Bifunctional proteins and pharmaceutical compositions comprising the same

Abstract

Bifunctional protein and pharmaceutical composition comprising the same. The present invention relates to a bifunctional protein comprising a bioactive protein and a fibroblast growth factor 21 (FGF21) mutein, and a pharmaceutical composition comprising the same. The present invention provides a bifunctional protein prepared by linking a bioactive protein and an FGF mutein to an Fc region of an immunoglobulin, which has improved pharmacological potency, in vivo duration, and protein stability. The bifunctional protein according to the present invention exhibits improved pharmacological potency, in vivo duration, and protein stability, and a pharmaceutical composition containing the same as an active ingredient can be effectively used as a therapeutic agent for diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or a cardiovascular disease.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680062638.5 entitled "Bifunctional Protein and Pharmaceutical Composition Containing the Thereof", which is a PCT international patent application filed on October 28, 2016, which entered the Chinese national phase. Technical Field

[0002] This invention relates to a bifunctional protein comprising a bioactive protein and a mutant fibroblast growth factor 21 (FGF21) protein, and pharmaceutical compositions comprising the same. Background Technology

[0003] Glucagon-like peptide-1 (GLP-1) is an enterohepatic hormone composed of 31 amino acids, secreted by L cells in the intestine in response to stimuli such as food. Its biological effects are generated through intracellular signaling via GLP-1 receptors, G protein-coupled receptors expressed in target tissues such as pancreatic β cells and the brain. GLP-1 secreted in the blood has a very short half-life of less than 2 minutes, caused by the loss of activity due to the cleavage of the N-terminal amino acid by the enzyme dipeptidyl peptidase-4 (DPP-4). Because GLP-1 stimulates insulin secretion from pancreatic β cells based on blood glucose levels, it has a strong effect on lowering blood glucose without inducing hypoglycemia. Furthermore, in various animal models and humans, administration of GLP-1 leads to weight loss, known to be due to its appetite-suppressing effect, resulting in reduced food intake. GLP-1 induces β-cell proliferation and inhibits cell death caused by glucolipolysis through GLP-1 receptors expressed in pancreatic β cells, thereby enhancing β-cell viability. Excessive secretion of glucagon increases blood glucose levels, which is known to be one of the causes of hyperglycemia in diabetic patients. Furthermore, GLP-1 is known to act on α-cells in the pancreas, inhibiting the secretion of glucagon-specific protein kinase A (PKA) to suppress fasting blood glucose elevation.

[0004] Exendin-4 is a clinically important GLP-1 receptor agonist. It is a 39-amino acid peptide normally produced in the salivary glands of the Gila Monster lizard. Exendin-4 is known to share 52% amino acid sequence homology with GLP-1 and interacts with the GLP-1 receptor in mammals (Thorens et al. (1993) Diabetes 42: 1678-1682). Exendin-4 has been shown to stimulate insulin secretion from insulin-producing cells in vitro, and to induce stronger insulin release from these cells under equimolar conditions than GLP-1. While exendin-4 strongly stimulates insulin secretion to lower blood glucose levels in both rodents and humans for a longer duration than GLP-1, it exhibits antigenicity in mammals lacking GLP-1 due to its epitope, which is unfamiliar in such animals.

[0005] GLP-1 and venomous lizard exopeptide-4 analogs (such as liraglutide and byetta) have been clinically demonstrated to improve glucose control in humans. GLP-1 has been reported to increase β-cell populations by inhibiting apoptosis and inducing proliferation. Furthermore, GLP-1 has been reported to act as an intestinal hormone that inhibits gastric acid secretion and gastric emptying, while simultaneously enhancing satiety signals, thereby reducing appetite. These effects of GLP-1 may explain the weight loss observed when GLP-1 analogs are administered to patients with type 2 diabetes. Additionally, GLP-1 exhibits cardioprotective effects in rodents following ischemia.

[0006] Several attempts have been made to develop long-acting GLP-1 analogs. Clinically proven 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; their pharmacokinetic profiles allow for once-weekly dosing. Both drugs, when administered once weekly, have excellent effects in lowering blood glucose and reducing weight, and offer significantly improved treatment convenience compared to byetida and liraglutide.

[0007] Meanwhile, fibroblast growth factor 21 (FGF21), synthesized in the liver, is a hormone known to play an important role in glucose and lipid homeostasis. FGF21 exhibits pharmacological effects in liver, adipocytes, pancreatic β-cells, the hypothalamus in the brain, and muscle tissue expressing both the FGF21-specific receptor (i.e., the FGF receptor) and the β-klotho complex. FGF21 has been reported to lower blood glucose levels, reduce weight, and decrease blood triglyceride and low-density lipoprotein (LDL) concentrations in a non-human primate and mouse models of various diabetes and metabolic diseases in an insulin-independent manner. Furthermore, due to its ability to improve insulin sensitivity, FGF21 has the potential to be developed as a novel therapeutic agent for diabetes and obesity (see WO2003 / 011213).

[0008] Therefore, in order to develop new antidiabetic drugs based on FGF21, attempts have been made to improve its biological activity and in vivo stability by constructing FGF21 mutants based on the wild-type FGF21 sequence through substitution, insertion, and deletion of some amino acids (see WO2010 / 065439). However, due to the very short half-life of FGF21, it has been shown to be problematic if used directly as a biological therapeutic agent (Kharitonenkov, A. et al. (2005) Journal of Clinical Investigation 115: 1627-1635). In mice, the in vivo half-life of FGF21 is 1 to 2 hours, and in monkeys it is 2.5 to 3 hours. Therefore, for FGF21 to be used as a therapeutic agent for diabetes in its current form, daily administration is required.

