Single fusion protein and pharmaceutical composition containing the same

Abstract

This invention relates to a fusion protein comprising the IF1 protein and derived peptides and the Fc region of an immunoglobulin, and a pharmaceutical composition containing the same. The fusion protein according to the present invention exhibits improved pharmacological efficacy, persistence in the body, and protein stability, and a pharmaceutical composition containing the fusion protein as an active ingredient can be usefully used as a therapeutic agent for cancer, neurological disorders, diabetes, obesity, dyslipidemia, metabolic disorders, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, prevention of hair loss or promotion of hair growth, muscle loss, and obesity-related muscle loss.

Description

Technical Field

[0001] The present invention relates to a fusion protein comprising an IF1 protein and a derived peptide and the Fc region of an immunoglobulin, and a pharmaceutical composition containing the same, and the pharmaceutical composition can be usefully used for therapeutic applications for cancer, nervous system diseases, diabetes, obesity diseases, dyslipidemia, metabolic diseases, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, prevention of hair loss or promotion of hair growth, sarcopenia, and obesity sarcopenia.

Background Art

[0002] Protein pharmaceuticals are pharmaceuticals composed of amino acids such as insulin, growth factors, and antibodies, and can be classified into a first generation that utilizes the same structure as natural proteins and a second generation that is a sustained preparation in which the efficacy is improved by applying protein engineering technology or the half-life is extended by binding an accompanying substance.

[0003] Protein pharmaceuticals are manufactured using biologically derived substances, so they have low inherent toxicity and a clear mechanism of action, and thus have the advantage of being more therapeutically effective than chemically synthesized pharmaceuticals. However, since they are macromolecular substances with a higher-order structure, they are inferior in physicochemical stability compared to low-molecular compounds, and therefore have the disadvantage of having a short half-life in vivo. In addition, since they are produced in organisms, the types and degrees of modifications that bind to proteins vary depending on the type of organism and culture conditions, and there is a problem that it is not easy to ensure homogeneity.

[0004] In particular, when the three-dimensional structure of a protein changes due to amino acid substitution or addition, the inherent activity of the protein changes. Therefore, it is still difficult at present to extend the half-life while maintaining the inherent activity or to increase the inherent activity itself.

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the circumstances described above, the inventors conducted research to extend the in vivo activity and half-life of the peptide. They found that by attaching an Fc sequence to the peptide, the half-life was extended and the activity of the peptide itself increased, thus completing the present invention.

[0006] Therefore, the object of the present invention is to provide a fusion protein having the following structure and a pharmaceutical composition containing the same: An amino acid sequence selected from the group consisting of SEQ ID NOs: 8 through 10 and 46; A linker ligated to the C-terminus of the aforementioned amino acid sequence; and An Fc array connected to the end of the aforementioned linker. [Means for solving the problem]

[0007] To achieve the above objective, one aspect of the present invention provides the following fusion protein: An amino acid sequence selected from the group consisting of SEQ ID NOs: 8 through 10 and 46; A linker ligated to the C-terminus of the aforementioned amino acid sequence; and An Fc array connected to the end of the aforementioned linker.

[0008] The amino acid sequence consisting of SEQ ID NO: 8 is that of the wild-type ATPIF1 protein, and the sequences of SEQ ID NOs: 9, 10, and 46 are sequences derived from the wild-type ATPIF1 protein. The sequence of SEQ ID NO: 9 consists of amino acids 1 through 60 of the wild-type ATPIF1 protein, and the sequence of SEQ ID NO: 10 consists of amino acids 1 through 47 of the wild-type ATPIF1 protein. The sequence of SEQ ID NO: 46 is the sequence of SEQ ID NO: 9 with the G20S and F34M mutations introduced.

[0009] ATPIF1 (ATPase inhibitory factor 1) is a major protein that is expressed in cells and binds to ATPase present in the mitochondrial membrane, inhibiting its rotational movement. As a result, it inhibits ATP synthesis / degradation by the electron transport chain, thereby affecting intracellular energy regulation and mitochondrial homeostasis.

[0010] According to one specific example of the present invention, the linker sequence may be the sequence of SEQ ID NO: 40 or SEQ ID NO: 41, but is not limited thereto. Any linker sequence used as a peptide linker in the art to which the present invention belongs can be used without limitation.

[0011] According to one specific example of the present invention, the Fc sequence may be the sequence of SEQ ID NO: 42 or SEQ ID NO: 43, but is not limited thereto, and can be used without restriction as long as it is used as an Fc sequence in the art to which the present invention belongs. The Fc sequence may be an Fc sequence of immunoglobulin (IgG1).

[0012] The sequence of Sequence ID No. 43 is a sequence in which a mutation has been introduced into the sequence of Sequence ID No. 42 in order to effectively suppress the effector function (immune response induction by antibodies) associated with Fc fusion, thereby preventing unwanted immune responses from occurring when the Fc fusion material functions as a drug.

[0013] According to one specific example of the present invention, the amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 10 and 46 may be one in which one or more amino acids selected from the group consisting of the 8th, 9th, 10th, 11th, 13th, 14th, 15th, 16th, 17th, 22nd, 23rd, 24th, 25th, 26th, 29th, 30th, 31st, 32nd, 33rd, 34th, and 31st amino acids are substituted with alanine. Even when the amino acids at the positions mentioned above were substituted with alanine, there was no significant difference in their ability to bind to the ATP5B protein. This result means that the protein structure does not change significantly by alanine substitution, and that the in vivo activity of the amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 10 and 46 is also maintained.

[0014] According to one specific example of the present invention, the fusion protein consists of a sequence selected from the group consisting of SEQ ID NOs: 1 to 3, 13, 14, and 15, but is not limited thereto.

[0015] According to one specific example of the present invention, the fusion protein comprises an additional sequence and has the following structure, consisting of the sequence of Sequence ID No. 7; An amino acid sequence selected from the group consisting of SEQ ID NOs: 8 through 10 and 46; A linker ligated to the C-terminus of the aforementioned amino acid sequence; A GLP-1 (Glucagon-like peptide-1) sequence consisting of sequence number 44 is ligated to the end of the linker; A GLP-1 linker consisting of sequence number 45 ligated to the end of the GLP-1 sequence; and The Fc sequence ligated to the terminal end of the GLP-1 linker showed a more significant change (decreased adipose tissue mass / increased muscle tissue mass) when administered to obesity-induced mice compared to the semaglutide monotherapy group (Figure 18B, C).

[0016] Meanwhile, the inventors of the present invention confirmed the diverse in vivo activities of the fusion protein.

