Dual-function protein and pharmaceutical composition containing the same

Abstract

To provide dual-function proteins comprising biologically active proteins and FGF21 mutant proteins with improved pharmacokinetic parameters, high stability, low likelihood of forming aggregate complexes, and reduced potential immunogenicity.SOLUTION: Provided is a dual-function protein produced by linking a biologically active protein and an FGF mutant protein to an Fc region of immunoglobulin. The dual-function protein of the present invention exhibits improved pharmacological efficacy, in vivo duration, and protein stability, and pharmaceutical compositions containing the dual-function protein as an active ingredient can be effectively used as therapeutic agents for diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cardiovascular disease.SELECTED DRAWING: Figure 20

Description

[Technical Field]

[0001] The present invention relates to a dual-function protein comprising a biologically active protein and a fibroblast growth factor 21 (FGF21) mutant protein, and a pharmaceutical composition containing the same. [Background technology]

[0002] Glucagon-like peptide-1 (GLP-1) is an incretin hormone consisting of 31 amino acids secreted by L cells in the intestinal tract in response to food stimulation. Its biological effects are mediated through intracellular signaling via the GLP-1 receptor, a G protein-coupled receptor expressed in target tissues such as β-cells in the pancreas and brain. GLP-1 secreted into the bloodstream has a very short half-life of less than 2 minutes, which is caused by loss of activity due to cleavage of the N-terminal amino acid by the enzyme dipeptidyl peptidase-4 (DPP-4). Because GLP-1 stimulates insulin secretion in pancreatic β-cells based on blood glucose levels, it has a strong effect of lowering blood glucose without inducing hypoglycemia. Furthermore, GLP-1 administration is known to cause weight loss in various animal models and in humans, due to reduced food intake caused by its appetite-suppressing effect. GLP-1 enhances the viability of β-cells by inducing β-cell proliferation and inhibiting cell death caused by glycosphingolipid toxicity via the GLP-1 receptor expressed in pancreatic β-cells. Excessive glucagon secretion increases blood glucose and is known to be one of the causes of hyperglycemia in diabetic patients. In addition, GLP-1 is known to act on pancreatic α-cells to inhibit the rise in fasting blood glucose by inhibiting the secretion of protein kinase A (PKA) protein-specific glucagon.

[0003] Exendin-4 is a clinically important GLP-1 receptor agonist. Exendin-4 is a polypeptide with 39 amino acid residues and is typically produced in the salivary glands of Gila Monster lizards. Exendin-4 has 52% amino acid sequence homology with GLP-1 and is known to interact with the GLP-1 receptor in mammals (Thorens et al. (1993) Diabetes 42:1678-1682). Exendin-4 has been shown to stimulate insulin secretion by insulin-producing cells in vitro, and the induction of insulin release by insulin-producing cells is stronger than that of GLP-1 under equimolar conditions. Exendin-4 strongly stimulates insulin secretion to reduce blood glucose levels in both rodents and humans with a longer duration of action than GLP-1, but because it has an unfamiliar epitope in GLP-1-deficient mammals, exendin-4 exhibits antigenicity in GLP-1-deficient mammals.

[0004] The ability of GLP-1 and exendin-4 analogs (e.g., liraglutide and Byetta) to improve glucose control in humans has been clinically confirmed. GLP-1 has been reported to increase β-cell mass through apoptosis inhibition and proliferation induction. Furthermore, GLP-1 has been reported to act as an intestinal hormone that inhibits gastric acid secretion and gastric emptying, enhances satiety signals, and thereby reduces appetite. Such effects of GLP-1 can explain the weight loss observed when GLP-1 analogs are administered to patients with type 2 diabetes. In addition, GLP-1 exhibits cardioprotective effects after ischemia in rodents.

[0005] Various attempts have been made to develop long-acting GLP-1 analogs. Clinically validated long-acting GLP-1 analogs include dulaglutide (WO 2005 / 000892) and albiglutide (WO 2003 / 059934). Dulaglutide is an Fc-fusion GLP-1 analog, and albiglutide is an albumin-fusion GLP-1 analog; both have pharmacokinetic profiles that allow for once-weekly administration. Both drugs have excellent effects in lowering blood glucose and reducing weight with once-weekly administration, and also offer significantly improved convenience in terms of administration compared to Byetta and liraglutide.

[0006] On the other hand, fibroblast growth factor 21 (FGF21), synthesized in the liver, is a hormone known to play a crucial role in glucose and lipid homeostasis. FGF21 exerts pharmacological effects in the liver, adipocytes, pancreatic β-cells, the hypothalamus of the brain, and muscle tissue where FGF21-specific receptors, i.e., both the FGF receptor and the β-Klotho complex, are expressed. In various non-human primate and mouse models of diabetes and metabolic diseases, FGF21 has been reported to lower blood glucose levels, reduce body weight, and decrease blood triglyceride and low-density lipoprotein (LDL) concentrations in an insulin-independent manner. Furthermore, due to its effect of improving insulin sensitivity, FGF21 has potential for development as a novel therapeutic agent for diabetes and obesity (see WO2003 / 011213).

[0007] Therefore, in order to develop novel antidiabetic drugs based on FGF21, attempts have been made to improve its biological activity and in vivo stability by constructing FGF21 variants based on the wild-type FGF21 sequence through several amino acid substitutions, insertions, and deletions (see WO2010 / 065439). However, it has been shown that FGF21 has a very short half-life, which poses problems when used directly as a biological agent (Kharitonenkov, A. et al. (2005) Journal of Clinical Investigation 115:1627-1635). The in vivo half-life of FGF21 is 1 to 2 hours in mice and 2.5 to 3 hours in monkeys. Therefore, daily administration is required for FGF21 to be used in its current form as a therapeutic agent for diabetes.

[0008] Various approaches have been reported in attempts to increase the in vivo half-life of recombinant FGF21 protein. One such example involves linking polyethylene glycol (PEG), an i.e., polymer material, to FGF21 to increase its molecular weight, thereby inhibiting renal excretion and increasing its in vivo retention time (see WO2012 / 066075). Another approach attempts to improve the half-life by fusing it with a fatty acid that binds to human albumin (see WO2012 / 010553). A further example attempts to increase the half-life while maintaining pharmacological activity equivalent to wild-type FGF21 by producing agonist antibodies that specifically bind to the human FGF receptor alone or in complex with β-Klotho (see WO2012 / 170438). In another example, the half-life was improved by producing a long-acting fusion protein in which the Fc region of IgG binds to the FGF21 molecule (see WO2013 / 188181).

[0009] Among the various technologies available for creating long-acting drugs, Fc fusion technology is widely used because it has fewer drawbacks than other approaches that induce immune responses or toxicity while increasing the in vivo half-life. The following conditions must be met for the development of Fc-fused FGF21 proteins as long-acting therapeutic agents.

[0010] Firstly, the reduction in in vitro activity caused by fusion should be minimized. Both the N-terminus and C-terminus of FGF21 are involved in FGF21 activity. In this regard, the activity of FGF21 fusion proteins is known to change significantly depending on the site of fusion. Therefore, the activity of Fc-fusion FGF21 fusion proteins in which mutations have been introduced into FGF21 may change depending on the presence / absence or site of fusion. Secondly, a pharmacokinetic profile that allows for once-weekly administration in humans should be achieved by increasing the in vivo half-life due to fusion. Thirdly, given that immunogenicity may be expected in a large number of patients after administration of biopharmaceuticals, the risk of immunogenicity due to fusion linkers or mutations should be minimized. Fourthly, there should be no stability issues arising from the site of fusion or the introduction of mutations. Fifthly, since undesirable immune responses may occur depending on the isotype of the fusion immunoglobulin, solutions are needed to prevent such responses.

[0011] Attempts to develop long-acting fusion proteins by ligating the Fc region of immunoglobulin G (IgG) to the FGF21 molecule have already been reported (see WO 2013 / 188181). In the case of a single Fc-FGF21 structure in which Fc is fused to the N-terminus of wild-type FGF21, there is no clear difference in in vitro activity compared to wild-type FGF21, but the half-life is known to be very short due to in vivo degradation of the protein. To address this problem, attempts have been made to improve the in vivo half-life by introducing several mutations at specific sites on FGF21 to resist proteolytic degradation. However, the risk of immunogenicity may increase with the introduction of multiple mutations. In contrast, in the case of an FGF21-Fc structure in which Fc is fused to the C-terminus of the FGF21 molecule, a significant decrease in activity caused by fusion at this site is known to exist compared to the Fc-FGF21 structure.

[0012] The combined administration of GLP-1 and FGF21 may exhibit synergistic effects compared to single administration, which depends on the mechanism of action and target tissue in the body, potentially leading to significant antidiabetic efficacy and further benefits. The effects of combined administration of GLP-1 and FGF21, or the GLP-1 / FGF21 dual-function protein, have already been investigated and reported (see WO 2010 / 142665 and WO 2011 / 020319).

[0013] Developing dual-function proteins containing GLP-1 and FGF21 requires addressing various challenges. Wild-type GLP-1 and FGF21 have very short in vivo half-lives, meaning that even if developed as therapeutic agents, they would need to be administered at least once daily. Therefore, long-acting technologies such as Fc fusion are necessary to develop long-acting dual-function proteins to improve patient convenience. In dual-function drugs targeting the two targets of GLP-1 and FGF21, introducing mutations is crucial to maintaining the activity and in vivo stability of each drug, and the resulting changes in activity, structure, or stability, along with related issues, must be addressed. The efficacy of the two targets, GLP-1 and FGF21, must be balanced, and drug design considering in vitro activity, pharmacokinetic profiles, pharmacological efficacy in animal models, and clinical efficacy evaluation in humans is necessary for this purpose. Dual-function proteins possess structures that cannot exist in the human body and are structurally more complex compared to fusion proteins targeting a single target. In addition, because mutations or linker manipulations are necessary to balance the two targets, the likelihood of aggregate complex formation may increase, potentially requiring further protein manipulation to prevent this. Furthermore, potential immunogenicity may be increased by novel mutant sequences or complex structures and should be addressed or avoided.

[0014] The inventors of the present invention have strived to improve the stability, pharmacokinetic profile, and pharmacological efficacy of dual-function proteins, including GLP-1 mutant protein and FGF21 mutant protein, and have found that the stability, pharmacokinetic profile, and pharmacological efficacy of dual-function proteins can be improved when the GLP-1 mutant protein is fused to the Fc region of immunoglobulin and a novel FGF21 mutant protein is fused to it, thereby completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0015] The object of the present invention is to provide a dual-function protein comprising a biologically active protein and an FGF21 mutant protein that has improved pharmacokinetic parameters, high stability, a low likelihood of aggregation complex formation, and reduced potential immunogenicity.

[0016] Another object of the present invention is to provide a pharmaceutical composition comprising a dual-function protein for preventing or treating FGF21-related disorders.

