. 6, 7, 8, 9, 10, or 11. [B6] The GDF15 region contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7. The dimer described in [B5] above. [B7] The two polypeptide chains are linked by a disulfide bond, as described above [B5 The dimer described in [B6] or [B6]. [B8] A pharmaceutical composition comprising the fusion protein described in [B1] above and a pharmaceutically acceptable carrier. thing. [B9] A pharmaceutical composition as described in [B8] above for treating or preventing metabolic diseases. [B10] Type 2 diabetes, elevated blood glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, Metabolic disorders selected from the group consisting of myocardial ischemic injury, congestive heart failure, and rheumatoid arthritis. A pharmaceutical composition for treating a disease, comprising the fusion protein or above [B1] described above. A pharmaceutical composition comprising the dimer described in [B5].

[0252] The sequences referenced in this application are shown in the table below.

[0253] Table 65-1

[0254] Table 65-2

[0255] Table 65-3

[0256] Table 65-4

[0257] Table 65-5

[0258] Table 65-6

[0259] Table 65-7

[0260] Table 65-8

[0261] Table 65-9

[0262] Table 65-10

[0263] Table 65-11

[0264] Table 65-12

[0265] Table 65-13

[0266] Table 65-14

[0267] Table 65-15

[0268] Table 65-16

[0269] Table 65-17

[0270] Table 65-18

[0271] Table 65-19

[0272] Table 65-20

[0273] Table 65-21

[0274] Table 65-22

[0275] Table 65-23

[0276] Table 65-24

[0277] Table 65-25

[0278] Table 65-26

[0279] Table 65-27

[0280] Table 65-28

[0281] Table 65-29

[0282] Table 65-30

[0283] Table 65-31

[0284] Table 65-32

[0285] Table 65-33

[0286] Table 65-34

[0287] Table 65-35

[0288] Table 65-36

[0289] Table 65-37

[0290] Table 65-38

[0291] Table 65-39

[0292] Table 65-40

[0293] Table 65-41

[0294] Table 65-42

[0295] Table 65-43

[0296] Table 65-44

[0297] Table 65-45

[0298] Table 65-46

[0299] Table 65-47

[0300] Table 65-48

Claims

1. a. Half-life extension proteins, b. Linker and, c. Contains GDF15 protein, A fusion protein with (a)-(b)-(c) arranged from the N-terminus to the C-terminus.

2. The GDF15 protein is a mature GDF15 protein or a functional variant thereof. The fusion protein according to claim 1.

3. The GDF15 protein is at least 9 of the sequence numbers 6, 7, 8, 9, 10, or 11. The fusion protein according to claim 1, comprising an amino acid sequence having 0% identity.

4. The GDF15 protein is selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, 10, and 11. The fusion protein according to claim 3, comprising a selected amino acid sequence.

5. The aforementioned half-life-extending protein is human serum albumin (HSA) or a functional variant thereof. A fusion protein according to any one of claims 1 to 4.

6. The aforementioned half-life extension protein has at least 90% identity with SEQ ID NO:

1. The fusion protein according to claim 5, comprising an acid sequence.

7. The half-life extension protein is an amino acid selected from the group consisting of SEQ ID NOs: 1, 2, and 3. The fusion protein according to claim 6, comprising an acid sequence.

8. The fusion type according to any one of claims 1 to 7, wherein the linker is a flexible linker. Protein.

9. The linker includes the sequence (GGGGS)n (where n is 2 to 20), The fusion protein described in item 8.

10. The fusion type according to any one of claims 1 to 7, wherein the linker is a structured linker. Protein.

11. The linker is an array (AP)n or (EAAAK)n (where n is between 2 and 20). The fusion protein according to claim 10, comprising )

12. The linker is (GGGGGA)n, (PGGGGS)n, (AGGGGS)n, or GG S - (EGKSGSGSESKST)n - GGS (where n is between 2 and 20) A fusion fusion according to any one of claims 1 to 7, comprising an amino acid sequence selected from the group. Protein.

13. Sequence IDs 5, 25-30, 36-37, 40, 48, 55-56, 59-60, 64- 75, 92, 113, 115, 117, 119, 121, 123, 125, or 127 A fusion protein containing an amino acid sequence with at least 90% sequence identity.

14. Sequence IDs 5, 25-30, 40, 55-56, 59-60, 70, 92, 113, 11 Select from the group consisting of 5, 117, 119, 121, 123, 125, and 127. The fusion protein according to claim 13, comprising a mino acid sequence.

15. Nucleotide sequence encoding the fusion protein according to any one of claims 1 to 14 Isolated nucleic acid molecules containing these molecules.

16. Sequence numbers 76-91, 95, 109, 110, 114, 116, 118, 120, 12 Claim 15, having at least 90% sequence identity with 2, 124, 126, and 128. Isolated nucleic acid molecules as described.

17. Sequence numbers 76-91, 95, 109, 110, 114, 116, 118, 120, 12 The nucleotide sequence includes a sequence selected from the group consisting of 2, 124, 126, and 128. The isolated nucleic acid molecule described in item 16.