[0017] Therefore, another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, which contains the fusion protein as an active ingredient.

[0018] According to one specific example of the present invention, the fusion proteins of SEQ ID NOs: 1 to 3, 7, and 13 to 15 are excellent in antitumor activity because they can effectively kill cancer cells and promote the generation of reactive oxygen species (FIGS. 3 and 4).

[0019] In the present invention, the cancer may be a solid cancer, specifically, selected from the group consisting of lung cancer, ovarian cancer, colorectal cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, renal cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, uterine cancer, and bladder cancer, but is not limited thereto.

[0020] The term "prevention" used in the present invention means any act of suppressing the progression of a disease or delaying its onset by administering the pharmaceutical composition according to the present invention.

[0021] The term "treatment" used in the present invention means any act of improving the symptoms of a disease or causing a beneficial change by administering the pharmaceutical composition according to the present invention.

[0022] In addition, the present invention provides a pharmaceutical composition for preventing or treating metabolic syndrome, which contains the fusion protein as an active ingredient.

[0023] The metabolic syndrome refers to a phenomenon in which various metabolic-related diseases such as arteriosclerosis, hypertension, obesity, diabetes, and hyperlipidemia occur simultaneously. Therefore, the metabolic syndrome includes diseases such as obesity, diabetes, and dyslipidemia.

[0024] According to one specific example of the present invention, the fusion protein can be usefully used for the treatment of metabolic syndrome to suppress fat accumulation (Example 3-3), improve glucose tolerance impairment (Example 4-5), and improve obesity (Example 4-).

[0025] Since the fusion protein of the present invention also has an excellent effect of improving sarcopenia (Example 4-1), it can be used as a pharmaceutical composition for preventing or treating sarcopenia-related diseases.

[0026] The sarcopenia-related diseases may be selected from the group consisting of sarcopenia, Duchenne muscular dystrophy, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, and muscular weakness.

[0027] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease containing the fusion protein as an active ingredient.

[0028] According to a specific example of the present invention, the fusion protein has a significantly excellent effect of improving non-alcoholic fatty liver disease by reducing liver damage, blood cholesterol, and neutral fat in an animal model induced with non-alcoholic fatty liver disease (Example 4-4).

[0029] In this specification, non-alcoholic fatty liver disease (NAFLD; non-alcoholic fatty liver disease) refers to a state in which fat accumulates in hepatocytes despite not drinking alcohol at all or only drinking a small amount. Rather than a single disease, it includes various forms of liver diseases ranging from simple fatty liver without inflammation to non-alcoholic steatohepatitis (NASH) and cirrhosis.

[0030] On the other hand, in recent years, instead of the term non-alcoholic fatty liver disease, steatohepatitis caused by abnormal metabolism such as obesity and diabetes is called metabolic dysfunction-associated steatohepatitis (MASH).

[0031] Another aspect of the present invention provides a pharmaceutical composition for preventing hair loss or promoting hair growth, comprising the fusion protein as an active ingredient.

[0032] According to one specific example of the present invention, the fusion protein promotes the survival and proliferation of hair follicle cells, and can therefore be usefully used for preventing / improving hair loss and promoting hair growth (Examples 3-5).

[0033] Another aspect of the present invention provides a pharmaceutical composition for neuroprotection containing the fusion protein as an active ingredient.

[0034] According to one specific example of the present invention, the fusion protein has an excellent effect in suppressing the death of brain cells caused by the neurotoxin rotenone, and therefore can be usefully used for the protection of brain nerve cells (Examples 3-4).

[0035] Meanwhile, the inventors of the present invention also confirmed the bioactivity of sequences consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, in addition to the fusion protein.

[0036] Accordingly, yet another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, for the prevention or treatment of muscle loss-related muscle diseases, for the prevention or treatment of metabolic syndromes, for the prevention or treatment of non-alcoholic fatty liver disease, for the prevention or promotion of hair loss, and for the protection of brain nerve cells, comprising a sequence selected from the group consisting of SEQ ID NOs: 10, 11, and 12 as an active ingredient.

[0037] A pharmaceutical composition according to one example of the present invention can be applied to all animals, including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs, or monkeys.

[0038] A pharmaceutical composition according to an example of the present invention may optionally contain additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, fragrances, or sweeteners. A pharmaceutical composition according to an example of the present invention can be manufactured by conventional methods of the art.

[0039] In the present invention, examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0040] A pharmaceutical composition according to one example of the present invention can be administered orally, rectally, percutaneously, intravenously, intramuscularly, intraperitoneally, intramedullarily, intradurally, or subcutaneously.

[0041] Preparations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, liquids, or emulsions. Preparations for parenteral administration may be, but are not limited to, injections, infusions, gels, suspensions, emulsions, suppositories, patches, or sprays.

[0042] The aforementioned pharmaceutical compositions may be in the form of sterile injectable formulations, such as sterile aqueous or oily suspensions for sterile injection. These suspensions may be formulated by art known techniques using appropriate dispersants or wetting agents (e.g., Tween 80) and suspending agents. Sterile injectable formulations may also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents (e.g., solutions in 1,3-butanediol). Acceptably usable vehicles and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solutions. Sterile non-volatile oils are also commonly used as solvents or suspension media. For this purpose, non-volatile oils containing synthetic mono- or diglycerides and having low irritancy may be used. Fatty acids, such as oleic acid and its glyceride derivatives, as well as pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), particularly their polyoxyethylated forms, are useful in injectable formulations.

[0043] Parenteral administration of the pharmaceutical compositions according to the present invention is particularly useful when relating to a site or organ where the target treatment can be easily performed by topical application. Carriers for topical administration of the compositions of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water.

[0044] The active ingredient of the pharmaceutical composition according to the present invention may vary depending on the age, sex, weight, pathological condition and its severity, route of administration, or the prescriber's judgment of the recipient. Determining the dosage based on these factors is within the realm of those skilled in the art, and the daily dose may be, for example, 10 ng / kg / day to 10 mg / kg / day, specifically 0.1 μg / kg / day to 1 mg / kg / day, more specifically 1 μg / kg / day to 100 μg / kg / day, and even more specifically 2 μg / kg / day to 50 μg / kg / day, but if differences in effect are observed depending on the dose, it can be appropriately adjusted. A pharmaceutical composition according to one example of the present invention may be administered once to three times a day, but is not limited thereto.

