A fusion protein of serum albumin and a physiologically active protein.

Abstract

The present invention aims to provide a novel fusion protein that can be used in treating adipose-related diseases. [Solution] The present invention provides a fusion protein comprising fibroblast growth factor 21 (FGF21), thioredoxin (Trx), and serum albumin (SA), wherein the FGF21 is FGF21 that does not contain a signal sequence.

Description

Technical Field

[0001] The present invention relates to a fusion protein of serum albumin and a protein having two kinds of biological activities. Specifically, it relates to a fusion protein having fibroblast growth factor 21 (FGF21), thioredoxin (Trx), and serum albumin.

Background Art

[0002] Fibroblast growth factor 21 (FGF21) is an endocrine hormone with a molecular weight of 19.5 kDa belonging to the FGF family. It is mainly expressed in adipose tissue and contributes to maintaining the homeostasis of glucose and lipid metabolism. From previous studies, it has been reported that in addition to improving glucose metabolism and insulin sensitivity, FGF21 has various effects such as reducing the level of blood triglycerides, suppressing weight gain associated with increased lipid utilization and energy consumption. Therefore, the clinical application of FGF21 is expected. However, since FGF21 is rapidly excreted outside the body by glomerular filtration after intravenous administration, its blood half-life is very short. In addition, due to its structural instability, protease degradation at the N-terminus, and lack of stability in the blood due to glycosylation, etc., it poses a problem in clinical application, and improvement of blood residence time and stability is desired.

[0003] Therefore, it has been proposed to bind polyethylene glycol to FGF21 to extend its stability and blood half-life. It has also been proposed to improve stability by preventing protease degradation by modifying the amino acid sequence of FGF21 itself. Furthermore, it has been proposed to increase the molecular size by fusing FGF21 with a high molecular weight protein such as human albumin, delay clearance in the kidney, and improve stability in the blood.

[0004] The inventors have previously reported creating a stabilized mutant FGF21 (mFGF21: △HPIP, P171G, A180E, L118C-A134C, S167A) to extend the pharmacological effects of FGF21, and then genetically fusing it with human albumin via a polypeptide linker to create a fusion compound (HSA-mFGF21) (Non-patent Literature 1: Watanabe et al, J Control Release 324, 522-531, 2020). Physicochemical analysis confirmed that the fusion compound (HSA-mFGF21) is formed from both complete HSA and mFGF21. Furthermore, pharmacokinetic studies showed that the half-life of HSA-mFGF21 was extended 20 times compared to FGF21. Furthermore, in a study using STZ-induced type 1 diabetes model mice with suppressed insulin secretion, a single intravenous administration of HSA-mFGF21 rapidly alleviated hyperglycemia, and twice-weekly administration improved adipose tissue abnormalities caused by STZ treatment and sustained blood glucose suppression.

[0005] The inventors have also administered HSA-mFGF21 to non-alcoholic fatty liver disease (NFLDA) model mice induced using two types of fatty diets (HFD and STHD) and reported the results (Non-patent Literature 2: Chikamatsu et al, J Control Release 355, 42-53, 2023). Administration of HSA-FGF21 to HFD-induced NAFLD model mice suppressed weight gain, improved blood glucose levels, improved insulin levels, and reduced accumulation of plasma and liver lipids. Furthermore, administration of HSA-FGF21 to STHD-induced NAFLD model mice suppressed plasma ALT and AST levels, oxidative stress, inflammatory cell infiltration, and fibrosis.

[0006] Thioredoxin (Trx) is an endogenous antioxidant with a molecular weight of 12 kDa that plays a crucial role in maintaining homeostasis by regulating molecular redox reactions. In addition to its antioxidant effects, Trx has anti-inflammatory effects by suppressing neutrophil migration, infiltration, and extravasation at inflammatory sites, and is also known to directly act on infiltrating macrophages to induce conversion to macrophage type M2. Furthermore, endogenous Trx has been reported to act as a negative regulator of ASK1 by suppressing its activation, exhibiting anti-inflammatory and anti-apoptotic effects. Clinically, elevated serum Trx levels have been reported in NASH patients compared to healthy controls, suggesting that Trx may exert a hepatoprotective function by increasing its expression during liver injury. In addition, Trx administration has been reported to remove reactive oxygen species (ROS) and suppress acetaminophenone (APAP)-induced liver injury. Trx, known to possess antioxidant, anti-inflammatory, and anti-apoptotic properties, is a low molecular weight protein of approximately 12 kDa. Because it is rapidly removed from the circulatory system by glomerular filtration, it exhibits a very short blood half-life.

[0007] The inventors have created and reported on an HSA-Trx fusion protein by fusing human serum albumin (HSA) with Trx (Non-Patent Literature 3: Murata R. et al., Pharmaceuticals 14, 562, 2022; Patent Literature 1: WO2022 / 196683). The inventors have also evaluated and reported on the therapeutic effect of the HSA-Trx fusion protein on a high-fat diet (HFD) induced non-alcoholic steatohepatitis (NASH) model mouse (Non-Patent Literature 4: Murata R. et al., Heliyon 10, e25485, 2024). The blood residence time of the HSA-Trx fusion protein was 10 times longer than that of Trx. Furthermore, when HSA-Trx was administered before the onset of NASH pathology, it prevented the progression to NASH by suppressing lipid accumulation, liver damage markers, and liver fibrosis.

[0008] Non-alcoholic fatty liver disease (NAFLD) is a condition in which triglycerides accumulate in the liver due to excessive energy intake. NAFLD is classified into either non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH). NASH is a progressive disease with a high risk of progressing to cirrhosis and liver cancer, and approximately 20% of NAFLD patients have NASH. Currently, NAFLD affects about 25% of the world's population, and this number is increasing further with the rise in obesity. However, there are currently no approved treatments for NAFLD / NASH, and a cure is urgently needed. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] WO2022 / 196683 [Non-patent literature]

[0010] [Non-Patent Document 1] Watanabe et al, J Control Release 324, 522-531, 2020 [Non-Patent Document 2] Chikamatsu et a, l J Control Release 355, 42-53, 2023 [Non-Patent Document 3] Murata R. et al., Pharmaceuticals 14, 562, 2022 [Non-Patent Document 4] Murata R. et al., Heliyon 10, e25485, 2024 [Overview of the project] [Problems that the invention aims to solve]

[0011] The objective of the present invention is to provide a novel fusion protein that can be used in adipose-related diseases. [Means for solving the problem]