[0009] Several methods have been reported to attempt to increase the in vivo half-life of recombinant FGF21 protein. One such example is linking polyethylene glycol (PEG) (i.e., the polymer material) to FGF21 to increase its molecular weight, thereby inhibiting renal excretion and increasing in vivo retention time (see WO2012 / 066075). Another method attempts to increase the half-life by fusing it with fatty acids bound to human albumin (see WO2012 / 010553). Another example attempts to increase the half-life while maintaining pharmacological activity equivalent to that in wild-type FGF21 by generating agonist antibodies that specifically bind to the human FGF receptor alone or as a complex with β-klotho (see WO2012 / 170438). In yet another example, the half-life is increased by preparing a long-acting fusion protein in which the Fc region of IgG binds to the FGF21 molecule (see WO2013 / 188181).

[0010] Fc fusion technology is widely used among various technologies available for producing long-acting drugs because it has fewer drawbacks seen with other methods, such as inducing immune responses or toxicity while increasing in vivo half-life. To develop an Fc-fused FGF21 protein as a long-acting therapeutic agent, the following conditions must be met.

[0011] First, 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 vary considerably depending on the fusion site. Therefore, the activity of FGF21 fusion proteins with Fc fusions to which mutations are introduced can be altered depending on the presence / absence or location of the fusion. Second, fusion can enhance the in vivo half-life, enabling pharmacokinetic profiles that can be administered weekly in humans. Third, considering the anticipated immunogenicity in most patients after administration of the biologic, the risk of immunogenicity caused by fusion linkers or mutations should be minimized. Fourth, the fusion site or the introduction of mutations should not cause stability problems. Fifth, solutions to prevent such responses are needed because undesirable immune responses may occur with isotypes of the fusion-dependent immunoglobulins.

[0012] Attempts to develop long-acting fusion proteins by linking the Fc region of immunoglobulin G (IgG) to the FGF21 molecule have been reported (see WO 2013 / 188181). In the case of an Fc-FGF21 structure in which the Fc region is fused to the N-terminus of wild-type FGF21, although there is no significant difference in in vitro activity compared to wild-type FGF21, a very short half-life is known due to in vivo protein degradation. To address this issue, attempts have been made to increase the in vivo half-life by introducing several mutations at specific sites in FGF21 to resist protein 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 the Fc region is fused to the C-terminus of the FGF21 molecule, fusion at this site is known to cause a significant reduction in activity compared to the Fc-FGF21 structure.

[0013] Depending on the mechanism of action and target tissue in vivo, combined administration of GLP-1 and FGF21 may have a synergistic effect compared to single administration, and is expected to have potentially superior antidiabetic efficacy and additional benefits. The effects of combined administration of GLP-1 and FGF21 or the GLP-1 / FGF21 bifunctional protein have been studied and reported (see WO2010 / 142665 and WO2011 / 020319).

[0014] Several challenges must be addressed to develop bifunctional proteins containing both GLP-1 and FGF21. Due to the very short in vivo half-lives of wild-type GLP-1 and wild-type FGF21, they require at least once-daily administration, even when developed as therapeutics. Therefore, long-acting technologies such as Fc fusion are needed to develop long-acting bifunctional proteins to improve patient convenience. In bifunctional drugs targeting both GLP-1 and FGF21, introducing mutations is important for maintaining the activity and in vivo stability of each drug, and the issues associated with changes in activity, structure, or stability caused by each mutation must be addressed. The drug effects targeting both GLP-1 and FGF21 should be well balanced, and drug design considering in vitro activity, pharmacokinetic profiles, pharmacological efficacy in animal models, and clinical evaluation of efficacy in humans is required for this purpose. Bifunctional proteins have structures not present in the human body and are structurally more complex than fusion proteins targeting a single target. Furthermore, the need for mutations or linker engineering to balance the two targets may increase the likelihood of aggregated complex formation, potentially requiring further protein engineering to prevent this. Furthermore, potential immunogenicity may be enhanced due to novel mutated sequences or complex structures, which should be addressed or avoided.

[0015] The inventors have dedicated themselves to improving the stability, pharmacokinetic profile, and pharmacological efficacy of bifunctional proteins comprising GLP-1 mutant protein and FGF21 mutant protein, and have discovered that when GLP-1 mutant protein is fused to the Fc region of an immunoglobulin and a novel FGF21 mutant protein is fused to it, the stability, pharmacokinetic profile, and pharmacological efficacy of the bifunctional protein can be improved, thus completing this invention. Summary of the Invention

[0016] Technical issues

[0017] One object of the present invention is to provide a bifunctional protein comprising a bioactive protein and an FGF21 mutant protein, which has improved pharmacokinetic parameters, high stability, low likelihood of forming aggregated complexes, and reduced potential immunogenicity.

[0018] Another object of the present invention is to provide a pharmaceutical composition comprising the bifunctional protein for the prevention or treatment of FGF21-related conditions.

[0019] Another object of the present invention is to provide an isolated nucleic acid molecule encoding the bifunctional protein, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector.