[0045] Another aspect of the present invention provides a functional health food composition containing the fusion protein as an active ingredient for the prevention or improvement of cancer, the prevention or improvement of muscle loss-related muscle diseases, the prevention or improvement of metabolic syndromes, the prevention or treatment of non-alcoholic fatty liver disease, the prevention or promotion of hair loss, and the protection of brain nerve cells.

[0046] As used in this invention, the term "improvement" means any action that at least reduces a parameter related to the state in which a disease is treated, such as the severity of symptoms.

[0047] Since the details regarding the fusion protein in the aforementioned health functional food composition are as described above, any redundant information will be omitted.

[0048] The formulation of a food composition according to one example of the present invention is not particularly limited and can be formulated as, for example, tablets, granules, powders, liquids, solid formulations, etc. Each formulation can be appropriately selected and blended with other ingredients commonly used in the field, in addition to the active ingredient, according to the formulation or intended use, without difficulty for those skilled in the art, and synergistic effects may occur when applied simultaneously with other raw materials.

[0049] The terms used herein are intended solely to describe specific embodiments and are not intended to limit the invention. Terms in which a quantity is omitted before a noun do not limit that quantity, but rather mean that there is one or more articles of the noun described. The terms “contains,” “possesses,” and “contains” are to be interpreted broadly (i.e., “contains, but is not limited to.”)

[0050] Numerical ranges are mentioned simply because it is a convenient alternative to mentioning each distinct numerical value within that range individually; unless otherwise explicitly stated, each numerical value applies herein as it is mentioned individually in the specification. The limit values ​​of all ranges are contained within that range and can be combined independently.

[0051] All methods described herein may be performed in an appropriate order unless otherwise explicitly stated or clearly contradicted by the context. The use of any one example or all examples, as well as exemplary language (e.g., "like"), is merely to facilitate the description of one aspect of the invention and does not limit the scope of the invention, unless otherwise included in the claims. No language herein should be construed as making any unclaimed component essential for the practice of the invention. Unless otherwise specified, the technical and scientific terms used herein have the meanings that would normally be understood by a person of ordinary skill in the art to which the invention pertains. [Effects of the Invention]

[0052] The fusion protein according to the present invention exhibits improved pharmacological efficacy, in vivo persistence, and protein stability, and pharmaceutical compositions containing the fusion protein as an active ingredient can be usefully used as therapeutic agents for cancer, neurological disorders, diabetes, obesity, dyslipidemia, metabolic disorders, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, prevention of hair loss or promotion of hair growth, muscle loss, and obesity-related muscle loss. [Brief explanation of the drawing]

[0053] [Figure 1] This shows the results of comparing the plasma protein binding rates of the fusion protein (SEQ ID NO: 13) or recombinant protein (SEQ ID NO: 5) of the present invention (F: Free, unbound free protein; B: Bound, bound protein). [Figure 2] This report compares the cellular activity of the fusion protein (SEQ ID NOs. 1, 2, 3, 7) or recombinant protein (SEQ ID NOs. 5) of the present invention in C2C12 differentiated or undifferentiated muscle cells, using AKT, a marker of cellular signaling pathway activation, and the degree of S6 phosphorylation. [Figure 3]The antitumor activity of the test substances of the present invention (SEQ ID NOs: 1, 2, 3, 4, 7, 9, 10, 11, 12, 14, 15) or recombinant protein (SEQ ID NO: 5) was compared in the MBA-MB-231 breast cancer cell line using cell viability measured by the MTT assay (*p value < 0.05 compared to control). [Figure 4] This is a comparison of the antitumor activity of the substances of the present invention (SEQ ID NOs: 1, 2, 3, 6, 7, 8, 9, 10, 11, 13, 14, 15) or recombinant protein (SEQ ID NO: 5) in the MBA-MB-231 breast cancer cell line, based on the amount of reactive oxygen species generated by ROS assay (*p value < 0.05 compared to control). [Figure 5] The results shown are a comparison of UCP-1 expression levels by RT-PCR after treating 3T3-L1 adipocyte cells with the fusion protein (SEQ ID NO: 13) or synthetic peptide (SEQ ID NO: 8) of the present invention (*p value < 0.05 compared to control). [Figure 6] The following are the results of comparing (A) the change in lipid droplet size, (B) the total lipid droplet area, and (C) the maximum diameter of each lipid droplet after treating the fusion protein (SEQ ID NO: 13) or synthetic peptide (SEQ ID NO: 8) of the present invention with the 3T3-L1 adipocyte line (*p value < 0.05 compared to control). [Figure 7] The results of comparing the lipid energy metabolic activity of the substances of the present invention (SEQ ID NOs: 1, 2, 3, 7, 9, 10, 12, 13, 14, 15) measured by cAMP assay in 3T3-L1 adipocytes (*p value < 0.05 compared to control). [Figure 8] The following are the results of comparing the cell viability of SH-SY5Y dopaminergic neuron cells after treatment with the substances of the present invention (SEQ ID NOs: 1, 2, 3, 7, 9, 10, 11) together with rotenone, using an MTT assay (*p value < 0.05 compared to control). [Figure 9]The results of treating SH-SY5Y dopaminergic neuron cells with the substances of the present invention (SEQ ID NOs: 1, 2, 3, 7, 9, 10, 11) together with rotenone, and then measuring and comparing the degree of inhibition of reactive oxygen species generation by ROS assay (*p value < 0.05 compared to control). [Figure 10] The results shown are from an MTT assay that examined the cellular effects after treating hair follicle cells with the substances of the present invention (SEQ ID NOs: 13, 15) (*p value < 0.05 compared to control). [Figure 11] The biological functions of the substances of the present invention (SEQ ID NOs: 13, 14, 15) against muscle loss and muscle-related diseases were quantified in a sciatic nerve transection mouse model by (A) changes in muscle tissue mass and (B) grip strength measurements, and compared with the control group (*p value < 0.05 compared to control). [Figure 12] The biological function of the substance of the present invention (SEQ ID NO: 2) against muscle loss and muscle-related diseases was quantified using grip strength measurements in a mouse model of hereditary muscular atrophy disease and compared with a control group (*p value < 0.05 compared to control). [Figure 13] The antitumor activity of the substance of the present invention (SEQ ID NO: 14) was evaluated by quantifying tumor growth size in a xenograft mouse model of breast cancer and comparing it to a control group (*p value < 0.05 compared to control). [Figure 14] The biological functions of the substance of the present invention (SEQ ID NOs: 1, 2) against obesity, lipids, glucose metabolism, and sarcopenic diseases were compared with a control group in a diet-induced obesity-inducing mouse model by measuring (A) body weight, (B) blood glucose, (C) lipid droplet size in liver tissue, (D) fat mass, and (E) muscle mass (ns, not significant, *p value < 0.05 compared to control). [Figure 15]The preventive biological functions of the substance of the present invention (SEQ ID NO: 14) against obesity, sarcopenic obesity, and muscle disease were compared with a control group in a diet-induced obesity-inducing mouse model by measuring (A) adipose tissue ratio, (B) muscle tissue ratio, and (C) grip strength (*p value < 0.05 compared to control). [Figure 16] The biological functions of the substance of the present invention (SEQ ID NO: 3) against obesity, sarcopenic obesity, and muscle disease were compared with a control group in a diet-induced obesity-inducing mouse model by measuring (A) body weight, (B) adipose tissue ratio, and (C) muscle tissue ratio (*p value < 0.05 compared to control). [Figure 17] The biological functions of the substance of the present invention (SEQ ID NO: 7) against obesity, sarcopenic obesity, and muscle disease were investigated in a diet-induced obesity-inducing mouse model. The results compare data before and after substance treatment by measuring (A) body weight, (B) adipose tissue ratio, and (C) muscle tissue ratio (*p value < 0.05 compared to control). [Figure 18] The biological functions of the substance of the present invention (SEQ ID NO: 14) against obesity, sarcopenic obesity, and muscle disease were compared with a control group in a hereditary leptin-deficient mouse model by measuring (A) body weight, (B) adipose tissue ratio, (C) muscle tissue ratio, and (D) grip strength (*p value < 0.05 compared to each group). [Figure 19] The biological functions of the substances of the present invention (SEQ ID NOs: 14, 15) against obesity, sarcopenic obesity, and glucose metabolism disorders were quantified and measured using glucose tolerance tests in diet-induced obesity mice, and the results were compared with a control group (*p value < 0.05 compared to control). [Figure 20] The biological functions of the substance of the present invention (SEQ ID NO: 2) against glucose metabolism disorders, muscle diseases, and their complications were quantified and measured in a mouse model of leptin receptor dysfunction, and compared with a control group (*p value < 0.05 compared to control). [Figure 21]This report compares the biological function of the substance of the present invention (SEQ ID NO: 2) against glucose metabolism disorders in a mouse model of leptin receptor dysfunction with that of a control group by measuring blood glucose levels (*p value < 0.05 compared to control). [Modes for carrying out the invention]