[0017] A further object of the present invention is to provide an isolated nucleic acid molecule encoding a dual-function protein, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector. [Means for solving the problem]

[0018] The present invention relates to a dual-function protein comprising an FGF21 mutant protein; a biologically active protein, or a mutant or fragment thereof; and an immunoglobulin Fc region, The FGF21 mutant protein contains at least one mutation selected from the group consisting of the following mutations (1)-(7): (1) Amino acid substitution in the EIRP amino acid sequence (SEQ ID NO: 68) at positions 98-101 from the N-terminus of the wild-type FGF21 protein; (2) Amino acid substitution in the TGLEAV amino acid sequence (SEQ ID NO: 69) at positions 170-174 from the N-terminus of the wild-type FGF21 protein; (3) Amino acid substitution in the TGLEAN amino acid sequence (SEQ ID NO: 70) at positions 170-174 from the N-terminus of the wild-type FGF21 protein; (4) Amino acid substitution at amino acid N at position 170 from the N-terminus of the wild-type FGF21 protein; (5) Amino acid substitution at amino acid N at position 174 from the N-terminus of the wild-type FGF21 protein; (6) together with one or more of the above mutations (1) to (5), an amino acid substitution at amino acid E at position 180 from the N-terminus of the wild-type FGF21 protein; and (7) 1 to 10 amino acid mutations for reducing the immunogenicity of the wild-type FGF21 protein, a dual-function protein is provided.

[0019] In addition, the present invention provides a pharmaceutical composition comprising the dual-function protein for treating diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or cardiovascular disease.

[0020] Further, the present invention provides an isolated nucleic acid molecule encoding the dual-function protein, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector. [Advantageous Effects of Invention]

[0021] The dual-function protein of the present invention, which is produced by linking a biologically active protein and an FGF mutant protein to the Fc region of an immunoglobulin, has improved pharmacological efficacy, in vivo duration and protein stability. In addition, the pharmaceutical composition having the dual-function protein as an active ingredient can be used as a therapeutic agent for diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or cardiovascular disease. [Brief Description of Drawings]

[0022] [Figure 1a] Figures 1A to 1C are graphs showing the in vitro activity of a fusion protein comprising an FGF21 mutant protein (hereinafter referred to as "FGF21 mutant fusion protein") using a HEK293 cell line overexpressing human β-Klotho. The FGF21 mutant fusion protein showed no significant decrease in activity due to the introduction of the mutations. [Figure 1b]Figures 1A to 1C show graphs illustrating the in vitro activity of fusion proteins containing the FGF21 mutant protein (hereinafter referred to as "FGF21 mutant fusion protein") using the HEK293 cell line overexpressing human β-Klotho. The FGF21 mutant fusion protein did not show a significant decrease in activity due to the introduction of the mutation. [Figure 1c] Figures 1A to 1C show graphs illustrating the in vitro activity of fusion proteins containing the FGF21 mutant protein (hereinafter referred to as "FGF21 mutant fusion protein") using the HEK293 cell line overexpressing human β-Klotho. The FGF21 mutant fusion protein did not show a significant decrease in activity due to the introduction of the mutation.

[0023] [Figure 2a] Figures 2A and 2B are graphs showing the in vitro activity of FGF21 mutant fusion proteins with various linkers ligating the N-terminus of FGF21 to the Fc region, using the HEK293 cell line overexpressing human β-Klotho. While slight differences in activity were observed depending on the linker sequence, the FGF21 mutant fusion proteins did not show a significant decrease in activity. [Figure 2b] Figures 2A and 2B are graphs showing the in vitro activity of FGF21 mutant fusion proteins with various linkers ligating the N-terminus of FGF21 to the Fc region, using the HEK293 cell line overexpressing human β-Klotho. While slight differences in activity were observed depending on the linker sequence, the FGF21 mutant fusion proteins did not show a significant decrease in activity.

[0024] [Figure 3] Figure 3 is a graph showing the in vitro activity of RGE(Amgen), Fc-FGF21(Lilly), and DFD1 using the HEK293 cell line overexpressing human β-Klotho. Fc-FGF21(Lilly) had twice the in vitro activity of the other proteins, while DFD1 and RGE(Amgen) had similar activity.

[0025] [Figure 4] Figure 4 shows the stability of DFD4 and DFD13 to confirm the effect of the EIRP mutation in FGF21 on the stability of the fusion protein. We confirmed that DFD13 was associated with a lower percentage of high molecular weight aggregates (HMW%) in the initial stage and at more than two weeks compared to DFD4, indicating that the introduction of the EIRP mutation improves the stability of the FGF21 mutant fusion protein, thereby significantly reducing HMW%.

[0026] [Figure 5] Figure 5 shows the blood concentrations of each protein over time, 96 hours after subcutaneous administration of the FGF21 mutant fusion protein. Data are shown as mean and standard deviation.

[0027] [Figure 6] Figure 6 shows blood glucose levels in ob / ob mouse models after a single subcutaneous injection of DFD18, DFD72, DFD74, or Fc-FGF21 (Lilly). All three, DFD18, DFD72, and DFD74, had a continuous lowering effect on blood glucose levels. Data are presented as mean and mean standard error (SEM).

[0028] [Figure 7] Figure 7 shows a graph illustrating the change in body weight in ob / ob mouse models 14 days after a single subcutaneous injection of DFD18, DFD72, DFD74, or Fc-FGF21 (Lilly). All three, DFD18, DFD72, and DFD74, had a body weight-reducing effect compared to the PBS-treated group. Data are presented as mean and standard error of the mean.

[0029] [Figure 8]Figure 8 shows a graph illustrating the changes in glycated hemoglobin levels in an ob / ob mouse model from day 1 to day 16 after a single subcutaneous injection of DFD18, DFD72, DFD74, or Fc-FGF21 (Lilly). All three, DFD18, DFD72, and DFD74, reduced glycated hemoglobin levels on day 16 compared to day 1. Data are presented as mean and standard error of the mean.

[0030] [Figure 9] Figure 9 shows blood glucose levels in an HFD / STZ mouse model after a single subcutaneous injection of DFD72 or DFD74. Both DFD72 and DFD74 had a continuous lowering effect on blood glucose levels. Data are shown as mean and standard error of the mean.

[0031] [Figure 10] Figure 10 shows the change in animal body weight in an HFD / STZ mouse model from day 14 after a single subcutaneous injection of DFD72 or DFD74. Both DFD72 and DFD74 had a body weight-reducing effect compared to the PBS-treated group. Data are shown as mean and standard error of mean.

[0032] [Figure 11] Figure 11 shows a graph illustrating the changes in glycated hemoglobin levels in an HFD / STZ mouse model on days 1 and 13 after a single subcutaneous injection of DFD72 or DFD74. Both DFD72 and DFD74 were observed to cause a significant decrease in glycated hemoglobin levels compared to the PBS-treated group. Data are presented as mean and standard error of the mean.

[0033] [Figure 12] Figure 12 shows the change in body weight measured in a diet-induced obese mouse model from the day of administration to day 14 after a single dose of DFD18. DFD18 had a significant effect on weight loss. Data are shown as mean and standard error of the mean.

[0034] [Figure 13] Figure 13 is a graph showing the in vitro GLP-1 activity of GLP-1 mutants and dual-function proteins that depend on the hinge linking the C-terminus of GLP-1 to the Fc region, using a CHO cell line overexpressing the human GLP-1 receptor. Generally, dual-function proteins containing the GLP-1(A2G) sequence (DFD23) showed 2 to 3 times lower activity than other dual-function proteins containing other GLP-1 mutant sequences. No significant differences in GLP-1 activity were observed among dual-function proteins containing mutant sequences other than the GLP-1(A2G) sequence.

[0035] [Figure 14] Figure 14 shows graphs illustrating the GLP-1 activity of DFD59, DFD69, DFD112, and DFD114, as well as the FGF21 activity of DFD69, DFD112, and DFD114. In vitro GLP-1 activity of three dual-function proteins (DFD69, DFD112, and DFD114) and an Fc-fusion GLP-1 mutant (DFD59) lacking FGF21 was measured using a CHO cell line overexpressing the human GLP-1 receptor. The three dual-function proteins showed similar EC50 values, with the Fc-fusion GLP-1 mutant (DFD59) exhibiting approximately twice the activity of the dual-function proteins. In vitro activity of the FGF21 mutant-dependent dual-function protein was measured using a HEK293 cell line overexpressing human β-Klotho. The in vitro activity of the FGF21 moiety was confirmed to be similar to that of the three dual-function proteins.

[0036] [Figure 15] Figure 15 shows the blood protein concentration versus time 240 hours after subcutaneous administration of the dual-function protein. Data are shown as mean and standard deviation.

[0037] [Figure 16]Figure 16 shows blood glucose levels in db / db mouse models after single subcutaneous injections of DFD114 or DFD59 and a combination of DFD59 and DFD74. The groups treated with dual-function proteins showed a stronger effect in lowering blood glucose levels than those treated with single-function proteins. Data are presented as mean and standard error of mean (SEM).

[0038] [Figure 17] Figure 17 shows a graph illustrating the change in body weight in db / db mouse models from day 14 after single subcutaneous injection of DFD114 or DFD59 and single subcutaneous injection of a combination of DFD59 and DFD74. The groups treated with dual-function proteins showed a stronger effect in reducing body weight than those treated with single-function proteins. Data are presented as mean and standard error of mean (SEM).

[0039] [Figure 18] Figure 18 shows graphs illustrating the changes in glycated hemoglobin levels in db / db mouse models on day 1 and day 16 after single subcutaneous injections of DFD114 or DFD59 and single subcutaneous injections of a combination of DFD59 and DFD74. The groups treated with dual-function proteins showed a stronger effect in reducing glycated hemoglobin levels than those treated with single-function proteins or combinations thereof. Data are presented as mean and standard error of the mean.

[0040] [Figure 19] Figure 19 shows blood glucose levels in HFD / STZ mouse models after single subcutaneous injections of DFD114, DFD59, DFD74, or DFD72, and single subcutaneous injections of a combination of DFD59 and DFD74. The groups treated with dual-function proteins showed a stronger effect in lowering blood glucose levels than those treated with single-function proteins. Data are presented as mean and mean standard error (SEM).

[0041] [Figure 20] Figure 20 shows the change in body weight in HFD / STZ mouse models from day 14 to day 14 after single subcutaneous injections of DFD59, DFD72, DFD74, or DFD114, and single subcutaneous injections of a combination of DFD59 and DFD74. The groups treated with dual-function proteins showed a stronger effect in reducing body weight than those treated with single-function proteins. Data are presented as mean and standard error of mean (SEM).

[0042] [Figure 21] Figure 21 shows the changes in glycated hemoglobin levels at day 1 and day 16 in HFD / STZ mouse models after single subcutaneous injections of DFD59, DFD72, DFD74, or DFD114, and single subcutaneous injections of a combination of DFD59 and DFD74. The groups treated with dual-function proteins showed a stronger effect in reducing glycated hemoglobin levels than those treated with single-function proteins or combinations thereof. Data are presented as mean and standard error of the mean. [Modes for carrying out the invention]

[0043] The present invention is described in more detail below.