[0020] Solution to the problem

[0021] This invention provides a bifunctional protein comprising: an FGF21 mutant protein; a bioactive protein or a mutant or fragment thereof; and the Fc region of an immunoglobulin, wherein the FGF21 mutant protein comprises at least one mutation selected from the following mutations (1) to (7):

[0022] (1) The amino acids from position 98 to 101, starting from the N-terminus of the wild-type FGF21 protein, are replaced with the amino acid sequence EIRP (SEQ ID NO: 68);

[0023] (2) The amino acids from position 170 to 174 of the wild-type FGF21 protein are replaced with the amino acid sequence TGLEAV (SEQ ID NO: 69).

[0024] (3) The amino acids from position 170 to 174 of the wild-type FGF21 protein are replaced with the amino acid sequence TGLEAN (SEQ ID NO: 70);

[0025] (4) Replace the 170th amino acid from the N-terminus of the wild-type FGF21 protein with amino acid N;

[0026] (5) Replace the 174th amino acid from the N-terminus of the wild-type FGF21 protein with amino acid N;

[0027] (6) The amino acid at position 180 from the N-terminus of the wild-type FGF21 protein is replaced with amino acid E, and one or more of the above mutations (1) to (5); and

[0028] (7) Mutations of 1 to 10 amino acids used to reduce the immunogenicity of wild-type FGF21 protein.

[0029] Furthermore, the present invention provides a pharmaceutical composition comprising a bifunctional protein for the treatment of diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cardiovascular disease.

[0030] Furthermore, the present invention provides an isolated nucleic acid molecule encoding the bifunctional protein, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector.

[0031] Beneficial effects of the invention

[0032] The bifunctional protein of the present invention, prepared by linking a bioactive protein and an FGF mutant protein to the Fc region of an immunoglobulin, exhibits improved pharmacological potency, duration of in vivo activity, and protein stability. Furthermore, pharmaceutical compositions comprising the bifunctional protein as an active ingredient can be used as therapeutic agents for diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cardiovascular disease. Attached Figure Description

[0033] Figures 1a to 1c This is a graph showing the in vitro activity of a fusion protein containing the FGF21 mutant protein (hereinafter referred to as the "FGF21 mutant fusion protein") in the HEK293 cell line overexpressing human β-klotho. The FGF21 mutant fusion protein variant did not show a significant reduction in activity due to the introduction of the mutation.

[0034] Figure 2a and 2b This figure illustrates the in vitro activity of an FGF21 mutant fusion protein, in which the N-terminus of FGF21 is linked to the Fc region using multiple adaptors, within a HEK293 cell line overexpressing human β-klotho. The FGF21 mutant fusion protein did not show a significant reduction in activity, although minor differences in activity were observed based on the adaptor sequence.

[0035] Figure 3 This figure shows the in vitro activities of RGE (Amgen), Fc-FGF21 (Lilly), and DFD1 in the HEK293 cell line overexpressing human β-klotho. DFD1 and RGE (Amgen) have similar activities, while Fc-FGF21 (Lilly) has twice the in vitro activity of the other proteins.

[0036] Figure 4 The stability of DFD4 and DFD13 was demonstrated to confirm the role of the EIRP mutation of FGF21 in the stability of the fusion protein. It was confirmed that, compared with DFD4, DFD13 was associated with a lower proportion of high molecular weight aggregates (HMW%) at both the initial stage and time points more than two weeks later, indicating that the introduction of the EIRP mutation improved the stability of the FGF21 mutant fusion protein, thereby significantly reducing HMW%.

[0037] Figure 5 The concentrations of each protein in the blood over time are shown within 96 hours following subcutaneous administration of the FGF21 mutant fusion protein. Data are presented as mean and standard deviation.

[0038] Figure 6 The blood glucose levels in ob / ob mouse models following a single subcutaneous injection of DFD18, DFD72, DFD74, or Fc-FGF21 (Lilly) are shown. DFD18, DFD72, and DFD74 all had a sustained effect in reducing blood glucose levels. Data are presented as mean and standard error of mean (SEM).

[0039] Figure 7The graph shows the changes in body weight in ob / ob mouse models from the day of administration to day 14 following a single subcutaneous injection of DFD18, DFD72, DFD74, or Fc-FGF21 (Lilly). DFD18, DFD72, and DFD74 all showed a weight-reducing effect compared to the PBS-treated group. Data are presented as mean and standard error of the mean.

[0040] Figure 8 A graph showing changes in glycated hemoglobin levels in ob / ob mouse models on day 1 and day 16 following a single subcutaneous injection of DFD18, DFD72, DFD74, or Fc-FGF21 (Lilly) is presented. On day 16, all three DFD18, DFD72, and DFD74 reduced glycated hemoglobin levels compared to the levels on day 1. Data are presented as mean and standard error of the mean.

[0041] Figure 9 This graph shows blood glucose levels in an HFD / STZ mouse model following a single subcutaneous injection of DFD72 or DFD74. Both DFD72 and DFD74 have a sustained effect in lowering blood glucose levels. Data are presented as mean and standard error of the mean.

[0042] Figure 10 The changes in body weight in HFD / STZ mouse models from the day of administration to day 14 following a single subcutaneous injection of DFD72 or DFD74 are shown. Both DFD72 and DFD74 had a weight-reducing effect compared to the PBS-treated group. Data are presented as mean and standard error of mean.