[0054] One or more specific examples will be described in more detail below by reference to embodiments. However, these embodiments are for illustrative purposes only, and the scope of the present invention is not limited to these embodiments.

[0055] Example 1: Production of transformed E. coli cells and production and validation of fusion proteins.

[0056] To produce fusion proteins of the wild-type ATPIF1 protein (SEQ ID NO: 8) and its fragments (SEQ ID NOs: 9, 10, and 12), a vector containing the sequence encoding the fusion protein was transformed into the expression strain, SHuffle. The transformed strain was cultured in LB medium at 37°C for 2 hours. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the medium, and the cells were cultured for a further 12 hours. After the culture period, only the E. coli cells were collected and separated into water-soluble and insoluble fractions by sonication and centrifugation. The expressed fusion proteins were purified from each water-soluble fraction using affinity resins: proteins of SEQ ID NOs: 1, 2, 3, 7, 13, 14, and 15: Protein A resin; proteins of SEQ ID NOs: 4 and 6: Sepharose resin.

[0057] Furthermore, we commissioned a specialized institution to synthesize the peptides used in this invention.

[0058] Tables 1 and 2 below describe the fusion protein and peptide used in this invention, along with their sequences. The fusion protein and synthetic peptide used as the control group are His tag Human ATPIF1 (SEQ ID NO: 5) and Human ATPIF1 (SEQ ID NO: 8).

[0059] [Table 1] TIFF2026513837000003.tif60168

[0060] [Table 2] TIFF2026513837000005.tif243165TIFF2026513837000006.tif241168TIFF2026513837000007.tif103165

[0061] Example 2: Evaluation of improved in vivo stability of fusion proteins

[0062] The fusion protein produced in Example 1 was evaluated to determine whether it was stably present in vivo as follows. Ten-week-old mice of the C57BL / 6 strain were used for the evaluation. The mice were raised in a specialized breeding facility that provided a normal diet and a 12 / 12 hour day / night cycle before being used in the experiment.

[0063] 2-1. Confirmation of Half-Life Mice were injected with a synthetic peptide (SEQ ID NO: 8) and a fusion protein (SEQ ID NO: 13) at a concentration of 2.5 mg / kg. Blood samples were collected at 0, 0.5, 2, 4, and 24 hours after injection. The collected blood was centrifuged, and the resulting plasma was analyzed. To ensure effective quantification of each substance, Dylight 680 Near-Infrared dye (NIR) was conjugated to each substance before injection. The final blood samples were then analyzed by observing the fluorescence signal using a SpectraMax i3 microplate reader. The results showed that the synthetic peptide (SEQ ID NO: 8) had a half-life of 1.6 hours, while the fusion protein (SEQ ID NO: 13) had a half-life of 18.9 hours, indicating an approximately 11.8-fold increase in half-life (Table 3). This result suggests that the fusion protein possesses greater stability in the biological system.

[0064] [Table 3]

[0065] The half-life of the fusion protein was further measured using ELISA (Enzyme-Linked Immunosorbent Assay). Each test substance was subcutaneously injected into C57BL6 / J mice at a concentration of 5 mg / kg, and then blood was separated at different time points.

[0066] Separated blood was added to a 96-well plate coated with capture antibody, and each well was washed three times with PBS (phosphate-buffered saline). Detection solution was added to each well, and the wavelength was read at 450 nm for analysis. The analysis results showed that the half-lives of the fusion proteins (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) increased to 39.6, 45.6, and 74.7 hours, respectively, compared to the results for SEQ ID NO: 8 and SEQ ID NO: 13 using the full-length ATPIF1 sequence (Table 3) (Table 4).

[0067] [Table 4]

[0068] This increase in half-life was further confirmed by measuring the plasma protein binding test (PPB). When bound to plasma proteins (e.g., albumin), it is relatively protected from proteolytic enzymes, its size increases, delaying its elimination, and consequently increasing its circulation time in the blood and thus its half-life.