[0012] The inventors diligently investigated novel candidate substances that could be used for adipose-related diseases and, as a result, discovered that a fusion protein containing fibroblast growth factor 21 (FGF21), thioredoxin (Trx), and serum albumin improves the blood retention of FGF21 and Trx and shows efficacy against a NAFLD mouse model exhibiting fibrosis, thus completing the present invention. This invention includes the following: [1] A fusion protein comprising fibroblast growth factor 21 (FGF21), thioredoxin (Trx), and serum albumin (SA), wherein the FGF21 is FGF21 that does not contain a signal sequence. [2] The FGF21 is an FGF21 selected from any of the following a1 to a7: a1: FGF21 having the amino acid sequence shown in Sequence ID No. 1, a2: FGF21 having the amino acid sequence shown in Sequence ID No. 2, a3: FGF21 having 90% or more identity (or 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more) with the amino acid sequence shown in Sequence ID No. 1, a4: FGF21 having the amino acid sequence shown in Sequence ID No. 1, with 1 to 20 (or 1 to 15, 1 to 10) amino acids substituted, deleted, and / or added. a5: FGF21 having 90% or more identity (or 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more) with the amino acid sequence shown in Sequence ID No. 2, FGF21 having the amino acid sequence shown, a6: FGF21 having an amino acid sequence in which 1 to 15 (or 1 to 10, 1 to 5) amino acids are substituted, deleted, and / or added to FGF21 having the amino acid sequence shown in Sequence ID No. 2, and a7: FGF21 having the amino acid sequence shown in Sequence ID No. 3, The aforementioned Trx is a Trx selected from any of the following b1 to b3, b1: Trx having the amino acid sequence shown in Sequence ID No. 4, b2: Trx having 90% or more identity (or 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) with Trx having the amino acid sequence shown in SEQ ID NO: 4, and b3: Trx having an amino acid sequence in which 1 to 10 (or 1 to 7, 1 to 5) amino acids are substituted, deleted, and / or added to the amino acid sequence shown in SEQ ID NO: 4, The aforementioned SA is an SA selected from any of the following c1 to c3. c1: HSA having the amino acid sequence shown in Sequence ID No. 5, c2: HSAs having 90% or more (or 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the HSA having the amino acid sequence shown in Sequence ID No. 5, and c3: HSAs having an amino acid sequence in which 1 to 20 (or 1 to 15, or 1 to 10) amino acids are substituted, deleted, and / or added to an HSA having the amino acid sequence shown in Sequence ID No. 5. The fusion protein described in [1] above, characterized by the above. [3] The fusion protein according to [1] or [2] above, wherein the FGF21 is a modified FGF21 having the amino acid sequence shown in Sequence ID No. 3. [4] The fusion protein according to any one of [1] to [3] above, wherein the SA is human serum albumin (HSA). [5] The fusion protein according to any one of [1] to [4] above, wherein the Trx is the Trx shown in Sequence ID No. 4. [6] The fusion protein according to [1] above, wherein FGF21 is a modified FGF21 having the amino acid sequence shown in SEQ ID NO: 3, Trx is Trx having the amino acid sequence shown in SEQ ID NO: 4, and SA is human serum albumin (HSA) having the amino acid sequence shown in SEQ ID NO: 5. [7] The fusion protein according to any one of [1] to [6] above, wherein the FGF21, Trx and SA are arranged in the order of Trx-SA-FGF21 or SA-Trx-FGF21 when viewed from the N-terminus (5'-side). [8] The fusion protein according to any one of [1] to [7] above, wherein the Trx, FGF21 and SA are bound to each other via a linker. [9] The fusion protein according to any one of [1] to [7] above, wherein the Trx, FGF21 and SA are Trx-SA-FGF21 when viewed from the N-terminus (5'-side), SA is bound to the C-terminus of the Trx via a linker, and FGF21 is bound to the C-terminus of the SA via a linker.

[10] The fusion protein according to any one of [1] to [7] above, wherein the Trx, FGF21 and SA are SA-Trx-FGF21 when viewed from the N-terminus (5'-side), Trx is bound to the C-terminus of the SA via a linker, and FGF21 is bound to the C-terminus of the Trx via a linker.

[11] The fusion protein according to any one of [8] to

[10] above, wherein the linker consists of an amino acid sequence selected from the group consisting of the following (a) to (g): (a) Gly; (b) Ser; (c) Gly-Ser; (d) Gly-Gly-Ser; (e) Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 6); (f) (Gly-Gly-Gly-Gly-Ser)2 (SEQ ID NO: 7); and, (g) (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 8).

[12] The fusion protein according to [1] above, wherein the fusion protein has the amino acid sequence shown in SEQ ID NO: 9.

[13] The fusion protein according to [1] above, wherein the fusion protein has the amino acid sequence shown in SEQ ID NO: 10.

[14] DNA containing a gene encoding the fusion protein according to any one of [1] to

[13] above.

[15] An expression vector containing the DNA described in

[14] above.

[16] The expression vector described in

[15] above, wherein the expression vector is a Pichia yeast expression system vector.

[17] A fat-reducing inhibitor containing any one of the fusion proteins described in [1] to

[13] above.

[18] A pharmaceutical composition for use in the prevention or treatment of adipose-related disease, comprising the fusion protein described in any one of [1] to

[13] above.

[19] The pharmaceutical composition according to

[18] above, characterized in that the fat-related disease is fatty liver disease, subcutaneous fat obesity, visceral fat obesity, hyperlipidemia, atherosclerosis, abdominal aortic aneurysm, coronary artery disease, macrovascular disease, cerebral infarction, or ischemic heart disease.

[20] The pharmaceutical composition according to

[19] above, wherein the fatty liver disease is non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), or liver disease due to metabolic disorder (NAFLD). [Effects of the Invention]

[0013] The present invention provides a novel fusion protein that can be used for adipose-related diseases, comprising fibroblast growth factor 21 (FGF21), thioredoxin (Trx), and serum albumin. [Brief explanation of the drawing]