[0043] Figure 11 The graph shows the changes in glycated hemoglobin levels in an HFD / STZ mouse model on days 1 and 13 following a single subcutaneous injection of DFD72 or DFD74. Both DFD72 and DFD74 were observed to cause a greater reduction in glycated hemoglobin levels compared to the PBS-treated group. Data are presented as mean and standard error of the mean.

[0044] Figure 12 The changes in body weight measured in a diet-induced obese mouse model from the day of administration to day 14 after a single administration of DFD18 are shown. DFD18 has a significant effect on weight loss. Data are presented as mean and standard error of the mean.

[0045] Figure 13This diagram illustrates the in vitro GLP-1 activity of bifunctional proteins using CHO cell lines overexpressing the human GLP-1 receptor, based on the hinge linking the C-terminus of GLP-1 and GLP-1 mutants to the Fc region. Typically, bifunctional proteins containing the GLP-1 (A2G) sequence (DFD23) exhibit 2 to 3-fold lower activity than other bifunctional proteins containing other GLP-1 mutant sequences. No significant differences in GLP-1 activity were observed among bifunctional proteins containing mutant sequences other than the GLP-1 (A2G) sequence.

[0046] Figure 14 A graph showing the GLP-1 activity of DFD59, DFD69, DFD112, and DFD114, as well as the FGF21 activity of DFD69, DFD112, and DFD114, is presented. In vitro GLP-1 activity of the three bifunctional 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 bifunctional proteins showed similar ECG activity. 50 The Fc-fused GLP-1 mutant (DFD59) showed approximately 2-fold higher activity than the bifunctional proteins. In vitro activity of the bifunctional proteins based on the FGF21 mutant was measured using the HEK293 cell line overexpressing human β-klotho. The in vitro activity of the FGF21 moiety was confirmed to be similar among the three bifunctional proteins.

[0047] Figure 15 The concentration of the bifunctional protein in the blood is shown as a function of time, 240 hours after subcutaneous administration. Data are presented as mean and standard deviation.

[0048] Figure 16 The blood glucose levels in the db / db mouse model are shown after a single subcutaneous injection of DFD114 or DFD59 and a combination of single subcutaneous injections of DFD59 and DFD74. The group treated with the bifunctional protein showed a stronger effect in reducing blood glucose levels compared to those treated with monofunctional proteins. Data are presented as mean and standard error of the mean (SEM).

[0049] Figure 17 A graph showing the changes in body weight in the db / db mouse model from the day of administration to day 14 following a single subcutaneous injection of DFD114 or DFD59 and a combination of single subcutaneous injections of DFD59 and DFD74 is shown. The group treated with the bifunctional protein showed a stronger effect on weight reduction compared to those treated with monofunctional proteins. Data are presented as mean and standard error of mean (SEM).

[0050] Figure 18 The graph shows the changes in glycated hemoglobin levels in the db / db mouse model on day 16 after administration (day 1) and after a single subcutaneous injection of DFD114 or DFD59 and a combination of single subcutaneous injections of DFD59 and DFD74. The group treated with the bifunctional protein showed a stronger effect in reducing glycated hemoglobin levels compared to those treated with monofunctional proteins or combinations thereof. Data are presented as mean and standard error of the mean.

[0051] Figure 19 The blood glucose levels in an HFD / STZ mouse model following a single subcutaneous injection of DFD114, DFD59, DFD74, or DFD72, and a combination of single subcutaneous injections of DFD59 and DFD74, are shown. The group treated with the bifunctional protein showed a stronger effect in lowering blood glucose levels compared to those treated with monofunctional proteins. Data are presented as mean and standard error of the mean (SEM).

[0052] Figure 20 The changes in body weight in HFD / STZ mouse models from the day of administration to day 14 following a single subcutaneous injection of DFD59, DFD72, DFD74, or DFD114, and a combination of single subcutaneous injections of DFD59 and DFD74, are shown. The group treated with bifunctional proteins showed a stronger effect on body weight reduction compared to those treated with monofunctional proteins. Data are presented as mean and standard error of the mean (SEM).

[0053] Figure 21 The changes in glycated hemoglobin (HbA1c) levels in the HFD / STZ mouse model on day 16 after administration (day 1) and after a single subcutaneous injection of DFD59, DFD72, DFD74, or DFD114, or a combination of single subcutaneous injections of DFD59 and DFD74. The group treated with the bifunctional protein showed a stronger effect in reducing HbA1c levels compared to those treated with monofunctional proteins or combinations thereof. Data are presented as mean and standard error of the mean.

[0054] Best way to carry out the invention

[0055] The invention will be described in more detail below.