[0069] Mouse plasma was incubated with either the fusion protein (SEQ ID NO: 13) or the His tag-wild-type substance (1-81) (SEQ ID NO: 5) at a concentration of 5 μM at 37°C for 4 hours. The mixtures were then centrifuged using a 100 kD filter to obtain fractions of substances bound to or not bound to plasma proteins. Each fraction was evaluated by Western blotting. The results showed that the fusion protein (SEQ ID NO: 13) had a 74% binding rate to plasma proteins, while the His tag-wild-type substance (1-81) (SEQ ID NO: 5) had a binding rate of only 5%, indicating that the fusion protein of the present invention possesses higher in vivo stability (Figure 1).

[0070] Example 3: In vitro activity evaluation of the fusion protein

[0071] To confirm whether the fusion protein produced in this invention functions as effectively as the original protein, its substance activity was compared with that of a control group in a cellular system.

[0072] 3-1. Muscle cells To evaluate muscle loss-related functions, each test substance was treated with either undifferentiated C2C12 mouse skeletal muscle cell lines (mouse myoblast cell line) or cell lines that had fully differentiated into myotubes. Differentiation of the cell lines into myotubes was performed by adding 2% horse serum to the culture medium and replacing it with fresh medium every two days for a total of four days.

[0073] Undifferentiated or differentiated cell lines were treated with the test substance, a fusion protein (SEQ ID NOs: 1, 2, 3, 7, 14, and 15), at a concentration of 100 nM (Control: PBS-treated group) for 2 hours. Immediately afterward, the cells were washed once with PBS and lysed with RIPA buffer (Radioimmunoprecipitation assay buffer). Akt and S6 activity (phosphorylation, p-Akt / p-S6) was observed by Western blotting. Akt phosphorylation is a well-known marker of cellular signaling pathway activation and an indicator of beneficial effects on muscle cells, such as insulin-mediated glucose metabolism promotion and muscle biosynthesis promotion. Similarly, S6 phosphorylation is a well-known marker of mTOR activity, and mTOR activation is an indicator of muscle biosynthesis and diverse cellular signaling pathways.

[0074] Results from the activity testing of Akt and S6 in differentiated cell lines confirmed that the fusion proteins (SEQ ID NOs: 1, 2, 3, 7, 14, and 15) showed significantly higher activity than the control and His tag-wild type substances (1-81) (SEQ ID NO: 5) (Figure 2 and Tables 5 and 6).

[0075] [Table 5]

[0076] [Table 6]

[0077] 3-2. Cancer cells Next, the MDA-MB-231 triple-negative breast cancer cell line was treated with the test substance, a fusion protein (SEQ ID NOs: 1, 2, 3, 7, 14, 15) and its truncated sequence (SEQ ID NOs: 9, 10, 11, 12), at a concentration of 1 μM (Control: PBS) for 48 hours. The cells were then washed once with PBS and suspended in serum-free buffer. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was added at a concentration of 0.5 mg / ml and incubated at 37°C for 2 hours. The cells were then washed once with PBS and lysed with DMSO for 15 minutes. The solution was transferred to a new plate, and the absorbance was measured at 590 nm using a microplate reader.

[0078] The measurement results showed that cell viability was significantly lower when treated with fusion proteins (sequences 1, 2, 3, 7, 14, 15) or truncated sequences (sequences 9, 10, 11, 12) compared to the control, GST tag-wild-type substance (1-81) (sequence number 4), and His tag-wild-type substance (1-81) (sequence number 5) (Figure 3). This result means that the anti-cancer effect was increased even when the wild-type substance (1-81) (sequence numbers 4, 5) was fused with other sequences or when only a portion of the sequence was used.

[0079] Furthermore, to confirm the generation of reactive oxygen species, which play a crucial role in cancer cell death, MDA-MB-231 cell lines were treated with each test substance at a concentration of 1 μM (Control: PBS) for 24 hours. Subsequently, the cells were stained with DCF-DA, and their fluorescence values ​​were observed and compared at 485 / 535 nm.

[0080] As a result, it was confirmed that reactive oxygen species were also generated in greater quantities in the fusion protein (sequences 1, 2, 3, 7, 13, 14, 15) and abbreviated sequence (sequences 9, 10, 11) treatment groups compared to the Control and His tag-wild type substance (sequence number 5) (Figure 4).

[0081] 3-3. Fat cells To observe the lipid metabolism-related functionality of the fusion protein, the 3T3-L1 mouse pre-adipocyte cell line was used either as the cell line itself (undifferentiated) or differentiated into adipocytes. Differentiation into adipocytes was performed by inducing primary differentiation of 3T3-L1 cells for 2 days in a medium containing DMEM, 10% FBS, 10 μg / ml insulin, 0.5 mM IBMX (3-Isobutyl-1-methylxanthine), and 1 μM dexamethasone, followed by secondary differentiation for the remaining 4 days in a medium containing DMEM, 10% FBS, and 10 μg / ml insulin. Finally, the differentiated adipocytes were treated with the test substance at a concentration of 100 nM for 30 hours (Control: PBS) before being used in each test.

[0082] UCP-1 (uncoupling protein 1) is a representative thermogenic biomarker that indicates browning of adipocytes and activation of energy metabolism. To observe changes in UCP-1 mRNA and protein expression induced by fusion protein treatment, mRNA and protein were isolated from cells treated with each test substance and performed by RT-PCR and Western blotting. As a result, it was confirmed that the mRNA level of UCP-1 significantly increased upon treatment with the fusion protein (SEQ ID NO: 13) (Figure 5). This result suggests that the fusion protein (SEQ ID NO: 13) has an energy regulatory function in adipocytes.

[0083] Furthermore, after staining adipocyte lipid droplets with BODIPY 493 / 503 stained samples, confocal image scanning was performed to analyze the size and diameter of the lipid droplets. As a result, it was found that treatment with the fusion protein (SEQ ID NOs: 8, 13) significantly reduced the size of the lipid droplets, the total area of ​​the lipid droplets, and the maximum lipid droplet diameter per cell (Figure 6).

[0084] Next, changes in cAMP (cyclic AMP), a signaling pathway molecule contributing to lipolysis and energy metabolism, were evaluated in undifferentiated adipocytes. Undifferentiated cells were treated with the test substance at a concentration of 100 nM (Control: PBS) along with 0.5 mM IBMX for 30 minutes. Subsequently, cAMP levels were evaluated using the hTRF (Homogeneous Time Resolved Fluorescence) technique (using the cAMP Gs dynamic kit from Cisbio). The results showed that cAMP levels were higher in the fusion protein (Sequence IDs 1, 2, 3, 7, 13, 14, 15) and abbreviated sequence (Sequence IDs 9, 10, 11) treatment groups compared to the control group and the His tag-wild type substance (1-81) (Sequence ID 5) (Figure 7).