[0014] [Figure 1] The figure shows the amino acid sequences of the newly discovered fusion proteins, Trx-HSA-FGF21 and HSA-Trx-FGF21. The underlined, bolded sequences represent Trx, HSA, or FGF21, while the ununderlined sequences represent the linker. [Figure 2] The figure shows the results of confirming the preparation of the fusion protein of the present invention by SDS-PAGE and Western blotting. [Figure 3] The figure shows the results of confirming the preparation of the fusion protein of the present invention, indicated by the silver line. [Figure 4] The figure shows the results of an insulin reduction assay using the fusion protein of the present invention that was prepared. [Figure 5] The figure shows the protocol for measuring adiponectin expression using 3T3-L1 cells. [Figure 6] The figure shows the results of measuring the adiponectin expression-promoting activity of the fusion protein of the present invention that was prepared. [Figure 7] The figure shows the results of measuring the GLUT1 expression-promoting activity of the fusion protein of the present invention that was prepared. [Figure 8] The figure shows the protocol for the in vivo kinetics study of the fusion protein of the present invention that was prepared. [Figure 9] The figure shows the changes in plasma levels of the administered fusion protein. [Figure 10] The figures show the results of imaging studies confirming the accumulation of the administered fusion protein in the liver. The left figure is a bright-field image, and the right figure is a fluorescence imaging image. [Figure 11] The figure shows the evaluation protocol using the choline-deficient, high-fat diet (CDAHFD) model. [Figure 12] The figure shows the change in body weight of mice in each treatment group. n=6. [Figure 13] The figure shows the weight of the liver extracted from mice in each treatment group. [Figure 14] The figure shows the measurement results of ALT and AST in plasma collected from mice in each treatment group. [Figure 15] The figure shows the measurement results of 4-hydroxyproline (OH-Pro) in the livers extracted from mice in each treatment group. [Figure 16] The figure shows the results of measuring the expression levels of TNFα, α-SAM, and Col1a2 in the livers extracted from mice in each treatment group. [Figure 17] The figure shows the results of H&E staining of liver sections extracted from mice in each treatment group. [Figure 18] The figure shows the results of Sirius Red staining of liver sections extracted from mice in each treatment group. [Modes for carrying out the invention]

[0015] The present invention will be described below, with illustrative embodiments as examples, along with preferred methods and materials that may be used in carrying out the invention, but the present invention is not limited to the embodiments described below. Unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any materials and methods equivalent to or similar to those described herein may be used in carrying out the present invention. Furthermore, all publications and patents cited herein in connection with the present invention are cited herein and constitute part of this specification, for example, as indicating methods, materials, and other matters that may be used in the present invention.

[0016] In this specification, the notation "A~B" indicating a numerical range means a numerical range that includes the endpoints A and B. Also in this specification, "approximately" means that a tolerance of ±10% is allowed.

[0017] In one embodiment, the present invention is a fusion protein comprising fibroblast growth factor 21 (FGF21), thioredoxin (Trx), and serum albumin (SA), preferably FGF21 that does not contain an N-terminal signal peptide.

[0018] In the present invention, "serum albumin" or "SA" means serum albumin from mammals other than humans, and includes, for example, serum albumin derived from mammals other than humans (e.g., monkeys, cattle, sheep, goats, horses, pigs, rabbits, dogs, cats, mice, rats, etc.). Preferably, human serum albumin (HSA) or bovine serum albumin (BSA), particularly preferably HSA. The serum albumin used in the present invention is not particularly limited as long as, when the fusion protein of the present invention containing the serum albumin is expressed in host cells, preferably yeast, the SA is not degraded and the fusion protein is secreted from the cells into the culture medium while maintaining a structure containing Trx, FGF21, and SA.

[0019] Human serum albumin (HSA) consists of 585 amino acid residues with the amino acid sequence shown in Sequence ID No. 5 (hereinafter referred to as wild-type HSA). The amino acid sequence is shown below. DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0020] It is known that there are several natural variants of HSA, and in this invention, when we refer to "human serum albumin" or "HSA," we mean these variants. For example, known natural variants include human serum albumin Redhill, human serum albumin Darakalia (J. Carlson et al., PNAS. 89(17):8225-9. 1992), and human serum albumin Casebrook (RJ Peach et al., Biochim Biophys Acta.;1097(1):49-54. 1991). Human serum albumin Redhill differs from the amino acid sequence of normal human serum albumin in that the 320th amino acid residue from the N-terminus is threonine instead of alanine, and there is an additional arginine residue at the N-terminus, and it consists of 586 amino acids. The above conversion of alanine to threonine results in the formation of the Asn-Tyr-Thr sequence in albumin redhill, where the Asn (asparagine) residues in this sequence can be N-linked glycosidized. Albumin dalacaria and albumin case blocks similarly have Asn (asparagine) residues in their sequences that can be N-linked glycosidized.

[0021] In the present invention, when we refer to "human serum albumin" or "HSA," we may include, in addition to the natural HSA described above, variants of the amino acid sequence shown in Sequence ID No. 5. Examples of variants include those in which 1 to 20, 1 to 15, or 1 to 10 amino acid residues are substituted, deleted, and / or added (in this specification, "addition" of amino acid residues means adding residues to the terminal or internal part of the sequence). Furthermore, the amino acid sequences of HSA mutants preferably show 90% or more identity with the amino acid sequence of normal wild-type HSA shown in SEQ ID NO: 5. Examples include those showing 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. Many HSA mutants have been reported, and these mutants are also included. The same can be said for variant forms of SA in non-human animal species as described above.

[0022] In the present invention, when "fibroblast growth factor 21" or "FGF21" is used, it means that FGF21 from mammals other than humans is included, for example, FGF21 derived from mammals other than humans (e.g., monkeys, cattle, sheep, goats, horses, pigs, rabbits, dogs, cats, mice, rats, etc.) is also included. Preferably, it is human-derived FGF21. The amino acid sequence of human FGF wild type contains 209 amino acid residues of NCBI reference sequence number NP_061986.1, and the sequence of mature FGF21 contains 181 amino acid residues (sequence number 1) excluding the N-terminal signal peptide. The FGF21 used in the present invention is not particularly limited as long as, when the fusion protein of the present invention containing the FGF21 is expressed in host cells, preferably yeast, the FGF21 is not degraded and the fusion protein is secreted from the cells into the culture medium while maintaining a structure containing Trx, FGF21, and SA.

[0023] One embodiment of FGF21 in the fusion protein of the present invention is FGF21 consisting of 181 amino acid residues represented by SEQ ID NO: 1, with the N-terminal signal peptide removed, and preferably, FGF21 consisting of 177 amino acid residues (SEQ ID NO: 2), with the N-terminal 4 amino acids (HPIP) of the 181 amino acid residues removed.

[0024] In the fusion of the present invention, "fibroblast growth factor 21" or "FGF21" may include, in addition to the above-described FGF21, variants of the amino acid sequence represented by SEQ ID NO: 1, consisting of 181 amino acid residues, or the amino acid sequence represented by SEQ ID NO: 2, consisting of 177 amino acid residues. The variants are not limited as long as they do not affect the activity of FGF21, but examples include variants in which 1 to 20, for example, 1 to 15, 1 to 10, or 1 to 5 amino acid residues are substituted, deleted, and / or added (in this specification, "addition" of amino acid residues means adding residues to the terminal or internal part of the sequence). For example, but not limited to this, we have created and reported a mutant FGF21 without the HPIP sequence (mFGF21: △HPIP, P171G, A180E, L118C-A134C, S167A: the amino acid numbers indicate the position in SEQ ID NO: 1 of mature FGF21) (SEQ ID NO: 3). The amino acid sequence of the mutant mFGF21 is shown below. The underlined parts indicate the mutated amino acids. DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEAACSFRELLLEDGYNVYQSEAHGLPLH C PGNKSPHRDPAPRGP C RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL A MVG G SQGRSPSY E S

[0025] Furthermore, the amino acid sequence of the FGF21 mutant preferably shows 90% or more identity with the amino acid sequence of mature FGF21 shown in SEQ ID NO: 1. Examples include sequences showing 92% or more identity, 93% or more identity, 94% or more identity, 95% or more identity, 96% or more identity, and 97% or more identity. Furthermore, the amino acid sequence of the FGF21 mutant preferably shows 90% or more identity with the amino acid sequence of HPIP-deleted mature FGF21 shown in SEQ ID NO: 2. Examples include sequences showing 92% or more identity, 93% or more identity, 94% or more identity, 95% or more identity, or 96% or more identity, and 97% or more identity.