[0056] On the one hand, the present invention provides a bifunctional protein comprising: a fibroblast growth factor 21 (FGF21) mutant protein; a bioactive protein or a mutant or fragment thereof; and the Fc region of an immunoglobulin, wherein the FGF21 mutant protein comprises at least one mutation selected from the following mutations (1) to (7):

[0057] (1) The amino acids from position 98 to 101 from the N-terminus of the wild-type FGF21 protein are replaced with the amino acid sequence EIRP (SEQ ID NO: 68) (hereinafter referred to as "EIRP");

[0058] (2) The amino acids from position 170 to 174 of the wild-type FGF21 protein are replaced with the amino acid sequence TGLEAV (SEQ ID NO: 69) (hereinafter referred to as "TGLEAV");

[0059] (3) The amino acids from position 170 to 174 of the wild-type FGF21 protein are replaced with the amino acid sequence TGLEAN (SEQ ID NO: 70) (hereinafter referred to as "TGLEAN");

[0060] (4) Replace the 170th amino acid from the N-terminus of the wild-type FGF21 protein with amino acid N;

[0061] (5) Replace the 174th amino acid from the N-terminus of the wild-type FGF21 protein with amino acid N;

[0062] (6) The amino acid at position 180 from the N-terminus of the wild-type FGF21 protein is replaced with amino acid E, and one or more of the above mutations (1) to (5); and

[0063] (7) Mutations of 1 to 10 amino acids used to reduce the immunogenicity of wild-type FGF21 protein.

[0064] Wild-type FGF21 protein (a hormone known to play an important role in glucose and lipid homeostasis) may be derived from mammals (e.g., humans, mice, pigs, monkeys, etc.), preferably from humans. More preferably, wild-type FGF21 protein may be wild-type human FGF21 protein having the amino acid sequence represented by SEQ ID NO: 1.

[0065] The mutations included in the FGF21 mutant protein may preferably be: any one of the EIRP, TGLEAV, TGLEAN, G170N, and G174N mutations; a combination of any one of the TGLEAV, TGLEAN, G170N, and G174N mutations with the EIRP mutation; a combination of any one of the EIRP, TGLEAV, TGLEAN, G170N, and G174N mutations with the A180E mutation; or a combination of any one of the TGLEAV, TGLEAN, G170N, and G174N mutations with both the EIRP and A180E mutations. Furthermore, the FGF21 mutant protein may have a conformation in which 1 to 10 amino acids are missing from the N-terminus or C-terminus compared to the wild-type FGF21 protein. More preferably, the FGF21 mutant protein may comprise an amino acid sequence represented by any one of SEQ ID NO: 6 to 23. More preferably, the FGF21 mutant protein may comprise an amino acid sequence represented by any one of SEQ ID NO: 6 to 23 and also have a conformation in which 1 to 10 amino acids are missing from the N-terminus or C-terminus compared to the wild-type FGF21 protein.

[0066] In bifunctional proteins, the amino acid residue N of the FGF21 mutant protein introduced through mutation can be glycosylated.

[0067] Bioactive proteins can be selected from the following: insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9), oxytocin, oxytocin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), irisin, fibronectin type III domain-containing protein 5 (FNDC5), apelin, liponelin, C1q and tumor necrosis factor-related proteins (CTRP family), resistin, endothelin, omentin, and retinol-binding protein-4. The bioactive proteins include protein-4 (RBP-4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, and growth hormone. Preferably, the bioactive protein can be a bioactive protein selected from GLP-1, its mutants, and exendin-4.

[0068] GLP-1 protein is an intestinal glucagon hormone composed of 31 amino acids, secreted by L cells in the intestine in response to stimuli such as food. For example, GLP-1 protein can be represented by the amino acid sequence of SEQ ID NO: 42.

[0069] Mutants of GLP-1 can be represented by amino acid sequences, for example, any one of SEQ ID NO: 43 to 46.

[0070] As used herein, the terms "Fc region," "Fc fragment," or "Fc" refer to a protein containing the heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3) of immunoglobulins, but excluding the heavy and light chain variable regions of immunoglobulins and the light chain constant region 1 (CL1). Furthermore, as used herein, the term "Fc region mutant" refers to a mutant prepared by substituting certain amino acids in the Fc region or by combining different types of Fc regions.

[0071] The Fc region of an immunoglobulin can be the complete Fc region constituting an antibody, a fragment thereof, or an Fc region mutant. Furthermore, the Fc region contains molecules in monomeric or multimeric form and may also include a hinge region of the heavy chain constant region. Fc region mutants can be modified to prevent cleavage at the hinge region. Additionally, the hinge sequence of the Fc can have substitutions in some amino acid sequences to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Furthermore, partial amino acid sequences of the Fc hinge sequence can be substituted to inhibit rearrangement in the Fab region. Lysine residues at the C-terminus of the Fc can be removed.

[0072] Preferably, the Fc region of the immunoglobulin can be any one of the IgG1, IgG2, IgG3, IgG4, and IgD Fc regions; or a hybrid Fc of these combinations. Furthermore, the hybrid Fc can contain both the IgG4 and IgD regions. Additionally, the hybrid Fc region can contain a portion of the hinge sequence and CH2 of the IgD Fc, and the CH2 and CH3 sequences of the IgG4 Fc.

[0073] Furthermore, the Fc fragment of the present invention can be in the form of a wild-type glycosylated chain, a chain with a higher degree of glycosylation compared to the wild-type, a chain with a lower degree of glycosylation compared to the wild-type, or a deglycosylated chain. The addition, reduction, 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.

[0074] Preferably, the immunoglobulin Fc region can be represented by an amino acid sequence selected from SEQ ID NO: 24 to 26, 47 and 48.