[0044] In one aspect, the present invention relates to a dual-function protein comprising a fibroblast growth factor 21 (FGF21) mutant protein; a biologically active protein, or a mutant or fragment thereof; and an immunoglobulin Fc region, wherein the FGF21 mutant protein comprises at least one mutation selected from the group consisting of the following mutations (1)-(7): (1) Amino acid substitution in the EIRP amino acid sequence (SEQ ID NO: 68) at positions 98-101 from the N-terminus of the wild-type FGF21 protein (hereinafter referred to as "EIRP"); (2) Amino acid substitution of the TGLEAV amino acid sequence (SEQ ID NO: 69) at positions 170-174 from the N-terminus of the wild-type FGF21 protein (hereinafter referred to as "TGLEAV"); (3) Amino acid substitution of the TGLEAN amino acid sequence (SEQ ID NO: 70) at positions 170-174 from the N-terminus of the wild-type FGF21 protein (hereinafter referred to as "TGLEAN"); (4) Amino acid substitution at amino acid N at position 170 from the N-terminus of the wild-type FGF21 protein; (5) Amino acid substitution at amino acid N at position 174 from the N-terminus of the wild-type FGF21 protein; (6) One or more of the above mutations (1) to (5), along with an amino acid substitution at amino acid E at position 180 from the N-terminus of the wild-type FGF21 protein; and (7) 1 to 10 amino acid mutations to reduce the immunogenicity of wild-type FGF21 protein, It provides a dual-function protein.

[0045] Wild-type FGF21 protein, a hormone known to play an important role in glucose and lipid homeostasis, may be derived from mammals such as humans, mice, pigs, and monkeys, preferably from humans. More preferably, the wild-type FGF21 protein may be wild-type human FGF21 protein having the amino acid sequence shown by Sequence ID No. 1.

[0046] The mutations contained in the FGF21 mutant protein may preferably be one of the mutations EIRP, TGLEAV, TGLEAN, G170N, and G174N; a combination of one of the mutations TGLEAV, TGLEAN, G170N, and G174N and the mutation EIRP; a combination of one of the mutations EIRP, TGLEAV, TGLEAN, G170N, and G174N and the mutation A180E; or a combination of one of the mutations TGLEAV, TGLEAN, G170N, and G174N, the mutation EIRP, and the mutation A180E. Furthermore, the FGF21 mutant protein may have a structure in which 1 to 10 amino acids are deleted at the N-terminus or C-terminus compared to the wild-type FGF21 protein. More preferably, the FGF21 mutant protein may contain an amino acid sequence indicated by any one of SEQ ID NOs: 6 to 23. More preferably, the FGF21 mutant protein may further have a structure in which 1 to 10 amino acids at the N-terminus or C-terminus are deleted compared to the wild-type FGF21 protein, comprising the amino acid sequence shown by any one of SEQ ID NOs: 6 to 23.

[0047] In dual-function proteins, amino acid residue N in mutant FGF21 proteins introduced by mutation can be glycosylated.

[0048] A biologically active protein may be one selected from the group consisting of insulin, C-peptide, leptin, glucagon, gastrin, gastric suppressor polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9), oxytomodulin, oxytocin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), irisin, fibronectin type III domain-containing protein 5 (FNDC5), apelin, adiponectin, C1q and tumor necrosis factor-related proteins (CTRP family), resistin, visfatin, omentin, retinol-binding protein-4 (RBP-4), glycentin, angiopoietin, interleukin-22 (IL-22), exendin-4, and growth hormone. Preferably, the biologically active protein may be one selected from GLP-1, its variants, and exendin-4.

[0049] GLP-1 protein is a 31-amino acid incretin hormone secreted by L cells in the intestinal tract in response to stimulation from food, etc. For example, the GLP-1 protein can be represented by the amino acid sequence of SEQ ID NO: 42.

[0050] GLP-1 variants can be identified, for example, by the amino acid sequences of sequence numbers 43 to 46.

[0051] As used in this specification, the terms “Fc region,” “Fc fragment,” or “Fc” refer to a protein that includes the immunoglobulin heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3), but does not include the immunoglobulin heavy and light chain variable regions or the light chain constant region 1 (CL1). Furthermore, as used in this specification, the term “Fc region variant” refers to a variant prepared by substituting some of the amino acids in the Fc region or by combining various types of Fc regions.

[0052] The Fc region of an immunoglobulin may be the complete Fc region constituting the antibody, a fragment thereof, or an Fc region variant. Furthermore, the Fc region may contain molecules in monomeric or multimeric form and may further contain a hinge region of the heavy chain constant region. Fc region variants may be modified to prevent cleavage at the hinge region. In addition, the hinge sequence of Fc may have substitutions in several amino acid sequences to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Moreover, some amino acid sequences of the Fc hinge sequence may be substituted to inhibit rearrangement of the Fab region. Lysine residues at the C-terminus of Fc may be removed.

[0053] Preferably, the Fc region of the immunoglobulin may be a hybrid Fc which is one of the Fc regions of IgG1, IgG2, IgG3, IgG4, and IgD; or a combination thereof. Furthermore, the hybrid Fc may include the IgG4 region and the IgD region. In addition, the hybrid Fc region may include part of the hinge sequence and CH2 of the IgD Fc, and the CH2 and CH3 sequences of the IgG4 Fc.

[0054] In addition, the Fc fragment of the present invention may be in the form of a wild-type glycosylated chain, a more glycosylated chain than the wild type, a less glycosylated chain than the wild type, or a deglycosylated chain. The increase, decrease, or removal of glycosylated chains can be carried out by conventional methods known in the art, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms.

[0055] Preferably, the immunoglobulin Fc region may be indicated by an amino acid sequence selected from SEQ ID NOs: 24 to 26, 47, and 48.

[0056] A dual-function protein may include a biologically active protein, the Fc region of an immunoglobulin, and an FGF21 variant protein, linked in this order from the N-terminus to the C-terminus. Furthermore, a dual-function protein may include an FGF21 variant protein, the Fc region of an immunoglobulin, and a biologically active protein, linked in this order from the N-terminus to the C-terminus. Preferably, a dual-function protein may include a biologically active protein, the Fc region of an immunoglobulin, and an FGF21 variant protein, linked in this order from the N-terminus to the C-terminus.

[0057] Furthermore, the dual-function protein may include a GLP-1 mutant protein, an immunoglobulin Fc region, and an FGF21 mutant protein, linked in this order from the N-terminus to the C-terminus. Preferably, the dual-function protein may include a GLP-1 mutant protein, an immunoglobulin Fc region, and an FGF21 mutant protein, linked in this order from the N-terminus to the C-terminus.

[0058] Furthermore, dual-function proteins may contain additional linkers.

[0059] Dual-function proteins may be formed in which the FGF21 mutant protein is directly linked to the N-terminus or C-terminus of an immunoglobulin Fc region, or in which the FGF21 mutant protein is linked to an immunoglobulin Fc region via a linker.

[0060] In such cases, the linker may be ligated to the N-terminus, C-terminus, or free radical of the Fc fragment, or to the N-terminus, C-terminus, or free radical of the FGF21 mutant protein. When the linker is a peptide linker, ligation can occur at any region. For example, the linker may be ligated to the C-terminus of the immunoglobulin Fc region and the N-terminus of the FGF21 mutant protein, forming a fusion protein of the immunoglobulin Fc region and the FGF21 mutant protein.

[0061] Furthermore, the dual-function protein of the present invention may be in a form in which a biologically active protein is ligated to the N-terminus of the Fc region of the immunoglobulin of the fusion protein.

[0062] When the linker and Fc are expressed separately and then linked together, the linker may be a crosslinking agent known in the art. Examples of crosslinking agents may include, but are not limited to, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g., 4-azidosalicylic acid and disuccinimidyl esters, e.g., 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides, e.g., bis-N-maleimide-1,8-octane.

[0063] Furthermore, the linker may be a peptide. Preferably, the linker may be a peptide consisting of 10 to 30 amino acid residues.

[0064] Furthermore, alanine may be further bound to the end of the linker. Preferably, the linker may be a peptide having the amino acid sequence shown by any one of SEQ ID NOs: 2 to 5.

[0065] A dual-function protein may be a form in which one or more FGF21 mutant proteins are linked together, forming a dimer or polymer of FGF21 mutant proteins, which is then linked to an immunoglobulin Fc region. Furthermore, a dual-function protein may be a form of a dimer or polymer in which two or more immunoglobulin Fc regions are linked, and which contains an FGF21 mutant protein to which the immunoglobulin Fc region is linked.

[0066] Furthermore, the dual-function protein may preferably be a peptide having the amino acid sequence indicated by any one of SEQ ID NOs: 58 to 67. More preferably, the dual-function protein may be a peptide having the amino acid sequence indicated by SEQ ID NOs: 65, 66, or 67.

[0067] FGF21 mutant proteins may contain 1 to 10 further amino acid mutations to reduce the immunogenicity of the wild-type FGF21 protein. Immunogenicity can be predicted by conventional methods known in this field. For example, the possible immunogenicity of a protein can be predicted by iTope, for instance. TM and TCED TM Screening can be performed by using the method.

[0068] Furthermore, mutations to minimize immunogenicity can be designed using conventional methods known in this field. For example, EpiScreen can be used to assess the immunogenicity at which immunogenicity may occur. TM When observed through analysis, immunogenicity-inducing amino acid sequences can be identified via T-cell epitope mapping, and variants with minimized immunogenicity can be designed via computer prediction.

[0069] In another aspect, the present invention provides a pharmaceutical composition comprising a dual-function protein for treating FGF21-related disorders.

[0070] As used in this specification, the term "FGF21-related disorder" may include obesity, type 1 and type 2 diabetes, pancreatitis, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral artery disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, gastroparesis, disorders associated with severe inactivating mutations in the insulin receptor, and other metabolic disorders. Preferably, the FGF21-related disorder may be diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cardiovascular disease.

[0071] Furthermore, the pharmaceutical composition may further contain a pharmaceutical carrier. The pharmaceutical carrier can be any non-toxic substance suitable for delivering antibodies to the patient. For example, distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition. In these formulations, the concentration of the dual-function protein can vary considerably.

[0072] Specifically, a pharmaceutical composition may include formulation substances for modifying, maintaining, or preserving the pH, osmotic pressure, viscosity, transparency, color, isotonicity, aroma, sterility, stability, dissolution or release rate, or adsorption or permeability of the composition. Examples of suitable formulation substances include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfate, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), fillers (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulin), colorants, flavorings, diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), and preservatives. The ingredients may include, but are not limited to, agents (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., Pluronic; PEG; sorbitan esters; polysorbates, e.g., polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol, or tyroxapol), stability enhancers (e.g., sucrose or sorbitol), growth enhancers (e.g., alkyl metal halides, preferably sodium chloride or potassium chloride; or mannitol, sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants.