[0085] 3-4. Nerve cells The neuroprotective functionality of the fusion protein was confirmed as follows:

[0086] SH-SY5Y dopaminergic neuron cell lines were simultaneously treated with 1 μM of the toxic substance rotenone and 100 nM of each test substance. After 24 hours, MTT cell viability was evaluated or the effect of reducing reactive oxygen species was observed.

[0087] Observations revealed that, in a rotenone-induced toxic environment, cell viability was significantly increased in the fusion protein (SEQ ID NOs. 1, 2, 3, 7) and abbreviated sequence (SEQ ID NOs. 9, 10, 11) treatment groups compared to the Control (rotenone-only treatment group) (Figure 8), and the generation of reactive oxygen species was significantly suppressed (Figure 9). In Figures 8 and 9, "Control" on the X axis indicates treatment with only the test substance, while "+Rotenone" indicates treatment with both rotenone and the test substance.

[0088] 3-5. Functions related to hair growth and hair loss The present invention's test substance was evaluated as follows to determine whether it has the effect of preventing hair loss or promoting hair growth. Specifically, dermal papilla cells were used to evaluate the proliferation of dermal papilla cells, and it was observed whether it improved the shedding of dermal papilla cells in the scalp tissue that causes hair loss, or whether it had a hair growth effect by increasing the number of hair follicles.

[0089] Hair follicle cells were treated with 100 nM of each test substance (Control: PBS) for 24 hours, and the degree of cell proliferation was evaluated by the MTT assay. As a result, cell viability was found to be significantly increased in the His tag-wild-type substance (1-81) (SEQ ID NO: 5) and fusion protein (SEQ ID NOs: 13, 15) treatment groups compared to the control group. In particular, the fusion protein treatment group showed an improved cell proliferation-promoting effect compared to the His tag-wild-type substance (1-81) treatment group, and its efficacy in treating hair loss was verified (Figure 10).

[0090] 3-6. Evaluation of binding ability Surface plasma resonance (SPR) testing was performed to evaluate the substitutable residues within the sequences. Specifically, ATP5B protein was bound to a CM5 chip via amine conjugation, and the contact of each test substance was observed at a flow rate of 50 μl / min, and the refractive index was measured. Subsequently, the binding capacity was compared and evaluated by converting each binding unit. For sequences 17-40, the contribution of each amino acid residue to the binding capacity was evaluated by substituting each amino acid residue with the inactive amino acid alanine in the shortened sequence of sequence 16. A decrease in binding capacity of 10% or more was considered to have a negative impact on substitution.

[0091] As a result of the evaluation, when the binding degree of each sequence (sequence numbers 16-39) was observed compared to the control shortened sequence (sequence number 12), the amino acid substitution efficacy of the other residues was confirmed, with the exception of sequences 25, 26, and 32, which showed a binding ability of 10% or more compared to sequence number 16 (Table 7).

[0092] [Table 7]

[0093] These results mean that even if some amino acid sequences are substituted with alanine, the peptide structure can be maintained normally, and the peptide's in vivo function can also be preserved.

[0094] Example 4: Evaluation of the in vivo activity of the fusion protein

[0095] To confirm whether the fusion protein created as described above can effectively perform its function, its substance activity was evaluated in a biological system using a mouse model by comparing its glucose metabolism processing ability with that of a control group.

[0096] Example 4-1: Efficacy evaluation for muscle disease - Mouse disease model

[0097] [Sciatic nerve section model]

[0098] The effects of test substances on muscle diseases causing muscle loss were evaluated using a mouse model of sciatic nerve transection.

[0099] Muscle atrophy in the sciatic nerve transection mouse model was induced as follows: The thigh muscle of male C57BL / 6 mice was transversely incised to expose the sciatic nerve. The sciatic nerve was then transsected to weaken the nerve within the muscle tissue and induce muscle atrophy. The incision site was sutured, and a one-week recovery period was given after the surgery.

[0100] After a recovery period, each mouse group received a subcutaneous injection of the assigned test substance (Control-PBS; and fusion protein) at a concentration of 5.0 mg / kg six times a week for nine weeks (Control: n=8, No.13: n=9, No.14: n=9, No.15: n=8).

[0101] The effect of improving muscle loss was measured using an X-ray body component analyzer (DXA assay) four weeks after administration of the test substance, and the effect of improving muscle function was compared by measuring grip strength.

[0102] As a result, muscle atrophy was normally induced in the surgical site muscles, and while muscle mass in the affected area decreased in the control group during body composition analysis, muscle mass significantly increased in the fusion protein (sequences 13, 14, 15) administration group (Figure 11A). Similarly, in grip strength tests, higher performance was observed in the fusion protein administration group compared to the control group, confirming improved muscle function (Figure 11B).

[0103] [Duchenne muscular dystrophy (DMD)]

[0104] The effects of test substances on muscle diseases causing muscle loss were evaluated using a Duchenne muscular dystrophy mouse model (C57BL / 10ScSn-Dmdmdx / J) that induces progressive muscle degeneration through genetic modification.

[0105] The mouse model described above was divided into a control group and an experimental group. The control group received PBS, while the experimental group received the fusion protein (SEQ ID NO: 2) at a concentration of 5 mg / kg six times a week for three weeks via subcutaneous injection. After three weeks, a grip strength test was performed to confirm whether the fusion protein (SEQ ID NO: 2) improved muscle function decline caused by muscle degeneration (Control: n=9, No.2: n=8).

[0106] The results confirmed that grip strength was significantly improved in the fusion protein administration group compared to the control group, demonstrating the muscle function-improving effect of the fusion protein (Figure 12).

[0107] Example 4-2: Cancer disease evaluation - mouse disease model

[0108] MDA-MB-231 cells were injected into immunodeficient BALB / c nude mice to induce tumor formation over one week, creating a xenograft tumor model. The tumor-forming mice were randomly divided into a control group and an experimental group (Control: n=8, No. 14: n=7). The control group received PBS, while the experimental group received the fusion protein (SEQ ID NO: 14) subcutaneously six times a week for a total of 7.5 mg / kg over six weeks. Comparing the final tumor sizes at the six-week mark, a significant tumor growth inhibition effect was observed in the fusion protein administration group (Figure 13). This result demonstrates that the fusion protein of the present invention exerts a sufficient anti-cancer effect within the biological system.