[0075] A bifunctional protein may comprise a bioactive protein, an Fc region of an immunoglobulin, and an FGF21 mutant protein, linked sequentially from the N-terminus to the C-terminus. Alternatively, the bifunctional protein may comprise an FGF21 mutant protein, an Fc region of an immunoglobulin, and a bioactive protein, linked sequentially from the N-terminus to the C-terminus. Preferably, the bifunctional protein may comprise a bioactive protein, an Fc region of an immunoglobulin, and an FGF21 mutant protein, linked sequentially from the N-terminus to the C-terminus.

[0076] Furthermore, the bifunctional protein may comprise, from N-terminus to C-terminus, a GLP-1 mutant protein, the Fc region of an immunoglobulin, and an FGF21 mutant protein. Preferably, the bifunctional protein may comprise, from N-terminus to C-terminus, a GLP-1 mutant protein, the Fc region of an immunoglobulin, and an FGF21 mutant protein.

[0077] Furthermore, bifunctional proteins can further include linkers.

[0078] Bifunctional proteins can take the form of FGF21 mutant proteins directly linked to the N-terminus or C-terminus of the Fc region of immunoglobulins, or FGF21 mutant proteins linked to the Fc region of immunoglobulins via adapters.

[0079] In such cases, the linker can connect to the N-terminus, C-terminus, or free radical of the Fc fragment, and it can also connect to the N-terminus, C-terminus, or free radical of the FGF21 mutant protein. When the linker is a peptide linker, the connection can occur in any region. For example, the linker can connect to the C-terminus of the immunoglobulin Fc region and the N-terminus of the FGF21 mutant protein to form a fusion protein of the immunoglobulin Fc region and the FGF21 mutant protein.

[0080] Furthermore, the bifunctional protein of the present invention may be in the form in which the bioactive protein is attached to the N-terminus of the immunoglobulin Fc region of the fusion protein.

[0081] When the linker and Fc are expressed and subsequently linked, the linker can be a crosslinking agent known in the art. Some examples of crosslinking agents may include 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, imine esters (including N-hydroxysuccinimide esters (e.g., 4-azidosalicylic acid) and disuccinimide esters (e.g., 3,3'-dithiobis(succinimide propionate))), and bifunctional maleimides, such as bis-N-maleimide-1,8-octane, but are not limited thereto.

[0082] Furthermore, the linker can be a peptide. Preferably, the linker can be a peptide composed of 10 to 30 amino acid residues.

[0083] In addition, alanine may be additionally attached to the end of the linker. Preferably, the linker may be a peptide having an amino acid sequence represented by any one of SEQ ID NO: 2 to 5.

[0084] Bifunctional proteins can be in the form of FGF21 mutant protein dimers or multimers with one or more FGF21 mutant proteins linked together, which are then linked to the Fc region of an immunoglobulin. Alternatively, bifunctional proteins can be in the form of dimers or multimers with two or more immunoglobulin Fc regions linked together, where the immunoglobulin Fc region is linked to an FGF21 mutant protein.

[0085] Furthermore, the bifunctional protein can preferably be a peptide having an amino acid sequence represented by any one of SEQ ID NO: 58 to 67. More preferably, the bifunctional protein can be a peptide having an amino acid sequence represented by SEQ ID NO: 65, 66 or 67.

[0086] The FGF21 mutant protein may further contain mutations of 1 to 10 amino acids to reduce the immunogenicity of the wild-type FGF21 protein. Immunogenicity can be predicted using conventional methods known in the art. For example, it can be predicted using methods such as iTope. TM and TCED TM Methods to screen for the potential immunogenicity of proteins.

[0087] Furthermore, mutations used to minimize immunogenicity can be designed using conventional methods known in the art. For example, when performing EpiScreen... TM When analyzing immunogenicity to evaluate potential immunogenicity, T-cell epitope mapping can be used to identify the amino acid sequences that induce immunogenicity, and computer prediction can be used to design mutants with minimal immunogenicity.

[0088] In another aspect, the present invention provides a pharmaceutical composition comprising the bifunctional protein for treating FGF21-related conditions.

[0089] As used herein, the term "FGF21-related conditions" may include obesity, type I and type II 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, nephropathy, diabetic complications, neuropathy, gastroparesis, conditions associated with severe inactivation mutations of the insulin receptor, and other metabolic conditions. Preferably, FGF21-related conditions may be diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cardiovascular disease.

[0090] In addition, the pharmaceutical composition may also contain a pharmaceutical carrier. The pharmaceutical carrier can be any carrier, as long as it is a non-toxic material suitable for delivering antibodies to the patient. For example, distilled water, alcohols, fats, waxes, and inactive solids may be included as carriers. Pharmaceutical adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition. In these formulations, the concentration of the bifunctional protein can vary considerably.

[0091] Specifically, the pharmaceutical composition may include formulation materials for altering, maintaining, or preserving the composition's pH, molar osmolarity, viscosity, transparency, color, isotropic properties, odor, sterility, stability, dissolution or release rate, adsorption, or permeability. Examples of suitable formulation materials may include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), and bulking agents. Agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), compounding 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 immunoglobulins), coloring agents, flavoring agents, diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight peptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, p-hydroxybenzoate, p-hydroxybenzoic acid, etc.). Propyl benzoate, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (e.g. glycerol, propylene glycol or polyethylene glycol), sugar alcohols (e.g. mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., puronic acid; PEG; sorbitan esters; polysorbates, such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapol)), stability improvers (e.g. sucrose or sorbitol), growth promoters (e.g. alkali metal halides, preferably sodium chloride or potassium chloride; or mannitol, sorbitol), delivery carriers, diluents, excipients and / or pharmaceutical adjuvants, but not limited to these.