[0073] In another aspect, the present invention provides a method for preventing or treating FGF21-related disorders, comprising administering a dual-function protein to a subject requiring such prevention or treatment. The method particularly comprises administering an effective amount of the dual-function protein of the present invention to a mammal having symptoms of FGF21-related disorders, such as diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cardiovascular disease.

[0074] The pharmaceutical compositions of the present invention may be administered via any route. The compositions of the present invention may be provided to animals directly (e.g., by local administration, by injection, implantation, or local administration to a tissue area) or systemically (e.g., by oral or parenteral administration) via any suitable means. When the compositions of the present invention are provided non-enterally via intravenous, subcutaneous, intraocular, intraperitoneal, intramuscular, oral cavity, rectum, orbital, intracerebral, intracranial, intrathecal, intraventricular, intrathecal, intrasacral, intracapsular, intrajoint capsule, intranasal, or aerosol administration, the compositions may preferably be aqueous or may contain a portion of a physiologically applicable body fluid suspension or solution. Accordingly, a carrier or vehicle may be added to the composition and delivered to the patient, as long as it is physiologically applicable. Accordingly, physiologically suitable saline solutions may generally be included as carriers, such as body fluids, for the formulation.

[0075] Furthermore, the frequency of administration may vary depending on the pharmacokinetic parameters of the dual-function protein in the formulation used. Generally, the physician will administer the composition until the dose necessary to achieve the desired effect is reached. Thus, the composition may be administered as a unit dose, in at least two doses at time intervals (with or without the same amount of the target dual-function protein), or by continuous injection via an implantation device or catheter. The accuracy of additional appropriate doses may be routinely performed by those skilled in the art, and will correspond to the scope of routine practice by those skilled in the art.

[0076] Furthermore, in humans, the preferred unit dose of the dual-function protein may range from 0.01 μg / kg to 100 mg / kg body weight, more preferably from 1 μg / kg to 10 mg / kg body weight. While this is the optimal amount, the unit dose may vary depending on the disease being treated or the presence or absence of side effects. Nevertheless, the optimal dose can be determined by conducting customary experiments. The dual-function protein may be administered by periodic bolus injection, from an external reservoir (e.g., an intravenous bag), or by continuous intravenous, subcutaneous, or intraperitoneal administration from an internal source (e.g., a biocompatible implant).

[0077] In addition, the dual-function protein of the present invention may be administered to the target recipient together with other biologically active molecules. The optimal combination of the dual-function protein and other molecules, the form of administration, and the optimal dose can be determined by conventional experiments well known in the art.

[0078] In yet another aspect, the present invention provides isolated nucleic acid molecules encoding a dual-function protein.

[0079] As used in this specification, the term “isolated nucleic acid molecule” refers to a nucleic acid molecule of the present invention that is isolated from at least about 50% of the proteins, lipids, carbohydrates, or other substances naturally found when total nucleic acids are isolated from source cells; operably linked to polynucleotides that are not naturally linked; or is part of a large polynucleotide sequence that does not occur naturally. Preferably, the isolated nucleic acid molecule of the present invention is substantially free of other contaminating nucleic acids or other contaminants found in the natural environment that would interfere with polypeptide production or the use of nucleic acids in treatment, diagnosis, prevention, or research.

[0080] In such cases, isolated nucleic acid molecules encoding dual-function proteins may have different sequences from each other due to codon duplication. Furthermore, as long as the isolated nucleic acid can produce a dual-function protein, it may be appropriately modified for the desired purpose, or nucleotides may be added to the N-terminus or C-terminus of the isolated nucleic acid.

[0081] The isolated nucleic acid may contain a nucleotide sequence, for example, one of the sequence numbers 71 through 80.

[0082] In another aspect, the present invention provides an expression vector containing isolated nucleic acid molecules.

[0083] As used in this specification, the term “expression vector” refers to a vector containing a nucleic acid sequence suitable for the transformation of host cells and which directs or controls the expression of a heterologous nucleic acid sequence to be inserted. Expression vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and their analogues. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0084] As used in this specification, the terms “expression of heterologous nucleic acid sequences” or “expression” of target proteins refer to the transcription of the inserted DNA sequence, the translation of the mRNA transcript, and the production of Fc fusion protein products, antibodies, or antibody fragments.

[0085] A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. A useful expression vector may include a human cytomegalovirus (CMV) promoter to promote the continuous transcription of the target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to enhance the level of post-transcriptional RNA stability. In a typical embodiment of the present invention, the expression vector is pAD15, a modified vector of RcCMV.

[0086] In yet another aspect, the present invention provides a host cell containing an expression vector.

[0087] As used in this specification, the term “host cell” refers to a prokaryotic or eukaryotic cell to which a recombinant expression vector can be introduced. As used in this specification, the terms “transformation” or “transfect” refer to the introduction of nucleic acids (e.g., vectors) into cells by various techniques known in the art.

[0088] Suitable host cells may be transformed or transfected with the DNA sequence of the present invention and used for the expression and / or secretion of the target protein. Examples of suitable host cells that may be used in the present invention include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, CAP cells (human amniotic fluid-derived cells), and COS cells.

[0089] Typical embodiments of the present invention are described in detail below, based on examples. However, the examples of the present invention can be modified in many different forms, and the scope of the present invention should not be limited to the examples described herein.

[0090] Form of the present invention Preparation Example 1. Preparation and purification of a fusion protein containing an FGF21 mutant protein.

[0091] Preparation Example 1-1. Preparation of an expression vector for the expression of FGF21 mutant protein.

[0092] To improve the stability, activity, and pharmacokinetic profile of FGF21 in the Fc-FGF21 structure, mutation studies of FGF21 were performed.

[0093] Specifically, mutant proteins were designed for the LLLE region (amino acids at positions 98-101 from the N-terminus of the FGF21 protein), the GPSQG region (amino acids at positions 170-174 from the N-terminus of the FGF21 protein), and the A180 site, which were expected to significantly affect protein activity based on a three-dimensional structural analysis of the FGF21 protein.

[0094] The location, sequence information, target, and expected effect of each mutation introduced into the FGF21 protein are listed in Table 1 below (in Table 1, N (This indicates glycosylated asparagine (N)). Furthermore, FGF21 mutant proteins containing the mutations described in Table 1 are listed in Table 2 below.

[0095] [Table 1]

[0096] [Table 2]

[0097] The expression vector was prepared to express the amino acids of the three components—fusion carrier, linker, and FGF21 mutant—in that order from the N-terminus to the C-terminus. The substance code of each FGF21 mutant fusion protein, the sequence of the mutation introduced into FGF21, the sequence of the fusion carrier, and the linker sequence are listed in Table 3 below (in Table 3, N (This indicates glycosylated asparagine (N)).

[0098] [Table 3]

[0099] To produce the FGF21 mutant fusion proteins, the nucleotide sequences encoding each of the FGF21 mutant proteins were synthesized by consulting with Bioneer Corporation (Korea) based on the amino acid sequences of each protein. NheI and NotI restriction enzyme sequences were added to the 5' and 3' ends of the nucleotide sequences encoding each of the FGF21 mutant proteins, and a start codon for protein translation and a leader sequence (MDAMLRGLCCVLLLCGAVFVSPSHA) capable of secreting the extracellularly expressed protein were inserted at the 5' end following the restriction enzyme sequences. A stop codon was inserted after the nucleotide sequences encoding each of the FGF21 mutant fusion proteins. The nucleotide sequences encoding each of the FGF21 mutant fusion proteins were cloned into a pTrans-empty expression vector using the two restriction enzymes, NheI and NotI. A pTrans-empty expression vector with a simple structure containing a CMV promoter, a pUC-derived origin of replication, an SV40-derived origin of replication, and an ampicillin-resistance gene was purchased from CEVEC Pharmaceuticals (Germany).

[0100] In the case of the DFD6 (E. coli) and RGE (Amgen) fusion protein, the nucleotide sequences encoding each fusion protein were inserted into the pET30a expression vector for expression in E. coli.

[0101] Preparation Examples 1-2. Construction of plasmid DNA for the expression of FGF21 mutant fusion protein. To obtain a large quantity of plasmid DNA for expression, Escherichia coli was transformed with each of the expression vectors constructed in Preparation Example 1-1. E. coli cells with weakened cell walls were transformed with each expression vector via heat shock, and the transformants were placed on LB plates to obtain colonies. These colonies were seeded in LB medium and cultured at 37°C for 16 hours to obtain 100 mL of each E. coli culture containing the respective expression vector. The resulting E. coli was centrifuged to remove the culture medium, and then P1, P2, and P3 solutions (QIAGEN, Cat No.: 12963) were added to disrupt the cell walls, thereby obtaining a DNA suspension from which proteins and DNA were separated. Plasmid DNA was purified from this DNA suspension using a Qiagen DNA purification column. The eluted plasmid DNA was identified by agarose gel electrophoresis, and its concentration and purity were measured using a nanodrop device (Thermo Scientific, Nanodrop Lite). The DNA thus obtained was used for expression.

[0102] Preparation Examples 1-3. Expression of Fusion Proteins in CAP-T Cells Human cell lines were transfected with each plasmid DNA type obtained in Preparation Examples 1-2. Each plasmid DNA type was transduced into CAP-T cells (CEVEC) cultured in PEM medium (Life Technologies) using PEI solution (Polyplus, Cat. No.: 101-10N). A mixture of DNA and PEI solution was mixed with the cell suspension using Freestyle293 expression medium (Invitrogen), cultured at 37°C for 5 hours, and then PEM medium was added. After culturing at 37°C for 5-7 days, the culture was centrifuged to remove cells and obtain the supernatant containing the FGF21 mutant fusion protein.

[0103] Preparation Examples 1-4. Expression and Purification of FGF21 Mutant Fusion Protein in Escherichia coli E. coli strain BL21(DE3) was transformed with plasmid DNA expressing DFD6(E. coli) and RGE(Amgen) fusion proteins. The transformed E. coli expressing each fusion protein were seeded in 20 ml of LB medium and cultured at 37°C for 15 hours with shaking. Then, a portion of the culture medium was seeded into 100 ml of LB medium and cultured at 37°C for 16 hours with shaking. After culturing was complete, the culture was centrifuged to obtain an E. coli pellet, and then the cells were destroyed using a high-pressure cell disruptor to obtain inclusion bodies.

[0104] The obtained inclusion bodies were washed and eluted, and then purified by protein refolding. Specifically, the obtained inclusion bodies were washed 2-3 times with a buffer solution (pH 8.0) containing 0.5% Triton X-100, 50 mM Tris, 1 mM EDTA, and 0.1 M NaCl to isolate the bacterial proteins, which were then resuspended in 8 M urea buffer containing 8 M urea, 50 mM Tris, and 1 mM DTT. Since the proteins in the 8 M urea buffer were completely denatured, protein refolding was performed as follows.

[0105] First, urea was removed by gradually diluting 8M urea buffer with 20mM glycine buffer solution (pH 9.0). CuSO4 was then added to a concentration of 2M, increasing to 80μM, to induce stable protein folding. The refolded protein was suspended in PBS buffer solution (pH 7.4), filtered through a 0.22μm filter to remove impurities, and then loaded onto a Protein A affinity chromatography column. The column was washed with 1X PBS buffer solution (pH 7.4), and the protein was then eluted using 100mM glycine buffer solution (pH 3.0) to prepare the DFD6 (E. coli) fusion protein.