[0026] In the present invention, when "thioredoxin" or "Trx" is used, it includes Trx derived from mammals other than humans (e.g., monkeys, cattle, sheep, goats, horses, pigs, rabbits, dogs, cats, mice, rats, etc.), but preferably it is human-derived Trx. In humans, Trx is present in the cytoplasm (Trx-1) and also in mitochondria (Trx-2), which have mitochondrial localization signals, but the Trx in the fusion protein of the present invention is preferably Trx1. Human Trx1 is a protein consisting of 105 amino acid residues having the amino acid sequence shown in SEQ ID NO: 4, and has five cysteine ​​residues, all of which are free thiol (SH) groups. The amino acid sequence is shown below. VKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV

[0027] The Trx used in the present invention is not particularly limited as long as, when the fusion protein of the present invention containing the Trx is expressed in host cells, preferably yeast, the Trx is not degraded and the fusion protein is secreted from the cells into the culture medium while maintaining a structure containing Trx, FGF21, and SA. Trx is a protein with a molecular weight of 12 kDa and an active site containing Cys-Gly-Pro-Cys. The form in which the two cysteine ​​molecules in this active site form a disulfide bond is called the oxidized form, and the form in which a dithiol is formed is called the reduced form. Trx has the function of reducing the disulfide bond of substrate proteins by converting from the reduced form to the oxidized form.

[0028] In the fusion of the present invention, "thioredoxin" or "Trx" may include, in addition to the native Trx1 described above, variants of the amino acid sequence shown in SEQ ID NO: 4. Examples of variants include those in which 1 to 10 amino acid residues, for example, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid residue are substituted, deleted, and / or added (in this specification, "addition" of an amino acid residue means adding a residue to the terminal or internal part of the sequence).

[0029] Examples of Trx variants include those that do not affect the redox activity of thioredoxin. Since the 32nd and 35th cysteine ​​residues from the N-terminus of thioredoxin are central to the redox activity, examples of variants include substitutions of amino acid residues other than these cysteine ​​residues, or amino acid residues from the 50th, 60th, or 70th positions onward, or cysteine ​​residues other than the 32nd and 35th cysteine ​​residues, with other amino acids. Examples of variants include, but are not limited to, those described in Patent Document 1 (WO2022 / 196683).

[0030] Furthermore, the amino acid sequence of the Trx mutant preferably shows 90% or more identity with the amino acid sequence of wild-type Trx1 shown in SEQ ID NO: 4. Examples include those showing 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. Thioredoxin is a protein that has Cys-Gly-Pro-Cys as its active site and has the function of reducing disulfide bonds of substrate proteins by converting from its reduced form to its oxidized form. Therefore, the above-mentioned mutants may be selected to retain this function.

[0031] The binding of thioredoxin, FGF21, and serum albumin in the fusion protein of the present invention may take any form of binding, as long as it does not affect the function and secondary structure of each protein. Specifically, the proteins may be directly bound, or bound via linkers or spacers (hereinafter collectively referred to as linkers, etc.), or two of the three proteins may be directly bound, while the other two are bound via linkers, etc. In the form where the proteins are bound via linkers, etc., the linkers, etc. that bind the proteins may be the same or different.

[0032] In the fusion protein of the present invention, the binding of Trx, FGF21, and SA may be in any order, but preferably from the N-terminus (5' terminus) it is SA-Trx-FGF21 or Trx-SA-FGF21, more preferably SA-Trx-FGF21. Even more preferably, it is HSA-Trx-FGF21 or Trx-HSA-FGF21, and most preferably a fusion protein containing HSA in the order HSA-Trx-FGF21.

[0033] In the fusion protein of the present invention, although not essential, each protein may be linked via a linker. If a linker is present, its chemical structure may be flexible as it primarily functions as a spacer. The linker may consist of one or more amino acids, for example, 1 to 40 amino acids, preferably about 7 to 20, linked by peptide bonds, but can be of any length or composition. For example, a linker consisting of 1 to 40 amino acids may be selected from 40 native amino acids. In some embodiments, the amino acids constituting the linker are selected from glycine, serine, methionine, alanine, proline, asparagine, glutamine, and lysine, but preferably selected from glycine, methionine, and serine, which have smaller residue sizes, and more preferably glycine as the basic component. However, linkers may preferably be G-rich polypeptides, such as "GGGS", "GGGGS", "GGGGGS", and multiple linked groups thereof, for example, "(GGGGS)2", "(GGGGS)3". In some other embodiments, the linker may contain multiple G-rich polypeptides, or even have alanine added to them.

[0034] The linkers that can be used in the present invention are not limited to those listed below, but the following (a) to (g) can be presented. (a) Gly; (b) Ser; (c) Gly-Ser; (d) Gly-Gly-Ser; (e)Gly-Gly-Gly-Gly-Ser(Sequence ID 6); (f)(Gly-Gly-Gly-Gly-Ser)2(Sequence ID 7); (g)(Gly-Gly-Gly-Gly-Ser)3(Sequence ID 8).

[0035] Furthermore, from the viewpoint of producing a vector for producing the fusion protein of the present invention, the amino acid sequence of the linker preferably includes restriction enzyme cleavage sites in the encoded base sequence, thereby facilitating ligation. The preparation of Trx-HSA-FGF21 is described below. In the ligation of Trx and HSA, sticky ends of restriction enzyme cleavage sites are added to the 3' end of the base sequence encoding Trx and the 5' end of the base sequence encoding HSA, and by ligating these sticky ends together, the genes encoding them can be joined. The joined sticky ends form a base sequence that encodes the amino acid sequence of the linker. The same applies to the ligation of HSA and FGF21. Specifically, when the restriction enzyme is AvaI, the base sequence (the base sequence of the restriction enzyme cleavage site of AvaI) is CCCGAG, CCCGGG, CTCGAG, or CTCGGG, and the amino acid sequence encoded by the base sequence CTCGGG is Leu-Gly.