[0092] In another aspect, the present invention provides a method for preventing or treating FGF21-related conditions, comprising administering the bifunctional protein to a subject in need of such prevention or treatment. The method specifically comprises administering an effective amount of the bifunctional protein of the present invention to mammals exhibiting symptoms of diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cardiovascular disease, which are FGF21-related conditions.

[0093] The pharmaceutical compositions of the present invention can be administered via any route. The compositions of the present invention can be administered to animals directly (e.g., locally, by injection, transplantation to a tissue region, or by surface application) or systemically (e.g., by oral or parenteral administration) by any suitable means. When the compositions of the present invention are administered parenterally via intravenous, subcutaneous, ocular, intraperitoneal, intramuscular, oral, rectal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intrasheathal, intracavitary, intrasacral, intranasal, or aerosol administration, the compositions are preferably aqueous or may contain a portion of a physiologically suitable suspension or solution of bodily fluids. Therefore, due to their physiological suitability, a carrier or delivery agent can be added to the composition and delivered to the patient. Thus, physiologically suitable saline solutions can typically be included as a carrier (e.g., bodily fluids) for the formulation.

[0094] Furthermore, the frequency of administration can vary depending on the pharmacokinetic parameters of the bifunctional protein in the formulation to be used. Typically, physicians will administer the composition until the desired effect is achieved at the prescribed dose. Therefore, the composition can be administered as a unit dose (at least two doses at intervals) (which may or may not contain the same amount of the target bifunctional protein) or via continuous injection through a transplantation device or catheter. The precision of adding the appropriate dose can be routinely performed by those skilled in the art and corresponds to their usual scope of work.

[0095] Furthermore, the preferred unit dose of the bifunctional protein in humans can be from 0.01 μg / kg to 100 mg / kg body weight, and more preferably from 1 μg / kg to 10 mg / kg body weight. While this is the optimal amount, the unit dose can vary depending on the disease being treated or the presence / absence of adverse reactions. Nevertheless, the optimal dosage can be determined by performing routine experiments. The bifunctional protein can be administered via periodic bolus injection, external reservoir (e.g., intravenous bag), or continuous intravenous, subcutaneous, or intraperitoneal administration from an internal source (e.g., a biodegradable implant).

[0096] Furthermore, the bifunctional protein of the present invention can be administered to the recipient along with other bioactive molecules. The optimal combination, dosage form, and optimal dosage of the bifunctional protein and other molecules can be determined through conventional experiments known in the art.

[0097] In another aspect, the present invention provides isolated nucleic acid molecules encoding bifunctional proteins.

[0098] As used herein, the term "isolated nucleic acid molecule" refers to the nucleic acid molecule of the present invention that, when total nucleic acids are isolated from source cells, is separated from approximately 50% of naturally occurring proteins, lipids, carbohydrates, or other substances; is effectively linked to non-naturally linked polynucleotides; or is part of a larger polynucleotide sequence and is not naturally occurring. Preferably, the isolated nucleic acid molecule of the present invention is substantially free of any other contaminating nucleic acids or other pollutants found in the natural environment that inhibit the use of nucleic acids in polypeptide production or in treatment, diagnosis, prevention, or research.

[0099] In this case, due to codon redundancy, isolated nucleic acid molecules encoding bifunctional proteins can have different sequences from each other. Furthermore, as long as the isolated nucleic acid can produce a bifunctional protein, it can be appropriately modified according to the desired purpose, or nucleotides can be added to the N-terminus or C-terminus of the isolated nucleic acid.

[0100] The isolated nucleic acid may contain, for example, a nucleotide sequence represented by any one of SEQ ID NO: 71 to 80.

[0101] In another aspect, the present invention provides an expression vector comprising isolated nucleic acid molecules.

[0102] As used herein, the term "expression vector" refers to a vector containing a nucleic acid sequence suitable for transforming host cells and directing or controlling the expression of an inserted heterologous nucleic acid sequence. Expression vectors include linear nucleic acids, plasmids, phage particles, granules, RNA vectors, viral vectors, and analogues. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0103] As used in this article, “expression of heterologous nucleic acid sequences” or “expression” of target proteins refers to the transcription of inserted DNA sequences, the translation of mRNA transcripts, and the production of Fc fusion protein products, antibodies, or antibody fragments.

[0104] The available expression vector can be RcCMV (Invitrogen, Carlsbad) or a mutant thereof. The available expression vector may include a human cytomegalovirus (CMV) promoter for promoting sustained transcription of the target gene in mammalian cells and a bovine growth hormone polyadenylation signal sequence for enhancing post-transcriptional RNA stability. In one exemplary embodiment of the invention, the expression vector is pAD15, which is a modified vector of RcCMV.

[0105] In another aspect, the present invention provides a host cell comprising an expression vector.

[0106] As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector may be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using a variety of techniques known in the art.

[0107] Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, CAP cells (human amniotic fluid-derived cells), and COS cells.