[0106] For RGE(Amgen) fusion proteins, the refolded proteins were suspended in 50 mM Tris buffer solution (pH 8.0), filtered through a 0.22 μm filter to remove impurities, and then loaded onto an anion exchange resin column (POROS® HQ 50 μm, Thermo Fisher Scientific). The column was washed with 50 mM Tris buffer solution (pH 8.0), and then 50 mM Tris buffer solution (pH 8.0) was administered along a concentration gradient to elute the RGE(Amgen) fusion proteins. The RGE(Amgen) fusion proteins obtained by the anion exchange resin were mixed with ammonium sulfate to a concentration of 1 M and then purified using a hydrophobic interaction chromatography column (Phenyl sepharose FF, GE Healthcare). Specifically, the column was washed with a 50 mM Tris buffer solution (pH 8.0) containing 1 M ammonium sulfate, and a 50 mM Tris buffer solution (pH 8.0) was administered along a concentration gradient. The eluted fraction was analyzed via 10% Tris-glycine gel electrophoresis. The gel was stained with Coomassie Brilliant Blue R while gently shaking, and the fraction containing high-purity FGF21 mutant fusion protein was collected. This fraction was then dialyzed overnight at 4°C using the final buffer solution (1X PBS, 1 mM EDTA, pH 7.4). After dialyzing, the resulting protein storage solution was concentrated at 3,000 rpm using a 30,000 MW cutoff centrifuge filter at 4°C. The concentration of FGF21 mutant fusion protein was measured via BCA quantitative analysis.

[0107] Preparation Examples 1-5. Purification of FGF21 mutant fusion protein A Protein A affinity chromatography column (GE Healthcare) was equilibrated with 1X PBS buffer (pH 7.4). The culture supernatants containing each FGF21 mutant fusion protein obtained in Preparation Examples 1-3 were filtered through a 0.2 μm filter and then loaded onto the Protein A affinity chromatography column. The column was washed with 1X PBS buffer (pH 7.4), and the proteins were eluted using 100 mM glycine buffer (pH 3.0). The fusion proteins obtained by affinity chromatography were purified using an anion exchange resin column (POROS® HQ 50 μm, Thermo Fisher Scientific). Before the FGF21 mutant fusion proteins were eluted from the column, the anion exchange resin column was equilibrated with 50 mM Tris buffer (pH 8.0). Specifically, after washing the column with 50 mM Tris buffer (pH 8.0), the 50 mM Tris buffer (pH 8.0) was distributed along a concentration gradient, and the eluted fractions were analyzed. Each eluted fraction was analyzed using size exclusion chromatography (SEC-HPLC) to recover the fraction containing the FGF21 mutant fusion protein with high purity. Concentration and quantitative analysis were performed according to the methods described in Preparation Examples 1-4.

[0108] Experimental Example 1. In vitro activity of fusion protein Experimental Example 1-1. Effect of FGF21 mutation on protein activity The in vitro activity of the fusion proteins DFD4, DFD5, DFD6, DFD6 (E. coli), DFD7, DFD9, DFD13, DFD18, DFD72, DFD73, and DFD74 prepared in Preparation Example 1 was measured.

[0109] Specifically, the in vitro FGF21 activity of the fusion protein was evaluated using HEK293 cell lines (Yuhan Corporation, Korea) modified to overexpress human β-Klotho, a co-receptor of FGF21. For activity evaluation, concentrates containing the fusion proteins prepared in Preparation Examples 1-4 and 1-5 were subjected to 3-fold serial dilutions at a concentration of 3 μM. After culturing in a serum-deficient state for 5 hours, the cell lines overexpressing human β-Klotho were treated with the diluted fusion protein for 20 minutes, and then lysed by adding cell lysis buffer (Cisbio / Cat# 64ERKPEG) while stirring at 60 rpm for 30 minutes at room temperature. The cell lysate solution was mixed with an antibody (Cisbio / Cat# 64ERKPEG) capable of detecting extracellular signal-regulated kinase (ERK) and phosphorylated ERK, and the mixture was maintained at room temperature for 2 hours. Fluorescence was detected using a fluorescence detector (TECAN / GENiosPro). The activity of the fusion protein was evaluated using EC 50 The measurement was performed by comparing the values.

[0110] As shown in Figures 1A to 1C, the in vitro activity of the fusion proteins prepared by introducing the mutant sequence into the wild-type FGF21 protein was not inhibited, confirming that the activity of each fusion protein was similar to that of the others. It was also confirmed that the in vitro activity of the fusion proteins prepared by introducing the N-glycosylation mutation into the wild-type FGF21 protein was not inhibited via DFD6 (Es. coli) samples expressed in E. coli and DFD6 samples expressed in animal cells.

[0111] Experimental Example 1-2. Effect of linker sequence on protein activity The in vitro activity of the fusion proteins DFD1, DFD3, DFD4, and DFD13 prepared in Preparation Example 1 was measured.

[0112] Specifically, the FGF21 activity of the fusion protein was measured using concentrates containing the fusion protein prepared in Preparation Example 1-5 according to the method described in Experimental Example 1-1. The results are shown in Figures 2A and 2B.

[0113] While slight differences in activity were observed depending on the linker sequence, as shown in Figures 2A and 2B, we confirmed that the FGF21 mutant fusion protein did not show a significant decrease in activity.

[0114] Experimental Examples 1-3. Experimental results for DFD1, RGE (Amgen), and Fc-FGF21 (Lilly) The in vitro activity of the fusion protein DFD1 prepared in Preparation Example 1, as well as the control proteins RGE(Amgen) and Fc-FGF21(Lilly), was measured.

[0115] Specifically, the FGF21 activity of the fusion protein was measured using a concentrate containing the fusion protein and control protein prepared in Preparation Example 1-5, according to the method described in Experimental Example 1-1. The results are shown in Figure 3.

[0116] As shown in Figure 3, Fc-FGF21(Lilly) exhibited twice the in vitro activity compared to other proteins, while DFD1 and RGE(Amgen) showed similar in vitro activity.

[0117] Experimental Example 2. Evaluation of the stability of fusion proteins Experimental Example 2-1. Experimental Method for Evaluating Stability To measure the amount of protein aggregates in the initial stages of sample preparation, high molecular weight aggregates (%HMW) were quantified using size exclusion chromatography (SEC-HPLC). The results are shown in Figure 4.

[0118] Specifically, the TosoHaas model TSK-GEL G3000SW XLThe column was used for the SEC-HPLC method. The column was equilibrated by flowing a buffer solution (1X PBS, 1 mM EDTA, pH 7.4) at a flow rate of 1 mL / min. The DFD4 and DFD13 protein storage solutions prepared in Preparation Examples 1-5 were concentrated to a target concentration of 20 mg / mL or higher at 3,000 rpm using a 30,000 MW cutoff centrifuge filter at 4°C. After measuring the concentration of each sample by BCA quantitative analysis, the samples were diluted to a final concentration of 20 mg / mL with a buffer solution (1X PBS, 1 mM EDTA, pH 7.4). To measure the initial %HMW of DFD4 and DFD13, the 20 mg / mL samples were diluted to a final concentration of 1 mg / mL with a buffer solution (1X PBS, 1 mM EDTA, pH 7.4), and each sample in a 100 μL volume was analyzed by the SEC-HPLC column.

[0119] For the stability assessment of each sample, the %HMW of the sample was measured using the SEC-HPLC method on days 4, 8, and 14 while the samples were stored at 5°C, 25°C, and 37°C for two weeks.

[0120] As shown in Figure 4, we confirmed that DFD13 had a lower amount of high molecular weight aggregates (HMW%) compared to DFD4 in the initial stages and at up to 2 weeks, demonstrating that the introduction of the EIRP mutation improved the stability of the FGF21 mutant fusion protein, thereby significantly reducing the HMW%.

[0121] Experimental Example 2-2. Stability Results To investigate the effect of the EIRP mutation introduced into the original FGF21 sequence LLLE(98-101) on stability, the stability of DFD4 (SEQ ID NO: 29) and DFD13 (SEQ ID NO: 35) was measured according to the method described in Experimental Example 2-1. The analytical results for zero-time samples (initial stage; day 0) and samples stored at 4, 8, and 14 days are summarized in Table 4 below (in Table 4, ND means "not detected").

[0122] [Table 4]

[0123] As shown in Table 4, the %HMW levels at the initial stage (day 0) were 0.91% for DFD4 and 0.56% for DFD13. After two weeks, under storage conditions at 25°C, the %HMW level increased to 8.83% for DFD4, but was not observed in DFD13. This indicates that DFD13 had a lower %HMW rate compared to DFD4 at both the initial stage and at two weeks, suggesting that the %HMW rate of the FGF21 mutant fusion protein was significantly reduced by the introduction of the EIRP mutation.

[0124] Experimental Example 3. Pharmacokinetic Evaluation of Fusion Proteins Experimental Example 3-1. Experimental Method for Pharmacokinetic Evaluation Six-week-old male ICR mice purchased from Orient BIO (Korea) were divided into groups (n=3 / at blood sampling time) to have similar mean values ​​relative to body weight one day prior to drug treatment, and each sample was administered subcutaneously once at 1 mg / kg (2 mg / kg relative to RGE). Blood samples were then collected at 1, 4, 8, 12, 24, 48, 72, and 96 hours post-injection. The concentration of intact full-length FGF21 protein in the blood was measured using the Intact human FGF21 ELISA kit (F1231-K01, Eagle Biosciences, USA), which involves an immune response to the N-terminus and C-terminus of the FGF21 protein. The concentrations of samples in blood collected up to 96 hours after subcutaneous injection of each fusion protein into the mice were measured, and the pharmacokinetic parameters of each sample were calculated.

[0125] Experimental Example 3-2. Evaluation of pharmacokinetic activity Pharmacokinetic parameters were calculated based on graphs showing the concentration of each protein in the blood versus time after subcutaneous administration of the fusion protein in mice (Figure 5). The data are shown in Table 5 below.

[0126] [Table 5]

[0127] The pharmacokinetic profiles of each fusion protein were compared and evaluated based on the area under the curve (AUC), which indicates the degree of drug exposure.

[0128] As shown in Table 5, comparing DFD4 with DFD13 and DFD6 with DFD73, we determined that the introduction of the EIRP sequence resulted in an increase of approximately 10 to 20% in AUC. Comparing DFD9 with DFD4, the introduction of TGLEAV resulted in an increase of approximately 6 times in AUC.