18. A nucleic acid molecule comprising a fusion protein according to any one of claims 1 to 14 Kuta.

19. Sequence numbers 76-91, 95, 109, 110, 114, 116, 118, 120, 12 The nucleotide sequence includes a sequence selected from the group consisting of 2, 124, 126, and 128. The vector described in item 18.

20. A nascent Principal cell.

21. A method for producing a fusion protein according to any one of claims 1 to 14, (1) Contains nucleic acid molecules encoding the fusion protein under conditions in which the fusion protein is generated. Culturing host cells, (2) Recovering the fusion protein produced by the host cell, method.

22. A fusion protein according to any one of claims 1 to 14, and a pharmaceutically acceptable carrier. A pharmaceutical composition containing the following:

23. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 14, and pharmaceutical A pharmaceutical composition comprising a suitably permissible carrier.

24. A method for treating or preventing metabolic diseases, wherein the subject requiring treatment or prevention of the disease, A method comprising administering an effective amount of the pharmaceutical composition according to claim 22 or 23.

25. In patients requiring treatment for a disease, type 2 diabetes, elevated blood glucose levels, elevated insulin levels, Obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, and rheumatoid arthritis A method for treating diseases selected from a group consisting of metabolic diseases selected from a group consisting of ci The subject contains a fusion protein comprising the amino acid sequence of SEQ ID NO: 60, and a pharmaceutically acceptable substance. A method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a carrier to be contained.

26. In patients requiring treatment for a disease, type 2 diabetes, elevated blood glucose levels, elevated insulin levels, Obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, and rheumatoid arthritis A method for treating diseases selected from a group consisting of metabolic diseases selected from a group consisting of ci The subject contains a fusion protein comprising the amino acid sequence of Sequence ID No. 92, and a pharmaceutically acceptable substance. A method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a carrier to be contained.

27. A dimer containing two polypeptide chains, where each chain extends from the N-terminus to the C-terminus. It includes an HSA region, a linker, and a GDF15 region, wherein the HSA region is sequence numbers 1 and 2. , or comprising an amino acid sequence having at least 90% identity with 3, the GDF15 region However, an ami having at least 90% identity with sequence numbers 6, 7, 8, 9, 10, or 11 A dimer containing an acid sequence.

28. The HSA region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3. The dimer according to claim 27, including.

29. The GDF15 region is selected from the group consisting of sequence numbers 6, 7, 8, 9, 10, and 11. The dimer according to claim 27, comprising the amino acid sequence.

30. The two polypeptide chains are linked by a disulfide bond, according to the claim. The dimer described in 27.

31. The HSA region and the GDF15 region are linked by a polypeptide linker. The dimer according to claim 27.

32. The HSA region and the GDF15 region are connected by a flexible linker. The dimer described in item 31.

33. The polypeptide linker is sequence (GGGGS)n (where n is 2 to 20) The dimer according to claim 32, comprising:

34. The HSA region and the GDF15 region are connected by a structured linker. The dimer described in item 31.

35. The polypeptide linker is sequence (AP)n or (EAAAAK)n (where n is 2 The dimer according to claim 34, which includes (up to 20).

36. The linker is (GGGGGA)n, (PGGGGS)n, (AGGGGS)n, or GG S - (EGKSGSGSESKST)n - GGS (where n is between 2 and 20) The dimer according to claim 31, comprising an amino acid sequence selected from the group.

37. (i) comprising a first HSA region at the N-terminus, a linker, and a first GDF15 region at the C-terminus (ii) a first polypeptide chain and a second polypeptide chain containing a second GDF15 region A dimer comprising and, wherein the HSA region comprises at least 9 with SEQ ID NOs: 1, 2, or 3. It contains an amino acid sequence having 0% identity, and the first GDF15 region is sequence number 6, It contains an amino acid sequence having at least 90% identity with 7, 8, 9, 10, or 11, The second GDF15 region is at least 9 of sequence numbers 6, 7, 8, 9, 10, or 11. A dimer containing an amino acid sequence with 0% identity.

38. The HSA region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3. The dimer according to claim 37.

39. The GDF15 region is selected from the group consisting of sequence numbers 6, 7, 8, 9, 10, and 11. The dimer according to claim 37, comprising the amino acid sequence.

40. The linker is (GGGGGS)n, (AP)n, (EAAAK)n, (GGGGGA) n, (PGGGGS)n, (AGGGGS)n and GGS-(EGKSGSGSESKST ) an amino acid sequence selected from the group consisting of n-GGS (where n is 2 to 20) The dimer according to claim 37.

41. The two polypeptide chains are linked by a disulfide bond, according to the claim. The dimer described in 37.

42. A poly(P) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11. Lipeptide.

43. Isolated nucleic acid molecules containing nucleotide sequences selected from the group consisting of SEQ ID NOs: 96-108 。

44. It contains nucleic acid molecules containing nucleotide sequences selected from the group consisting of SEQ ID NOs: 96 to 108. Vector.

45. It contains nucleic acid molecules containing nucleotide sequences selected from the group consisting of SEQ ID NOs: 96 to 108. host cells.

Images