[0109] Example 4-3: Efficacy evaluation for obesity and sarcopenia obesity disease - Mouse disease model

[0110] [Dietary Obesity Induction Model]

[0111] To confirm the disease-targeting efficacy of the fusion protein in obesity and sarcopenic obesity, disease induction was performed in mice through dietary obesity. C57BL / 6N mice were used as an animal model. After a one-week acclimatization period with a normal diet, the mice were introduced into the experiment. The living environment was maintained at 18-24°C and 50-60% humidity, and ad libitum feeding was allowed, with mice having free access to food and water during both the acclimatization and experimental periods. After one week of acclimatization, obesity was induced in the mice using a high-fat diet (Research Diet Co., Ltd. D12492) to induce disease.

[0112] [Dietary Obesity Induction Model Study I]

[0113] Mice that had been successfully induced to be obese were divided into control and experimental groups (Control: n=8, No.1: n=6, No.2: n=6). The control group received PBS, and the experimental group received a fusion protein (SEQ ID NO: 1,2) via subcutaneous injection at a dose of 5 mg / kg six times a week for 10 weeks, and their effects were observed.

[0114] A comparison of final body weights revealed a significant decrease in body weight in the fusion protein administration group compared to the control group (Figure 14A), and furthermore, a decrease in blood glucose levels was also observed (Figure 14B).

[0115] Compared to the control group, the fusion protein administration group showed a significant reduction in the size of lipid droplets in liver tissue (Figure 14C). Body composition analysis by DXA at ​​the intermediate point (3 weeks after administration) showed a significant decrease in adipose tissue volume (Figure 14D), while muscle mass was maintained (Figure 14E). These results suggest that the fusion protein has an anti-obesity effect in obese patients, along with improvements in glucose metabolism, lipid metabolism, and body composition.

[0116] [Dietary Obesity Induction Model Study II]

[0117] We investigated whether the fusion protein prevents obesity and the muscle loss induced by obesity during the process of obesity induction. Mice were given a high-fat diet and administered the fusion protein (SEQ ID NO: 14) subcutaneously eight times at a dose of 5 mg / kg for a total of six weeks, and its efficacy was observed (Control: n=11, No. 14: n=9).

[0118] A comparison of final body weights revealed that weight gain due to obesity progression was significantly suppressed in the fusion protein administration group compared to the control group (34.61±2.33 vs 32.99±2.55g; Control vs fusion protein administration group).

[0119] DXA analysis revealed a relative increase in absolute muscle mass in the fusion protein administration group compared to the control group (18.57±1.26 vs 20.46±2.71g; Control vs fusion protein administration group), and significant changes in body composition (decreased adipose tissue ratio / increased muscle tissue ratio) (Figures 15A, B) were also observed. Furthermore, grip strength tests conducted at 5 weeks after administration of the test substance confirmed improvements in muscle function (Figure 15C).

[0120] [Dietary Obesity Induction Model Study III]

[0121] Mice that had been successfully induced to be obese were divided into a control group and an experimental group (Control: n=8, No.3: n=7). For two weeks, the control group received PBS, and the experimental group received a fusion protein (SEQ ID NO: 3) at a dose of 5 mg / kg subcutaneously six times a week, and the effects were observed.

[0122] A comparison of final body weights revealed a significant decrease in body weight in the fusion protein administration group compared to the control group (Figure 16A). Significant changes in body composition (decreased adipose tissue ratio / increased muscle tissue ratio) (Figures 16B, C) were observed, confirming the efficacy of the fusion protein.

[0123] [Dietary Obesity Induction Model Study IV]

[0124] Mice that had been successfully induced to be obese (No. 7: n=7) were subcutaneously injected with the fusion protein (SEQ ID NO: 7) at a dose of 5 mg / kg six times a week for 10 days, and the efficacy before and after administration was compared. When the experimental results were compared using paired t-test statistical analysis, a significant reduction in body weight was confirmed (Figure 17A), and at the same time, a significant reduction in adipose tissue-specific effects (decrease in adipose tissue ratio / increase in muscle tissue ratio) (Figures 17B, C) was observed, confirming its efficacy.

[0125] [Leptin-deficiency obesity disease model study]

[0126] To confirm the efficacy of the fusion protein in a genetically inherited obesity condition characterized by hormonal dysregulation and inability to suppress appetite, caused by genetically induced mutations in the leptin gene, and to simultaneously confirm the possibility of co-administration with the GLP-1 substance (Semaglutide), we used the ob / ob mouse model (C57BL / 6J-ob / ob).

[0127] Mice that had undergone a one-week acclimatization period were divided into two groups (Control: n=7, Semaglutide (30 nmol / kg): n=7, Semaglutide (30 nmol / kg) + No. 14: n=6). They were fed a normal diet, and for 10 weeks, the Control group received PBS, while each experimental group received either semaglutide or semaglutide + fusion protein (SEQ ID NO: 14) at a dose of 5 mg / kg subcutaneously every two days. The effects were then observed.

[0128] A comparison of final body weight revealed a significant decrease in weight compared to the control group (Figure 18A). In particular, significant changes in body composition were observed in the combination therapy group compared to semaglutide monotherapy (decreased adipose tissue mass / increased muscle tissue mass) (Figures 18B, C), confirming its efficacy. Furthermore, grip strength tests confirmed improvements in muscle function (Figure 18D).

[0129] Example 4-4: Evaluation of fatty liver and lipid metabolism disorders - Mouse disease model

[0130] We received 5-week-old C57BL / 6J female mice and, after a one-week acclimatization period, induced fatty liver disease. All mice were kept in a constant environment of 18-24°C and 50-60% humidity, and were allowed free feeding during both the acclimatization and fatty liver induction periods. After the one-week acclimatization period, the mice were fed a GAN diet (Gubra Amylin NASH diet) for 38 weeks to induce non-alcoholic fatty liver disease. The detailed components of the product are shown in Table 8 below.

[0131] [Table 8]

[0132] Mice in which non-alcoholic fatty liver disease (NAL) was induced were divided into a control group (n=7) and an experimental group (n=6). The control group received PBS subcutaneously at a dose of 2.5 mg / kg six times a week for five weeks, while the experimental group received a fusion protein (SEQ ID NO: 14) at a dose of 2.5 mg / kg. At the end of the study, the weight of adipose tissue and liver tissue was measured, and biochemical tests (OT, PT, Cholesterol, TG) and histological staining (Masson's trichrome) were performed. Masson's trichrome staining was used to analyze the degree of collagen accumulation, which causes liver fibrosis induced in fatty liver disease, and inflammatory cell infiltration, which causes inflammation, because collagen fibers are stained blue, cytoplasm red, and cell nuclei dark brown.