[0129] Furthermore, the TGLEAN, G170N, and G174N mutations were designed to extend the half-life of FGF21 by introducing N-glycosylation to the C-terminus, which is known to be proteolytically degraded in vivo. The increase in AUC due to the introduction of N-glycosylation was confirmed by comparing the mutants with each control substance. To confirm the effect of N-glycosylation on improving AUC, the AUC value of DFD6 (Escherichia coli) produced by E. coli without glycosylation was compared with DFD6 produced by human cell lines. DFD6 produced by human cell lines showed a three-fold or more increase in AUC value compared to DFD6 (Escherichia coli) produced by E. coli, demonstrating the improvement in the pharmacokinetic profile due to glycosylation.

[0130] A180E is a mutation described in WO 2009 / 149171, owned by Amgen Inc. When the A180E mutation was further introduced into mutants DFD13 or DFD73, which contain the TGLEAV or G170N mutations respectively, the resulting mutants DFD18 or DFD74 each showed a further increase of approximately 2 to 3 times in AUC value.

[0131] In summary, we confirmed that pharmacokinetic parameters were improved by introducing various mutations and combinations thereof compared to the wild-type FGF21 fusion protein, DFD9. The fusion protein showing the most improved AUC value was DFD74, which included the EIRP, G170N, and A180E mutations, showing an approximately 45-fold improvement in AUC value compared to DFD9. Furthermore, considering RGE(Amgen) at a dose of 2 mg / kg body weight, DFD74 may have a higher degree of drug exposure compared to RGE. The overall effect of the pharmacokinetic improvements due to mutations is summarized in Table 6 below.

[0132] [Table 6]

[0133] Experimental Example 4. Evaluation of fusion protein activity in ob / ob mice Experimental Example 4-1. Experimental method for evaluating activity in ob / ob mice Ob / ob mice, characterized by genetic leptin deficiency and exhibiting hyperglycemia, insulin resistance, hyperphagia, fatty liver, and obesity, are widely used in the study of type 2 diabetes. Male ob / ob mice (Harlan, USA) were purchased from Raonbio (Korea). These mice were 5 to 6 weeks old upon arrival and 8 to 9 weeks old at drug treatment, 3 weeks after adaptation. The mice were divided into groups (n=8 / group) to have similar mean values ​​for body weight and tail blood glucose levels one day before drug treatment (day 0), and samples were administered subcutaneously once according to each of these respective doses. Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) was administered as the vehicle treatment, and blood glucose concentrations were measured using a GlucoDr glucose meter (All Medicus, Korea). Non-fasting glucose levels and body weight were measured daily until day 14 after administration. Glycated hemoglobin levels were also measured in each group before administration and after the study. Glycated hemoglobin levels were calculated using the DCA 2000 HbA1c kit (Siemens, 5035C).

[0134] Experimental Example 4-2. Evaluation of activity in ob / ob mice Changes in non-fasting blood glucose levels and body weight in male ob / ob mice were observed after a single subcutaneous injection of 30 or 100 nmol / kg of DFD18 and DFD72, or 10, 30, or 100 nmol / kg of DFD74.

[0135] We confirmed that all three drugs, DFD18, DFD72, and DFD74, had a dose-dependent effect in lowering blood glucose levels. When comparing the three drugs at a high dose of 100 nmol / kg, DFD72 and DFD74 showed improved effects in lowering blood glucose levels compared to DFD18 (Figure 6). In addition, Fc-FGF21 (Lilly), which was used as a control substance in the study, was less effective in lowering blood glucose levels compared to DFD18, DFD72, and DFD74 at the same dose level (30 nmol / kg).

[0136] Regarding the effect of weight loss, when comparing the three drugs at a high dose of 100 nmol / kg, DFD72 was the most effective in ob / ob mice, causing a weight loss of approximately 6%, followed by DFD18, and then DFD74 (Figure 7).

[0137] After the study, glycated hemoglobin levels, which indicate the mean blood glucose level, were measured, and the change in mean blood glucose was analyzed in each study group. With the exception of the control group treated with the control protein Fc-FGF21 (Lilly), all treatment groups showed negative values ​​in the difference between pre- and post-study periods, confirming the effectiveness of the test protein compared to the control substance in lowering blood glucose (Figure 8).

[0138] Experimental Example 5. Evaluation of fusion protein activity in HFD / STZ mice Experimental Example 5-1. Experimental method for evaluating activity in HFD / STZ mice The effects of an FGF21 mutant fusion protein in lowering blood glucose and body weight were evaluated in comparison to another diabetes model, the HFD / STZ mouse model. Conventional diet-induced obesity mouse models (induced by feeding C57BL / 6 mice a 60 kcal% high-fat diet for 8 weeks or more) induce insulin resistance but have weak hyperglycemia and diabetic characteristics. HFD / STZ mice, which can compensate for the shortcomings of conventional diet-induced obesity mouse models, can produce dysfunctional β-cells in the pancreas and reduce insulin secretion as a result of administration of a high-fat diet (HFD) and low levels of streptozotocin (STZ), and are therefore useful for pharmacological testing of type 2 diabetes.

[0139] Specifically, to induce an HFD / STZ mouse model, C57BL / 6 mice (Japan SLC) were fed a 60 kcal% high-fat diet for 4 weeks, followed by daily intraperitoneal administration of 50 mg / kg of STZ (Sigma, 85882) for 3 days to induce dysfunction in pancreatic β-cells. After a further 2 weeks of high-fat dieting, mice with non-fasting blood glucose levels of 200 mg / dL or higher were used for the study. Mice were divided into groups (n=6 / group) to have similar mean values ​​for body weight and tail blood glucose levels one day before drug treatment (day 0), and samples were administered subcutaneously once according to each of these respective doses. Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) was administered as a vehicle treatment, and blood glucose concentrations were measured using a GlucoDr glucose meter (All Medicus, Korea). Non-fasting glucose levels and body weight were measured daily until day 14 after administration. Glycated hemoglobin levels were also measured in each group before administration and after the study. Glycated hemoglobin levels were calculated using the DCA 2000 HbA1c kit (Siemens, 5035C).

[0140] Experimental Example 5-2. Evaluation of activity in HFD / STZ mice Changes in non-fasting blood glucose levels and body weight in male HFD / STZ mice were observed after a single subcutaneous injection of 10 nmol / kg of DFD72 or DFD74.

[0141] Regarding changes in non-fasting blood glucose levels, DFD72 and DFD74 had similar effects in lowering blood glucose levels, maintaining this blood glucose-lowering effect until day 10 after administration, after which it was lost due to drug metabolism (Figure 9). DFD72 showed a longer-lasting effect than DFD74 in terms of changes in non-fasting blood glucose levels 10 days after administration.

[0142] Regarding the effect of administering FGF21 mutant proteins on weight reduction, both DFD72 and DFD74 had a similar effect of reducing body weight by approximately 5%, and the effect disappeared 10 days after administration (Figure 10).

[0143] After the trial, glycated hemoglobin levels, which indicate the mean blood glucose level, were measured, and the change in mean blood glucose was analyzed for each trial group. The vehicle group showed an increase of 0.25 in glycated hemoglobin levels, the group treated with DFD74 showed an increase of 0.1, and the group treated with DFD72 showed a decrease of 0.27 (Figure 11).

[0144] Experimental Example 6. Activity of fusion proteins in diet-induced obese mice. Experimental Example 6-1. Experimental method for evaluating activity in diet-induced obese mice The weight-reducing effect of DFD18, an FGF21 mutant fusion protein, was evaluated in diet-induced obese mice. For the diet-induced obesity model, C57BL / 6J mice were purchased from Central Lab. Animal Inc. and fed a high-fat diet (Research diet) containing 60 kcal% fat for 8 to 12 weeks. Mice were divided into groups (n=8 / group) to have similar mean body weights one day before drug treatment (day 0), and then administered a single subcutaneous dose of 30 nmol / kg. Changes in body weight were compared to a group treated with a vehicle (PBS).

[0145] Experimental Example 6-2. Protein activity in diet-induced obese mice In a diet-induced obese mouse model, the change in body weight over time after a single dose of 30 nmol / kg of DFD18 was observed. The weight-reducing effect continued until day 10 post-administration, with the maximum weight loss (approximately 18%) occurring on day 11, which was maintained until day 14 (Figure 12).

[0146] Preparation Example 2. Preparation and Purification of Dual-Functional Proteins Preparation Example 2-1. Preparation of an expression vector for the expression of a dual-function protein. To identify the effects of GLP-1 mutant protein sequences and the sequences of the Fc hinges fused to them on in vitro activity, pharmacokinetic profiles, and pharmacological efficacy, various sequences for Fc-fused GLP-1 mutant proteins were designed. The sequences of GLP-1 mutant proteins are listed in Table 7 below, and the sequences of Fc-fused GLP-1 mutants are listed in Table 8.

[0147] [Table 7]

[0148] [Table 8]

[0149] In Table 8, HyFc5 is represented by sequence number 47, and HyFc40 is represented by sequence number 48.

[0150] To investigate the effects of the sequences of GLP-1 mutant proteins and FGF21 mutant proteins, the sequence of the Fc hinge fused to the GLP-1 mutant, and the sequence of the linker linked between the Fc regions of the FGF21 mutant protein on in vitro activity, pharmacokinetic profile, and pharmacological efficacy, various sequences were designed for dual-function proteins. The sequences of dual-function proteins, including GLP-1 mutant proteins and FGF21 mutant proteins, are listed in Table 9 below. Each dual-function protein contains a GLP-1 mutant protein, an immunoglobulin Fc region, a linker, and an FGF21 mutant protein, linked in this order from the N-terminus to the C-terminus.

[0151] [Table 9]

[0152] Specifically, the nucleotide sequences encoding each of the dual-function proteins were synthesized after consulting with Bioneer Corporation (Korea) based on the amino acid sequences of each protein. NheI and NotI restriction enzyme sequences were added to the 5' and 3' ends of the nucleotide sequences encoding each of the dual-function proteins, and a start codon for protein translation and a leader sequence (MDAMLRGLCCVLLLCGAVFVSPSHA) capable of secreting the extracellularly expressed protein were inserted at the 5' end following the restriction enzyme sequences. A stop codon was inserted after the nucleotide sequences encoding each of the dual-function proteins. The nucleotide sequences encoding each of the dual-function proteins were cloned into a pTrans-empty expression vector using the two restriction enzymes, NheI and NotI. A pTrans-empty expression vector with a simple structure containing a CMV promoter, pUC-derived origin of replication, SV40-derived origin of replication, and an ampicillin-resistance gene was purchased from CEVEC Pharmaceuticals (Germany).

[0153] Preparation Example 2-2. Construction of plasmid DNA for the expression of Fc-fusion GLP-1 mutants and dual-function proteins. To obtain a large quantity of plasmid DNA for expression, Escherichia coli was transformed with each of the expression vectors constructed in Preparation Example 2-1. E. coli cells with weakened cell walls, obtained by heat shock, were transformed with each expression vector, and the transformants were placed on LB plates to obtain colonies. These colonies were seeded in LB medium and cultured at 37°C for 16 hours to obtain 100 mL of each E. coli culture containing the respective expression vector. The resulting E. coli was centrifuged to remove the culture medium, and then P1, P2, and P3 solutions (QIAGEN, Cat No.: 12963) were added to disrupt the cell walls, thereby obtaining a DNA suspension from which proteins and DNA were separated. Plasmid DNA was purified from this DNA suspension using a Qiagen DNA purification column. The eluted plasmid DNA was identified by agarose gel electrophoresis, and its concentration and purity were measured using a nanodrop device (Thermo Scientific, Nanodrop Lite). The DNA thus obtained was used for expression.