[0036] Examples of such restriction enzyme cleavage sites include AccI, AfaI, ApaI, AvaI, AvaII, BalI, BamHI, BbeI, BcnI, BglI, BlnI, ClaI, CpoI, DraI, EaeI, EcoRI, EcoRV, FbaI, FokI, FseI, HaeI, HaeII, HaeIII, HapII, HhaI, HinfI, HpaI, KpnI, MboI, MboII, MluI, MspI, NaeI, NcoI, NotI, SacI, SacII, SalI, ScaI, SmaI, SpeI, StuI, TaqI, XbaI, and XhoI, which can be selected as appropriate. Since restriction enzyme cleavage sites directly encode a part of the linker, the base sequence encoding the linker can be designed by appropriately adding several bases to the 5' and / or 3' ends of the restriction enzyme cleavage sites. In particular, as mentioned above, it is preferable to design the enzyme to contain a large amount of glycine, methionine, and serine, preferably glycine. Specifically, if the restriction enzyme is AvaI, the base sequence CTCGGG can be selected, and by adding GG to the 5' end and one suitable base to the 3' end, a base sequence encoding Gly-Ser-Gly can be obtained.

[0037] One preferred embodiment of the fusion protein of the present invention is a fusion protein containing Trx-HSA-FGF21 in the order shown in SEQ ID NO: 9 in Figure 1. This amino acid sequence is such that the amino acid sequence of Trx (amino acid sequence of SEQ ID NO: 4) is bound to the amino acid sequence of HSA (amino acid sequence of SEQ ID NO: 5) via the amino acid sequence of a linker ([Gly-Gly-Gly-Gly-Ser]2), and the amino acid sequence of HSA is bound to the FGF21 variant (mFGF21) (amino acid sequence of SEQ ID NO: 3) via the amino acid sequence of a linker ([Gly-Gly-Gly-Gly-Ser]2). A fusion protein consisting of the amino acid sequence shown in Sequence ID No. 9 may have one or more amino acids deleted, substituted, or added in these amino acid sequences, as long as the function of the fusion protein having the amino acid sequence shown in Sequence ID No. 9 is not substantially impaired.

[0038] Another preferred embodiment of the fusion protein of the present invention is a fusion protein containing HSA-Trx-FGF21 in the order shown in SEQ ID NO: 10. This amino acid sequence is such that the amino acid sequence of HSA (amino acid sequence of SEQ ID NO: 5) is bound to the amino acid sequence of Trx (amino acid sequence of SEQ ID NO: 4) via the amino acid sequence of a linker ([Gly-Gly-Gly-Gly-Ser]2), and the amino acid sequence of Trx is bound to the FGF21 variant (mFGF21) (amino acid sequence of SEQ ID NO: 3) via the amino acid sequence of a linker ([Gly-Gly-Gly-Gly-Ser]3). A fusion protein consisting of the amino acid sequence shown in Sequence ID No. 10 may have one or more amino acids deleted, substituted, or added in these amino acid sequences, as long as the function of the fusion protein having the amino acid sequence shown in Sequence ID No. 10 is not substantially impaired.

[0039] Furthermore, for example, a polypeptide corresponding to any of the following (1) to (3) may be added to any end of the amino acid sequence of the fusion protein of the present invention. (1) A polypeptide encoded by a restriction enzyme cleavage site (or its sticky end) used in the production of the vector, (2) A polypeptide encoded by a nucleotide sequence for initiating protein synthesis and a nucleotide sequence for terminating synthesis, which are pre-provided in the vector, in order to produce a protein (3) Polypeptides used to separate and purify the produced proteins. For example, polypeptides having an amino acid sequence that can be bound to a column.

[0040] The fusion protein of the present invention can be produced by recombinant DNA methods and / or chemical synthesis methods known to the present invention. For example, it can be produced by incorporating DNA having a base sequence encoding the amino acid sequence of the fusion protein of the present invention into a vector, transforming host cells with this vector, culturing these transformed cells under conditions suitable for the expression of the fusion protein, expressing the protein, and recovering the resulting fusion protein from the cells or from the culture medium.

[0041] Any vector known in the field can be used, and can be appropriately selected considering an appropriate combination with the host cell to be used. While not limited to these, vectors can include viral vectors, plasmids, phages, or cosmids, and are not particularly limited as long as they can autonomously replicate within the introduced host. The vector may preferably contain appropriate selection markers and / or promoters, and various other expression regulators. Plasmids constructed for recombinant vector purposes are preferred as vectors due to their ease of handling. The host cell can be any cell that can stably and autonomously replicate the vector and express the recombinant protein. Examples include prokaryotic cells such as E. coli, eukaryotic cells such as yeast (e.g., Saccharomyces cerevisiae), insects, plant cells, and animal cells, but yeast is preferred. When yeast is used as the host cell, for example, a Pichia yeast expression system can be used as the vector.

[0042] Methods for introducing the vector into host cells include known methods such as electroporation, competent cell method, and conjugation method, and these methods can be used as appropriate. Appropriate conditions for the expression of the fusion protein of the present invention (e.g., culture medium, culture temperature, etc.) can be appropriately determined considering the host cells, the vector used for expression, etc.

[0043] The fusion protein of the present invention can be used, for example, as a pharmaceutical composition containing the fusion protein of the present invention as an active ingredient. For example, a pharmaceutical composition containing the fusion protein of the present invention can be administered to mammals, including humans, for the purpose of preventing or treating adipose-related diseases. Adipose-related diseases include, but are not limited to, fatty liver disease, subcutaneous fat obesity, visceral fat obesity, hyperlipidemia, atherosclerosis, abdominal aortic aneurysm, coronary artery disease, macrovascular disease, cerebral infarction, or ischemic heart disease. Preferably, a pharmaceutical composition containing the fusion protein of the present invention can be used for the treatment or prevention of non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), or liver disease due to metabolic disorders (NAFLD).

[0044] When using the fusion protein of the present invention as a pharmaceutical composition, the fusion protein of the present invention may be used as is, but preferably, a pharmaceutical composition containing the fusion protein of the present invention as an active ingredient (hereinafter sometimes referred to as the pharmaceutical composition of the present invention) is prepared and used using pharmaceutical and pharmacologically acceptable additives, excipients, etc.

[0045] The administration route of the pharmaceutical composition of the present invention is not particularly limited and may be intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, or oral, but parenteral administration routes, particularly by injection, are preferred. In one preferred embodiment, the fusion protein of the present invention is administered as a single intravenous injection, taking into consideration the maximum concentration in the plasma of the subject (patient). In one embodiment, the fusion protein of the present invention is administered in a dose of about 0.1 mg to about 100 mg per kg of body weight of the subject. In another embodiment, the fusion protein of the present invention is administered in one or more doses containing about 0.1 mg to about 100 mg per kg of body weight of the subject.