[0133] As a result, compared to the control group, the experimental group showed a significant reduction in body weight and adipose tissue, as well as suppression of liver weight increase associated with fatty liver induction. Furthermore, compared to the control group, the experimental group showed a significant decrease in OT (GOT, AST (aspartate aminotransferase)) and PT (GPT, ALT (alanine aminotransferase)) levels, which are detected in response to liver tissue damage, as well as a significant decrease in blood cholesterol and triglycerides (TG), confirming positive effects on lipid metabolism as a whole. In addition, a decrease in the degree of collagen accumulation was confirmed by Masson's trichrome staining (Table 9), verifying the efficacy of the fusion protein against fatty liver and lipid metabolism disorders.

[0134] [Table 9]

[0135] Example 5-5: Evaluation of glucose metabolism disorders - Mouse disease model

[0136] [Study on a glucose tolerance disorder model induced by dietary obesity]

[0137] To secure a glucose tolerance impairment model, we planned conditions to induce glucose metabolism disorders in mice through dietary obesity. As the animal model, we received 5-week-old male C57BL / 6N mice, which were fed a normal diet for one week to acclimate before being used. The rearing environment was maintained at 18-24°C and 50-60% humidity, and the mice were allowed free access to feed and water during both the acclimatization and experimental periods.

[0138] Mice that had undergone a one-week acclimatization period were fed a high-fat diet to induce glucose intolerance and obesity. The high-fat diet was prepared and provided as shown in Table 10 below.

[0139] [Table 10]

[0140] Obesity-induced mice were divided into a control group (negative control group; n=14) and an experimental group (fusion protein sequence numbers 14 and 15; No. 14: n=9, No. 15: n=9), and fasted for 8 hours. Then, the control group was administered PBS, and the experimental group was administered the corresponding fusion protein at a concentration of 5.0 mg / kg. One hour later, a 20% glucose solution was administered intraperitoneally. Blood samples were collected at each time point and analyzed using a blood glucose meter.

[0141] Analysis revealed a significant difference in glucose tolerance when comparing the time-dependent blood glucose changes in the fusion protein administration group with the control group (GLUAUC) (Figure 19), indicating that the fusion protein had an effect in improving glucose tolerance.

[0142] [Leptin Receptor Dysfunction Diabetic Disease Model Study I]

[0143] To confirm the efficacy of the fusion protein in diabetic mice with impaired glucose tolerance induced by genetically induced mutations in the leptin receptor gene, a 7-week-old db / db mouse model (C57BLKS / J-db / db) was used. After one week of acclimatization, the mice were divided into a control group (n=7) and an experimental group (fusion protein SEQ ID NO: 2; n=8). The mice were fed a normal diet, and for four weeks, the control group received PBS, while the experimental group received the corresponding fusion protein at a dose of 5 mg / kg six times per week via subcutaneous injection. The efficacy was then observed.

[0144] Observations revealed that changes in body composition caused by impaired metabolism due to diabetes were improved by the administration of fusion protein (decreased adipose tissue ratio / increased muscle tissue ratio) (Table 11). Furthermore, grip strength tests confirmed a significant improvement in muscle function (Figure 20).

[0145] [Table 11]

[0146] In summary, the fusion protein of the present invention is expected to exhibit more effective material functions based on its high stability within biological systems and improved activity verified in cellular systems.

[0147] [Leptin Receptor Dysfunction Diabetic Disease Model Study II]

[0148] To confirm the efficacy of the fusion protein in diabetic mice with impaired glucose tolerance induced by genetically induced mutations in the leptin receptor gene, a 9-week-old db / db mouse model (C57BLKS / J-db / db) was used. After one week of acclimatization, the mice were divided into a control group (n=9) and an experimental group (fusion protein SEQ ID NO: 2; n=7). The mice were fed a normal diet, and for 11 weeks, the control group received PBS, while the experimental group received the corresponding fusion protein at a dose of 5 mg / kg six times per week via subcutaneous injection. The efficacy was then observed.

[0149] Observations revealed that blood glucose levels altered due to impaired glucose metabolism caused by diabetes were significantly reduced by administration of the fusion protein, confirming the efficacy of the fusion protein against glucose metabolism disorders (Figure 21).

Claims

1. Amino acid sequences selected from the group consisting of SEQ ID NOs: 8 to 10 and 46; A linker attached to the C-terminus of the aforementioned amino acid sequence; and A fusion protein comprising an Fc sequence linked to the end of the linker.

2. The fusion protein according to claim 1, wherein the linker is the sequence of SEQ ID NO: 40 or SEQ ID NO:

41.

3. The fusion protein according to claim 1, wherein the Fc sequence is the sequence of SEQ ID NO: 42 or SEQ ID NO:

43.

4. The fusion protein according to claim 1, wherein the amino acid sequence selected from the group consisting of Sequence IDs 8 to 10 and 46 has one or more amino acids selected from the group consisting of the 8th, 9th, 10th, 11th, 13th, 14th, 15th, 16th, 17th, 22nd, 23rd, 24th, 25th, 26th, 29th, 30th, 31st, 32nd, 33rd, 34th and 31st amino acids substituted with alanine.

5. The fusion protein according to claim 1, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 3, 13, 14, and 15.

6. The fusion protein according to claim 1, wherein the fusion protein has the following configuration. Amino acid sequences selected from the group consisting of SEQ ID NOs: 8 to 10 and 46; A linker attached to the C-terminus of the aforementioned amino acid sequence; A GLP-1 (Glucagon-like peptide-1) sequence consisting of sequence number 44 is linked to the end of the linker; A GLP-1 linker consisting of sequence number 45 attached to the end of the GLP-1 sequence; and Fc array connected to the end of the GLP-1 linker

7. The fusion protein according to claim 6, wherein the fusion protein consists of the sequence of Sequence ID No.

7.

8. A pharmaceutical composition for the prevention or treatment of cancer, comprising the fusion protein described in claim 1 as an active ingredient.

9. A pharmaceutical composition for the prevention or treatment of muscle loss-related muscle diseases, comprising the fusion protein described in claim 1 as an active ingredient.

10. A pharmaceutical composition for the prevention or treatment of metabolic syndromes, comprising the fusion protein described in claim 1 as an active ingredient.

11. A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising the fusion protein described in claim 1 as an active ingredient.

12. A pharmaceutical composition for preventing hair loss or promoting hair growth, comprising the fusion protein described in claim 1 as an active ingredient.

13. A pharmaceutical composition for protecting brain nerve cells, comprising the fusion protein described in claim 1 as an active ingredient.

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