[0154] Preparation Example 2-3. Expression of Fc-fusion GLP-1 mutants and dual-function proteins in CAP-T cells Human cell lines were transformed with each plasmid DNA obtained in Preparation Example 2-2. Each plasmid DNA type was transduced into CAP-T cells (CEVEC) cultured in PEM medium (Life Technologies) using PEI solution (Polyplus, Cat. No.: 101-10N). A mixture of DNA and PEI solution was mixed with the cell suspension using Freestyle293 expression medium (Invitrogen), cultured at 37°C for 5 hours, and then PEM medium was added. After culturing at 37°C for 5-7 days, the culture was centrifuged to remove cells and obtain the supernatant containing each protein.

[0155] Preparation Examples 2-4. Purification of Fc-fusion GLP-1 mutants and dual-function proteins. A Protein A affinity chromatography column (GE Healthcare) was equilibrated with 1X PBS buffer (pH 7.4). The culture supernatants containing the Fc-fusion GLP-1 mutant and dual-function protein obtained in Preparation Example 2-3 were filtered through a 0.2 μm filter and then loaded onto the Protein A affinity chromatography column. The column was washed with 1X PBS buffer (pH 7.4), and the proteins were eluted using 100 mM glycine buffer (pH 3.0). The proteins obtained by affinity chromatography were purified using an anion exchange resin column (POROS® HQ 50 μm, Thermo Fisher Scientific). Before loading the proteins eluted from affinity chromatography, the anion exchange resin column was equilibrated with 50 mM Tris buffer (pH 8.0).

[0156] After washing the column with 50 mM Tris buffer solution (pH 8.0), the eluted fractions were analyzed by distributing 50 mM Tris buffer solution (pH 8.0) along a concentration gradient. Each eluted fraction was analyzed using size exclusion chromatography (SEC-HPLC) to recover fractions containing high purity Fc-fusion GLP-1 mutants and dual-function proteins, and dialyzed overnight at 4°C using final buffer solution (1X PBS, 1 mM EDTA, pH 7.4). After dialyzing, the resulting protein storage solution was concentrated at 3,000 rpm using a 30,000 MW cutoff centrifuge filter at 4°C. The concentration of each protein was measured via BCA quantitative analysis.

[0157] Experimental Example 7. In vitro activity of dual-function proteins Experimental Example 7-1. Activity of DFD23, DFD24, DFD25, DFD26, DFD27, DFD28, and DFD29 The in vitro GLP-1 activity of the dual-function proteins DFD23, DFD24, DFD25, DFD26, DFD27, DFD28, and DFD29 was measured. Specifically, a CHO cell line overexpressing the human GLP-1 receptor (Eurofins, HTS163C2) was purchased and used to evaluate the GLP-1 activity of the dual-function proteins. For activity evaluation, samples containing the fusion proteins (protein storage solutions prepared in Preparation Example 2-4; hereinafter referred to as "samples") were subjected to 4-fold serial dilutions at a concentration of 25 nM. After treating the CHO cell line overexpressing the human GLP-1 receptor for 30 minutes, the intracellular cAMP produced was measured (Cisbio, 62AM4PEB). The activity of each protein was measured using EC2. 50 The measurement was performed by comparing the values.

[0158] As shown in Figure 13, dual-function proteins containing the GLP-1(A2G) sequence exhibited approximately 2–3 times lower activity than dual-function proteins containing other GLP-1 mutant sequences. No significant differences in GLP-1 activity were observed among dual-function proteins containing mutant sequences, except for the GLP-1(A2G) sequence.

[0159] Experimental Example 7-2. Activity of DFD59, DFD69, DFD112, and DFD114 In vitro GLP-1 activity was measured for the dual-function proteins DFD69, DFD112, DFD114, and DFD59 (Fc-fusion GLP-1 mutants) prepared in Preparation Example 2. Specifically, a CHO cell line (Eurofins, HTS163C2) overexpressing the human GLP-1 receptor was purchased and used to evaluate the GLP-1 activity of the dual-function proteins. For activity evaluation, samples containing each fusion protein were subjected to 4-fold serial dilutions at a concentration of 25 nM. After treating the CHO cell line overexpressing the human GLP-1 receptor for 30 minutes, the intracellular cAMP produced was measured (Cisbio, 62AM4PEB).

[0160] As shown in Figure 14, the activity of each protein was measured by EC 50Evaluation was performed by comparing the values. The three dual-function proteins exhibited similar EC 50 values, and DFD59 (which does not contain the FGF21 mutant) exhibited approximately 2-fold higher activity than the dual-function proteins.

[0161] Next, the in vitro activity of the FGF21 moiety in DFD69, DFD112 and DFD114 was measured. Specifically, the in vitro activity of the FGF21 moiety in the dual-function proteins was evaluated using a HEK293 cell line overexpressing human β-klotho (a co-receptor of FGF21). For activity evaluation, samples containing each of the dual-function proteins were subjected to 3-fold serial dilution at a concentration of 3 μM. After being cultured for 5 hours under serum deprivation, the HEK293 cell line overexpressing human β-klotho was treated for 20 minutes, and the cells were lysed by adding cell lysis buffer (Cisbio / Cat# 64ERKPEG) while agitating at 60 rpm for 30 minutes at room temperature. The cell lysate solution was mixed with antibodies capable of detecting ERK and phosphorylated ERK, and the mixture was maintained at room temperature for 2 hours. Fluorescence was detected using a fluorescence detector (TECAN / GENiosPro). The activity was measured by comparing the EC 50 values.

[0162] It was confirmed that the in vitro activities of the FGF21 moieties of the dual-function proteins DFD69, DFD112 and DFD114 were similar as shown in Figure 14.

[0163] Experimental Example 8. Pharmacokinetic Evaluation of Dual-Function Proteins Experimental Example 8-1. Experimental Method for Pharmacokinetic Evaluation Six-week-old male ICR mice purchased from Orient BIO (Korea) were divided into groups (n=3 / at blood sampling time) to have similar mean body weights one day prior to drug treatment, and each sample was administered subcutaneously once at a dose of 1 mg / kg. Blood samples were collected at 1, 4, 8, 12, 24, 48, 72, 96, 144, 192, and 240 times post-injection. The concentrations of each dual-function protein in the blood were measured separately based on the FGF21 and GLP-1-Fc moieties. The concentration of the intact full-length FGF21 moiety of the dual-function protein in the blood was measured using the Intact human FGF21 ELISA kit (F1231-K01, Eagle Biosciences, USA), which involves an immune response to the N-terminus and C-terminus of the FGF21 protein. Furthermore, the concentration of the active GLP-1-Fc moiety of the dual-function protein in the blood was determined via ELISA analysis, and measured using antibodies with an immune response to the N-terminus of GLP-1 and Fc. The concentrations of FGF21 and GLP-1-Fc moieties of each protein were measured in blood samples collected up to 240 hours after single subcutaneous injection of each protein into mice, and the pharmacokinetic parameters of each protein were calculated.

[0164] Experimental Example 8-2. Pharmacokinetic Activity Results Based on the concentrations of each active substance in the blood over time after a single subcutaneous administration of each protein in mice (Figure 15), pharmacokinetic parameters for the FGF21 and GLP-1-Fc moieties of the dual-function protein were calculated. The data are shown in Table 10 below.

[0165] [Table 10]

[0166] The pharmacokinetic profiles of each dual-function protein were compared and evaluated based on the area under the curve (AUC), which indicates the degree of drug exposure.

[0167] As shown in Table 10, for the pharmacokinetic parameters of the FGF21 portion, DFD114 showed the highest levels of drug exposure (AUC) and half-life, followed by DFD112 with the next highest AUC value, and then DFD69. DFD114 showed approximately a twofold or greater increase in AUC value compared to DFD69. For the pharmacokinetics of the GLP-1-Fc portion, the four proteins containing the same GLP-1 variant sequence (DFD59, DFD69, DFD112, and DFD114) showed similar AUC values.

[0168] Experimental Example 9. Activity Evaluation in db / db Mice Experimental Example 9-1. Method for evaluating activity in db / db mice db / db mice, characterized by a genetic deficiency of the leptin receptor resulting in hyperglycemia, insulin resistance, hyperphagia, fatty liver, and obesity, and exhibiting more severe hyperglycemia and obesity than ob / ob mice, are widely used for the study of type 2 diabetes. Male db / db mice (Harlan, USA) were purchased from Raonbio (Korea). These mice were 5 to 6 weeks old upon arrival and 8 to 9 weeks old at drug treatment, 3 weeks after adaptation. The mice were divided into groups (n=6 / group) to have similar mean values ​​for body weight and tail blood glucose levels one day before drug treatment (day 0), and samples were administered subcutaneously once according to each of these respective doses. Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) was administered as the vehicle treatment, and blood glucose concentrations were measured using a glucose meter GlucoDr (All Medicus, Korea). Non-fasting glucose levels and body weight were measured daily until day 14 after administration. Glycated hemoglobin levels were also measured in each group before administration and after the study. Glycated hemoglobin levels were calculated using the DCA 2000 HbA1c kit (Siemens, 5035C).

[0169] Experimental Example 9-2. Evaluation of activity in db / db mice Changes in non-fasting blood glucose levels and body weight in male db / db mice were observed after single subcutaneous injections of 10 or 30 nmol / kg of the dual-function protein DFD114, single subcutaneous injections of 30 nmol / kg of the long-acting GLP-1-Fc single-function protein DFD59, and combined administrations of 30 nmol / kg of DFD59 and DFD74 (which are GLP-1-Fc and Fc-FGF21 single-function proteins, respectively) to compare the effects of the dual-function protein DFD114 with the combined administration of Fc-FGF21 and GLP-1-Fc single-function proteins.

[0170] The long-acting GLP-1-Fc protein DFD59 caused a sharp decrease in blood glucose levels up to day 1 post-administration, but the decrease in blood glucose decreased after day 2, and blood glucose levels were similar to those of the vehicle-treated group after day 4. On the other hand, the group treated with DFD114 showed a superior effect in lowering blood glucose up to day 3 post-administration, and the effect of lowering blood glucose levels disappeared more rapidly after day 4 from the 10 nmol / kg dose than from the 30 nmol / kg dose, showing a dose-dependent difference in the duration of the blood glucose-lowering effect. The groups treated with combinations of proteins showed the most sustained effect in lowering blood glucose levels compared to the other groups, indicating that the combination of GLP-1 and FGF21 had a superior effect in controlling blood glucose levels (Figure 16).

[0171] Regarding the effect on weight loss, the group treated with a combination of DFD59 and DFD74 showed the best effect on weight loss, and the group treated with 30 nmol / kg of DFD114 also showed a significant effect on weight loss (Figure 17).