[0046] The treatment plan, including the dosage and administration schedule, can be determined by the physician who diagnosed the patient, taking into consideration the patient's symptoms, age, condition, route of administration, etc., and referring to guidelines provided by various organizations as needed.

[0047] The route of administration of the pharmaceutical composition of the present invention may be arbitrarily determined in consideration of the target symptoms, condition, and other conditions. The routes of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical composition may be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through the epithelium or the lining of the skin or mucous membrane (e.g., oral mucosa, rectum, and intestinal mucosa), and may also be administered together with other biologically active agents. Administration may be systemic or topical.

[0048] Various delivery systems are known for administering pharmaceutical active ingredients, and these can be used to administer the pharmaceutical composition of the present invention. Examples include encapsulation in liposomes, microparticles, nanoparticles, and microcapsules. In some embodiments, the pharmaceutical composition can also be delivered by a controlled-release system. In a controlled-release system, the composition is placed close to the target, and therefore only a small systemic dose is required.

[0049] When the pharmaceutical composition of the present invention is administered by injection, the injectable preparations include forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injection, or forms for drip infusion. These injectable preparations can be prepared by reference to known methods. The injectable preparations can be prepared, for example, by dissolving, suspending, or emulsifying the fusion protein of the present invention or a salt thereof in a sterile aqueous or oily medium commonly used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose, and other adjuvants, and can be used in combination with appropriate solvents such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, and can be used in combination with solvents such as benzyl benzoate and benzyl alcohol. Furthermore, the prepared injection is preferably filled into a suitable ampoule.

[0050] When the pharmaceutical composition of the present invention is an injectable preparation, it can be prepared as a ready-to-use injectable preparation or as a pre-filled syringe preparation. The pharmaceutical composition of the present invention is administered subcutaneously or intravenously using a standard needle and syringe. A pen delivery device may also be used for subcutaneous administration. The pen delivery device may be reusable or disposable.

[0051] The pharmaceutical composition is prepared in a dosage form that is adjusted to a unit dose suitable for the target dose of the active ingredient, whether for oral or parenteral administration. Dosage forms containing a unit dose include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The amount of fusion protein contained is generally preferably 0.1 mg to approximately 100 mg per dosage form in a unit dose. [Examples]

[0052] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. (Example 1) Production of a fusion protein containing Trx, FGF21, and HSA Fusion proteins containing Trx, FGF21, and HSA were prepared as follows. Two types of fusion proteins were created: a Trx-HSA-FGF21 fusion and an HSA-Trx-FGF21 fusion.

[0053] Preparation of Trx-HSA-mFGF21 and HSA-Trx-mFGF21 fragments Each fragment was prepared as follows: FGF21 underwent mutations in multiple regions (S167A, ΔHPIP, P171G, A180E, L118C-A134C) to enhance molecular stability (referred to as mFGF21). mFGF21 has the amino acid sequence shown in Sequence ID No. 3. DNA fragments containing XhoI and EcoRI restriction enzyme recognition sites at the 5' and 3' ends, respectively, of the DNA sequence encoding Trx-HSA-mFGF21 were purchased from GENEWIZ (Tokyo, Japan) and used. For HSA-Trx-mFGF21, DNA fragments containing AvaI and EcoRI restriction enzyme recognition sites at the 5' and 3' ends, respectively, of the DNA sequence encoding Trx-mFGF21 were purchased from GENEWIZ (Tokyo, Japan) and used after ligating with an HSA-containing plasmid vector.

[0054] The amino acid sequence of the constructed Trx-HSA-FGF21 is shown in Sequence ID No. 9. This amino acid sequence is formed when the amino acid sequence of Trx (amino acid sequence of Sequence ID No. 4) is linked to the amino acid sequence of HSA (amino acid sequence of Sequence ID No. 5) via the linker amino acid sequence ([Gly-Gly-Gly-Gly-Ser]2), and the amino acid sequence of HSA is linked to the FGF21 variant (mFGF21) (amino acid sequence of Sequence ID No. 3) via the linker amino acid sequence ([Gly-Gly-Gly-Gly-Ser]2). The sequence is described below. The underlined parts are, in order, the amino acid sequence of Trx, the amino acid sequence of HSA, and the amino acid sequence of mFGF21. VKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV GGGGSGGGGS DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL GGGGSGGGGS DSPLL

[0055] The amino acid sequence of the constructed HSA-Trx-FGF21 is shown in Sequence ID No. 10. This amino acid sequence is formed when the amino acid sequence of HSA (amino acid sequence of Sequence ID No. 5) is linked to the amino acid sequence of Trx (amino acid sequence of Sequence ID No. 4) via the linker amino acid sequence ([Gly-Gly-Gly-Gly-Ser]2), and the amino acid sequence of Trx is linked to the FGF21 variant (mFGF21) (amino acid sequence of Sequence ID No. 3) via the linker amino acid sequence ([Gly-Gly-Gly-Gly-Ser]3). The sequence is described below. The underlined parts are, in order, the amino acid sequence of HSA, the amino acid sequence of Trx, and the amino acid sequence of mFGF21. DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLERTYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDMPADLPSLAADFVESKDVCNKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL GGGGSGGGGS VKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV GGGGSGGGGSGGGGS DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYVNYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGGSQGRSPYES

[0056] Furthermore, as comparative compounds, a fusion protein of Trx and HSA (5'-HSA-Trx-3') (HSA-Trx) and a fusion protein of mFGF21 and HSA (5'-HSA-mFGF21-3') (HF21) were prepared as follows. HSA-Trx was linked via the linker's amino acid sequence ([Gly-Gly-Gly-Gly-Ser]2). HF21 was linked via the linker's amino acid sequence ([Gly-Gly-Gly-Gly-Ser]2). Specifically, DNA fragments with AvaI and EcoRI restriction enzyme recognition sites added to the 5' and 3' ends of the DNA sequence, respectively, were purchased from GENEWIZ (Tokyo, Japan) and ligated with a plasmid vector containing HSA to produce the desired results.

[0057] (3) Preparation and verification of plasmid DNA Fusion protein expression was performed using the Pichia Expression Kit (Invitrogen), purchased and following the manufacturer's protocol. Using a yeast expression vector (pPIC9), the two fusion fragments prepared above, the Trx-HSA-FGF21 fragment or the HSA-Trx-FGF21 fragment, were ligated to pPIC9 to create recombinant plasmids (pPIC9-fusion fragments). Cells (Pichia yeast) were transformed by electroporation and cultured in LB plates. Subsequently, the pPIC9-fusion protein was recovered using the QIAprep spin miniprep kit. The obtained DNA was cut with BamH1, and agarose gel electrophoresis was performed to confirm that the target DNA fragment had been introduced. Furthermore, the DNA was recovered from the agarose gel and sequenced to confirm that it was the target plasmid.

[0058] (4) Preparation of Trx-HSA-FGF21(THF) and HSA-Trx-FGF21(HTF) fusion proteins Large-scale culture of transformants was first performed in 5 mL test tubes using BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen source, ammonium sulfate, 4 × 10⁻⁵ biotin, 1% glycerol), followed by 1.25 L flask culture. Next, the culture medium was changed to 200 mL of BMMY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen source (amino acid-free), 4 × 10⁻⁵ -5 The mixture was changed to % biotin and 1% methanol, and methanol was used as a carbon source to induce protein expression. Cells were cultured continuously at 30°C for 3 days. In addition, methanol was added every 24 hours to maintain the induction of protein expression until the final concentration reached 1%. Yeast was precipitated by centrifugation to obtain the culture supernatant containing the fusion protein. The supernatant was then replaced with acetate buffer (pH 5.5) and applied to a Blue Sepharose 6-Fast Flow column (GE Healthcare Japan). The eluted fraction was collected using 2M KSCN in 200mM sodium acetate buffer (pH 5.5). After dialysis and concentration, the protein was purified by hydrophobic interaction chromatography using a HiTrap Phenyl HP column (GE Healthcare Japan). Confirmation was performed by SDS-PAGE, and a high-purity protein eluate was collected. The eluate was concentrated using AquaSide and then dialyzed. The results of SDS-PAGE and Western blotting are shown in Figure 2. The staining results of the final product are shown in Figure 3.

[0059] (Example 2) Activity evaluation of THF and HTF fusion protein The activity of the THF and HTF fusion proteins purified in Example 1 was measured as follows. (1) Insulin reduction assay The ability of a fusion protein to catalyze the reduction of human insulin in the presence of DTT (dithiothreitol) was tested. Protein-mediated catalysis of insulin reduction was measured by spectrophotometer at 650 nm as an increase in turbidity due to precipitation of free insulin chains at 25°C. An assay mixture containing 100 mM potassium phosphate, 2 mM EDTA (pH 7.0), 0.13 mM insulin (0.75 mg / ml), and various concentrations of the fusion protein (final concentration 5 μM) was used. The reaction was initiated by adding 5 mM DTT. The total reaction volume was 1 ml. The results are shown in Figure 4. THF showed similar insulin-reducing activity to HSA-Trx. On the other hand, HTF showed significantly greater insulin-reducing activity compared to the control, but it was weaker than HSA-Trx.

[0060] (2) Evaluation of the effect of promoting adiponectin secretion FGF21 is known to act on adipocytes to promote the secretion of adiponectin (Lin Z., et al., Cell Metab., 2013). Adiponectin is a protein secreted by adipocytes that improves insulin sensitivity, promotes lipid metabolism, and has anti-inflammatory effects. The adiponectin secretion-promoting effect of the fusion protein of the present invention was tested using 3T3-L1 cells, which are mouse embryonic fibroblasts that differentiate into adipocytes. Specifically, as shown in Figure 5, after differentiating 3T3-L1 cells into adipocytes, 50 and 100 nM of each protein were added to the culture medium, and adiponectin expression was measured at the mRNA level. The results are shown in Figure 6. Both THF and HTF, at a concentration of 100 nM, promoted adiponectin secretion, similar to HF21.

[0061] (3) Evaluation of the effect of promoting sugar uptake FGF21 is known to act on adipocytes, activating mTORC1 via MAPK and promoting glucose uptake by GLUT1 (A Minard., et al., Cell Rep., 2016). Using 3T3-L1 cells, the glucose uptake-promoting effect of the fusion protein of the present invention was evaluated by measuring GLUT1 expression. Specifically, after differentiating 3T3-L1 cells into adipocytes in the same manner as shown in Figure 5, each protein was added to the culture medium at concentrations of 50 and 100 nM, and GLUT1 expression was measured at the mRNA level. The results are shown in Figure 7. Both THF and HTF promoted GLUT1 expression at concentrations of 50 and 100 nM, similar to HF21.

[0062] (Example 3) In vivo pharmacokinetic study of THF and HTF fusion protein The in vivo dynamics of the fusion protein of the present invention were evaluated according to the protocol shown in Figure 8. THF and HTF were labeled with Cy5 and administered to mice. Cy5-labeled HSA was used as a control. Specifically, 0.3 mg each of the fluorescently labeled proteins was intravenously administered to 4-week-old male ICR mice, and blood was collected from the tail vein immediately after administration, 15 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours later. The mice were then euthanized, the livers were removed, and ImageQuant TM Imaging was performed using LAS 4000. Figure 9 shows the results of measuring plasma levels indicating residual activity in the blood, and Figure 10 shows the distribution in the liver. The blood half-lives were similar for HSA, THF, and HTF. In addition, strong fluorescence was observed after 24 hours for all fusion proteins, indicating high translocation to the liver.

[0063] (Example 4) Evaluation using a choline-deficient high-fat diet (CDAHFD) model The CDAHFD model is used as a model for non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). This model mimics the progression from fatty liver disease to NASH, including the processes of fat accumulation, inflammation, and fibrosis in the liver. The CDAHFD model can be induced by feeding a choline-deficient, high-fat diet for 4-6 weeks. The disease-preventive effect of the fusion protein of the present invention was evaluated using the CDAHFD model according to the protocol shown in Figure 11. PBS or HSA were administered as controls. Specifically, 4-week-old C57BL / 6J male mice were given a choline-deficient, high-fat diet, and simultaneously administered THF or HTF intravenously twice a week at 200 nmol / kg. After 4 weeks, blood was collected, the mice were euthanized, and their livers were removed. The weight of the removed livers was measured. Figure 12 shows the changes in the mice's body weight over 4 weeks. The control group consisted of mice fed a normal diet. Mice fed the choline-deficient, high-fat diet showed a decrease in body weight.

[0064] The excised livers were visually inspected and weighed. Visual inspection revealed that mice administered PBS or HSA had enlarged and whitish livers, while mice administered THF or HTF exhibited a color similar to the control. The results of the excised liver weights are shown in Figure 13. Liver weight was reduced in mice administered THF or HTF compared to mice administered PBS or HSA.