[0172] After the trial, glycated hemoglobin levels, which indicate the mean blood glucose level, were measured, and the change in mean blood glucose was analyzed in each trial group. As shown in Figure 18, the vehicle-treated group showed an increase in glycated hemoglobin levels after the trial compared to the pre-treatment group, and the DFD59-treated group showed a similar increase. The group treated with 30 nmol / kg of DFD114 showed the best reduction in glycated hemoglobin levels, followed by the group receiving combination therapy, and then the group treated with 10 nmol / kg of DFD114. When evaluating the proteins by comparing them based on the reduction in glycated hemoglobin levels in each treated group, it was confirmed that the dual-function protein DFD114 showed a stronger effect in lowering blood glucose levels than GLP-1-Fc or Fc-FGF21 single-function protein alone.

[0173] Experimental Example 10. Activity of the fusion protein in HFD / STZ mice Experimental Example 10-1. Experimental method for evaluating activity in HFD / STZ mice The effects of dual-function proteins on lowering blood glucose and body weight were compared and evaluated in another diabetes model, the HFD / STZ mouse model.

[0174] Conventional diet-induced obesity mouse models (induced by feeding C57BL / 6 mice a 60 kcal% high-fat diet for 8 weeks or more) induce insulin resistance but have weak hyperglycemia and diabetic characteristics. The HFD / STZ mouse model, which can compensate for the shortcomings of conventional diet-induced obesity mouse models, can produce pancreatic β-cell dysfunction, reducing insulin secretion after administration of a high-fat diet (HFD) and low levels of streptozotocin (STZ), and is used for pharmacological studies of type 2 diabetes. To induce the HFD / STZ mouse model, C57BL / 6 mice were fed a 60 kcal% high-fat diet for 4 weeks, followed by intraperitoneal administration of 50 mg / kg of STZ (Sigma, 85882) daily for 3 days to induce pancreatic β-cell dysfunction. After a further 2 weeks of high-fat dieting, mice with non-fasting blood glucose levels of 200 mg / dL or higher were selected for testing. Mice were divided into groups (n=6 / group) to have similar mean values ​​for body weight and tail blood glucose levels one day before drug treatment (day 0), and samples were administered subcutaneously once according to each of these respective doses. Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) was administered as a vehicle treatment, and blood glucose concentrations were measured using a GlucoDr glucose meter (All Medicus, Korea). Non-fasting glucose levels and body weight were measured daily until day 14 after administration. Glycated hemoglobin levels were also measured in each group before administration and after the test. Glycated hemoglobin levels were calculated using the DCA 2000 HbA1c kit (Siemens, 5035C).

[0175] Experimental Example 10-2. Activity in HFD / STZ mice Changes in non-fasting blood glucose levels and body weight over time were observed in male HFD / STZ mice after single subcutaneous injections of the dual-function protein DFD114 at 3 nmol / kg or 10 nmol / kg, the Fc-fusion GLP-1 mutant DFD59 at 10 nmol / kg, or the Fc-fusion FGF21 mutants DFD72 and DFD74 at 10 nmol / kg, respectively. DFD59 and DFD74 were also administered once subcutaneously at 10 nmol / kg each to compare the effects of combined administration of single-function proteins with those of dual-function proteins.

[0176] As shown in Figure 19, regarding changes in blood glucose levels up to day 4, administration of DFD72 and DFD74 (long-acting FGF21 monofunctional protein) resulted in a slower decrease in blood glucose, while administration of DFD114 (long-acting protein containing GLP-1), DFD59, and combinations of DFD59 and DFD74 showed a more rapid decrease in blood glucose from day 1 of administration. Similar to the results in db / db mice, DFD59 showed a sharp decrease in blood glucose early on, but the decrease in blood glucose disappeared more slowly after day 4. DFD114 showed a similar pattern at a low dose of 3 nmol / kg. Similar non-fasting blood glucose profiles were observed in the groups treated with 10 nmol / kg of DFD114, DFD72, DFD74, and combinations.

[0177] Regarding the effect on weight loss, the group treated with a combination of DFD59 and DFD74 showed the best effect on weight loss (7 to 8%), and the group treated with 10 nmol / kg of DFD114 also showed a significant effect on weight loss (approximately 6%) (Figure 20). The group treated with DFD59 showed a 5% weight loss on day 1 after administration, but the effect disappeared after day 2 and became similar to the vehicle group after day 7. The groups treated with the long-acting FGF21 monofunctional proteins DFD72 and DFD74, respectively, showed a slower weight loss of 4 to 5% up to day 7 after administration, and the effect disappeared after day 10.

[0178] After the trial, glycated hemoglobin levels, which indicate the mean blood glucose level, were measured, and the change in mean blood glucose was analyzed in each trial group (Figure 21). The vehicle group showed an increase in glycated hemoglobin levels after the trial compared to before administration, and the group treated with DFD59 showed a similar increase. In contrast, the group treated with DFD114 showed a dose-dependent decrease in glycated hemoglobin levels, and the group treated with 10 nmol / kg of DFD114 showed the best effect in terms of the decrease in glycated hemoglobin levels (-0.42%). The group treated with a combination of DFD59 and DFD74 showed a similar decrease in glycated hemoglobin levels as DFD114 (-0.38%). For the long-acting FGF21 monofunctional protein, DFD72 was observed to be superior to DFD74. Based on the reduced levels of glycated hemoglobin in each group, we compared the proteins and confirmed that the dual-function protein DFD114 was superior to both the GLP-1-Fc and Fc-FGF21 single-function proteins.

[0179] Experimental Example 11. Prediction and Evaluation of Immunogenicity Experimental Example 11-1. Method and Results for Predicting Immunogenicity To predict the potential immunogenicity of dual-function proteins, we performed computer-based immunogenicity analyses on each protein.

[0180] Specifically, iTope TM and TCED TM Rapid screening was performed using the method (Prediction of immunogenicity of therapeutic proteins: validity of computational tools, BioDrugs, 2010). According to the two methods, T cell epitopes can be predicted more accurately compared to in-computer analysis methods that rely solely on MHC class II binding analysis.

[0181] Experimental Example 11-2. Ex vivo evaluation method and results for immunogenicity. To evaluate the potential immunogenicity of dual-function proteins, EpiScreen TM The analysis (Increased brain bio-distribution and chemical stability and decreased immunogenicity of an engineered variant of GDNF, Exp Neurol, 2015) was performed. When immunogenicity is detected, the amino acid sequence that induces immunogenicity can be identified via T cell epitope mapping, and deimmunized variants with minimal immunogenicity can be designed and prepared via computer prediction to reassess immunogenicity.

Claims

1. A dual-function protein comprising a fibroblast growth factor 21 (FGF21) mutant protein; a biologically active protein; an immunoglobulin Fc region; and a linker, The FGF21 mutant protein is (1) A mutation involving amino acid substitution in the amino acid sequence of EIRP (SEQ ID NO: 68) at positions 98-101 from the N-terminus of the wild-type FGF21 protein, and (6) A mutation in the wild-type FGF21 protein involving an amino acid substitution at amino acid E at position 180 from the N-terminus, and (2) an amino acid substitution in the TGLEAV amino acid sequence (SEQ ID NO: 69) at positions 170-174 from the N-terminus of the wild-type FGF21 protein; (3) an amino acid substitution in the TGLEAN amino acid sequence (SEQ ID NO: 70) at positions 170-174 from the N-terminus of the wild-type FGF21 protein; and (4) a single substitution of amino acid N selected from the group consisting of an amino acid substitution at position 170 from the N-terminus of the wild-type FGF21 protein. Includes, Biologically active proteins include insulin, C-peptide, leptin, glucagon, gastrin, gastric suppressor polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9), oxytomodulin, oxytocin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), irisin, and fibronectin type III domain-containing proteins. One selected from the group consisting of variants of GLP-1 having an amino acid sequence indicated by one of the following: nitrate 5 (FNDC5), apelin, adiponectin, C1q and tumor necrosis factor-related proteins (CTRP family), resistin, visfatin, omentin, retinol-binding protein-4 (RBP-4), glycentin, angiopoietin, interleukin-22 (IL-22), exendin-4, growth hormone, and sequence numbers 43 to 46. The wild-type FGF21 protein has the amino acid sequence shown by SEQ ID NO: 1, and the immunoglobulin's Fc region is a hybrid Fc containing one or a combination thereof of the Fc regions of IgG1, IgG2, IgG3, IgG4, and IgGD. Dual-function protein.

2. FGF21 mutant protein, (3) A mutation in the amino acid sequence of TGLEAN (SEQ ID NO: 70) at positions 170-174 from the N-terminus of the wild-type FGF21 protein, involving an amino acid substitution. A dual-function protein according to claim 1, comprising:

3. The dual-function protein according to claim 1, wherein amino acid residue N of the FGF21 mutant protein introduced by mutation is glycosylated.

4. The dual-function protein according to claim 1, wherein the biologically active protein is one selected from GLP-1, exendin-4, and a variant of GLP-1 having an amino acid sequence represented by any one of sequence numbers 43 to 46.

5. The dual-function protein according to claim 4, wherein the biologically active protein is a variant of GLP-1 having the amino acid sequence indicated by any one of sequence numbers 43 to 46.

6. The dual-function protein according to claim 1, wherein the FGF21 mutant protein has an amino acid sequence indicated by any one of sequence numbers 20 to 22.

7. The dual-function protein according to claim 1, wherein the linker ligates the FGF21 mutant protein to the Fc region of the immunoglobulin.

8. The dual-function protein according to claim 7, wherein the linker is ligated to the C-terminus of the Fc region of the immunoglobulin and to the N-terminus of the FGF21 mutant protein.

9. The dual-function protein according to claim 7, wherein the linker is a peptide consisting of 10 to 30 amino acid residues.

10. The dual-function protein according to claim 9, wherein the linker has an amino acid sequence indicated by any one of sequence numbers 2 to 5.

11. The dual-function protein according to claim 1, wherein the hybrid Fc comprises an IgG4 region and an IgD region.

12. The dual-function protein according to claim 1, comprising a biologically active protein, an immunoglobulin Fc region, a linker, and an FGF21 mutant protein, all linked in this order from the N-terminus to the C-terminus.

13. The dual-function protein according to claim 12, wherein the linker is ligated to the C-terminus of the Fc region of the immunoglobulin and to the N-terminus of the FGF21 mutant protein.

14. The dual-function protein according to claim 12, wherein the linker is a peptide consisting of 10 to 30 amino acid residues.

15. The dual-function protein according to claim 12, wherein the linker has an amino acid sequence indicated by any one of sequence numbers 2 to 5.

16. A pharmaceutical composition comprising a dual-function protein according to any one of claims 1 to 15 for treating diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cardiovascular disease.

17. An isolated nucleic acid molecule encoding a dual-function protein according to any one of claims 1 to 15.

18. An expression vector comprising the nucleic acid molecule described in claim 17.

19. A host cell comprising the expression vector described in claim 18.

Images