[0065] Blood collected from mice was centrifuged to obtain plasma. The activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of liver damage, was measured using the obtained plasma samples. The results are shown in Figure 14. Administration of the fusion protein significantly suppressed liver damage markers. Furthermore, the removed liver was evaluated for the following items. A portion of the excised liver was homogenized, hydrochloric acid was added, and the sample was left overnight at 110°C. Chloramine T and Ehnoh's reagent were then added to the sample, and the amount of 4-hydroxyproline (OH-Pro) was measured. The measurement results are shown in Figure 15. Total RNA was extracted from the liver, and the expression of the inflammatory marker TNFα and the fibrosis markers α-SAM and Col1a2 was quantified by RT-qPCR. The results are shown in Figure 16. H&E staining and Sirius Red staining were performed according to previous reports ( ). In short, liver sections were prepared to a thickness of 4 μm and observed using a BZ-X710 microscope. The results of H&E staining are shown in Figure 17, and the results of Sirius Red staining are shown in Figure 18. Administration of the fusion protein of the present invention significantly suppressed the amount of 4-hydroxyproline in the liver. Furthermore, administration of the fusion protein of the present invention significantly suppressed the expression of inflammatory markers and fibrosis markers, confirming that the fusion protein of the present invention suppresses inflammation and fibrosis. In addition, H&E staining and Sirius Red staining results confirmed that administration of the fusion protein of the present invention reduced the size of lipid droplets and showed a reduction in the fibrotic area. The results above clearly demonstrate that the fusion protein of the present invention is effective against MAFLD mice. In particular, HSA-Trx-FGF21 was shown to significantly prevent fibrosis in MAFLD mice.

[0066] The above detailed description merely illustrates the object and subject matter of the present invention and does not limit the scope of the appended claims. Various modifications and substitutions to the embodiments described without departing from the scope of the appended claims will be apparent to those skilled in the art from the teachings described herein.

[0067] This invention provides a novel fusion protein that can be used for adipose-related diseases.

Claims

1. A fusion protein comprising fibroblast growth factor 21 (FGF21), thioredoxin (Trx), and serum albumin (SA), wherein the FGF21 is FGF21 that does not contain a signal sequence.

2. The FGF21 is an FGF21 selected from any of the following a1 to a7, a1: FGF21 having the amino acid sequence shown in SEQ ID NO: 1, a2: FGF21 having the amino acid sequence shown in Sequence ID No. 2, a3: FGF21 having 90% or more identity with FGF21 having the amino acid sequence shown in Sequence ID No. 1, a4: FGF21 having an amino acid sequence in which 1 to 20 amino acids are substituted, deleted, and / or added to FGF21 having the amino acid sequence shown in SEQ ID NO: 1, a5: FGF21 having 90% or more identity with FGF21 having the amino acid sequence shown in Sequence ID No. 2, FGF21 having the amino acid sequence shown, a6: FGF21 having an amino acid sequence in which 1 to 15 amino acids are substituted, deleted and / or added to FGF21 having the amino acid sequence shown in SEQ ID NO: 2, and a7: FGF21 having the amino acid sequence shown in Sequence ID No. 3, The aforementioned Trx is a Trx selected from any of the following b1 to b3, b1: Trx having the amino acid sequence shown in Sequence ID No. 4, b2: Trx having 90% or more identity with Trx having the amino acid sequence shown in Sequence ID No. 4, and b3: A Trx having an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, and / or added to a Trx having the amino acid sequence shown in Sequence ID No. 4, The SA is selected from any of the following c1 to c3. c1: HSA having the amino acid sequence shown in Sequence ID No. 5, c2: HSA having 90% or more identity with HSA having the amino acid sequence shown in Sequence ID No. 5, and c3: HSA having an amino acid sequence in which 1 to 20 amino acids are substituted, deleted, and / or added to the amino acid sequence shown in Sequence ID No.

5. The fusion protein according to feature 1.

3. The fusion protein according to claim 1, wherein the FGF21 is a modified FGF21 having the amino acid sequence shown in SEQ ID NO:

2.

4. The fusion protein according to claim 1, wherein the SA is human serum albumin (HSA).

5. The fusion protein according to claim 1, wherein the aforementioned Trx is the Trx shown in Sequence ID No.

4.

6. The fusion protein according to claim 1, wherein the FGF21 is a modified FGF21 having the amino acid sequence shown in SEQ ID NO: 2, the Trx is a Trx having the amino acid sequence shown in SEQ ID NO: 4, and the SA is human serum albumin (HSA) having the amino acid sequence shown in SEQ ID NO:

5.

7. The fusion protein according to claim 1, wherein the FGF21, Trx, and SA are arranged in the order of Trx-SA-FGF21 or SA-Trx-FGF21 when viewed from the N-terminus (5' side).

8. The fusion protein according to claim 1, wherein the Trx, FGF21, and SA are linked to each other via a linker.

9. The fusion protein according to claim 1, wherein the Trx, FGF21, and SA are Trx-SA-FGF21 when viewed from the N-terminus (5' side), with SA bound to the C-terminus of Trx via a linker, and FGF21 bound to the C-terminus of SA via a linker.

10. The fusion protein according to claim 1, wherein the Trx, FGF21, and SA are SA-Trx-FGF21 when viewed from the N-terminus (5' side), with Trx bound to the C-terminus of SA via a linker, and FGF21 bound to the C-terminus of Trx via a linker.

11. The fusion protein according to claim 8, wherein the linker described above consists of an amino acid sequence selected from the group consisting of (a) to (g) below: (a) Gly; (b) Ser; (c) Gly-Ser; (d) Gly-Gly-Ser; (e) Gly-Gly-Gly-Gly-Ser (Sequence No. 6); (f) (Gly-Gly-Gly-Gly-Ser) ２ (Sequence ID 7); and, (g) (Gly-Gly-Gly-Gly-Ser) ３ (Sequence number 8).

12. The fusion protein according to claim 1, wherein the fusion protein is a fusion protein having the amino acid sequence shown in Sequence ID No.

9.

13. The fusion protein according to claim 1, wherein the fusion protein is a fusion protein having the amino acid sequence shown in Sequence ID No.

9.

14. DNA containing a gene encoding a protein fusion according to any one of claims 1 to 13.

15. An expression vector comprising the DNA described in claim 14.

16. The expression vector according to claim 15, wherein the expression vector is a Pichia yeast expression system vector.

17. A fat-regulating inhibitor comprising a protein fusion according to any one of claims 1 to 13.

18. A pharmaceutical composition for use in the prevention or treatment of adipose-related diseases, comprising a protein fusion according to any one of claims 1 to 13.

19. The pharmaceutical composition according to claim 18, characterized in that the fat-related disease is fatty liver disease, subcutaneous fat obesity, visceral fat obesity, hyperlipidemia, atherosclerosis, abdominal aortic aneurysm, coronary artery disease, macrovascular disease, cerebral infarction, or ischemic heart disease.

20. The pharmaceutical composition according to claim 19, wherein the fatty liver disease is non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), or liver disease due to metabolic disorder (NAFLD).

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