[0108] In the following, exemplary embodiments of the present invention will be described in detail with reference to examples. However, these embodiments of the present invention can be modified in many different forms, and the scope of the invention should not be construed as limited to the embodiments set forth herein. Detailed Implementation

[0109] Preparation Example 1. Preparation and purification of a fusion protein containing the FGF21 mutant protein

[0110] Preparation Example 1-1. Preparation of expression vector for expressing FGF21 mutant protein

[0111] To improve the stability, activity, and pharmacokinetic profile of FGF21 in the Fc-FGF21 structure, mutation studies of FGF21 were conducted.

[0112] Specifically, mutants were designed for the following regions: the LLLE region (amino acids 98 to 101 from the N-terminus of the FGF21 protein), the GPSQG region (amino acids 170 to 174 from the N-terminus of the FGF21 protein), and the A180 site, which are expected to significantly affect protein activity based on the three-dimensional structural analysis of the FGF21 protein.

[0113] 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 refers to glycosylated asparagine (N). Furthermore, FGF21 mutant proteins containing the mutations described in Table 1 are listed in Table 2 below.

[0114] [Table 1]

[0115]

[0116] [Table 2]

[0117] 6 FGF21(EIRP) 7 FGF21(TGLEAV) 8 FGF21(TGLEAN) 9 FGF21(G170N) 10 FGF21(G174N) 11 FGF21 (EIRP, TGLEAV) 12 FGF21 (EIRP, TGLEAN) 13 FGF21(EIRP, G170N) 14 FGF21(EIRP, G174N) 15 FGF21(EIRP, A180E) 16 FGF21 (TGLEAV, A180E) 17 FGF21 (TGLEAN, A180E) 18 FGF21(G170N, A180E) 19 FGF21(G174N, A180E) 20 FGF21 (EIRP, TGLEAV, A180E) 21 FGF21 (EIRP, TGLEAN, A180E) 22 FGF21 (EIRP, G170N, A180E) 23 FGF21 (EIRP, G174N, A180E)

[0118] Expression vectors were prepared to express the following three amino acid components sequentially from N-terminus to C-terminus: fusion vector, adapter, and FGF21 mutant. The material code of each FGF21 mutant fusion protein, the mutated sequence introduced into FGF21, the fusion vector sequence, and the adapter sequence are listed in Table 3 below (in Table 3, ...). N It refers to glycosylated asparagine (N).

[0119] [Table 3]

[0120]

[0121] To generate FGF21 mutant fusion proteins, nucleotide sequences encoding each FGF21 mutant protein were synthesized by consulting Bioneer Corporation (Korea) based on the amino acid sequence of each protein. NheI and NotI restriction enzyme sequences were added to the 5′ and 3′ ends of the nucleotide sequences encoding each FGF21 mutant protein, and a start codon for protein translation and a leader sequence (MDAMLRGLCCVLLLCGAVFVSPSHA) immediately following the 5′ end were inserted. A stop codon was immediately inserted following the nucleotide sequence encoding each FGF21 mutant fusion protein. The nucleotide sequences encoding each FGF21 mutant fusion protein were cloned into pTrans-empty expression vectors using both NheI and NotI restriction enzymes. pTrans-empty expression vectors 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 were purchased from CEVEC Pharmaceuticals (Germany).

[0122] In the case of the fusion proteins DFD6 (E. coli) and RGE (Amgen), the nucleotide sequence encoding each fusion protein was inserted into the pET30a expression vector for expression in E. coli.

[0123] Preparation Examples 1-2. Construction of plasmid DNA for expressing FGF21 mutant fusion protein

[0124] E. coli were transformed with each expression vector constructed in Preparation Example 1-1 to obtain a large quantity of plasmid DNA for expression. E. coli cells with weakened cell walls were transformed with each expression vector by heat shock, and the transformed cells were plated on LB plates to obtain colonies. The colonies thus obtained were inoculated into LB medium and incubated at 37°C for 16 hours, yielding 100 mL of each E. coli culture containing each expression vector. The resulting E. coli were centrifuged to remove the medium, and then P1, P2, P3 solutions (QIAGEN, catalog number: 12963) were added to rupture the cell walls, resulting in a DNA suspension in which the protein and DNA phases were separated. Plasmid DNA was purified from the DNA suspension thus obtained 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 (Thermoscientific, Nanodrop Lite). The DNA thus obtained was used for expression.

[0125] Preparation Examples 1-3. Expression of Fusion Proteins in CAP-T Cells

[0126] 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) already cultured in PEM medium (Lifetechnologies) using PEI solution (Polyplus, catalog number: 101-10N). The mixture of DNA and PEI solution was mixed with the cell suspension using Freestyle293 expression medium (Invitrogen), incubated at 37°C for 5 hours, and then PEM medium was added. After incubation at 37°C for 5 to 7 days, the culture was centrifuged to remove cells, yielding a supernatant containing the FGF21 mutant fusion protein.

[0127] Preparation Examples 1-4. Expression and purification of FGF21 mutant fusion protein in Escherichia coli

[0128] E. coli strain BL21(DE3) was transformed with DNA from each plasmid expressing the DFD6 (E. coli) and RGE (Amgen) fusion proteins. The transformed E. coli expressing each fusion protein were inoculated into 20 mL of LB medium and cultured at 37°C with shaking for 15 hours. A portion of the culture was then inoculated into 100 mL of LB medium and cultured at 37°C with shaking for 16 hours. After incubation, the culture was centrifuged to obtain an E. coli pellet, and the cells were then disrupted using a high-pressure cell disruptor to obtain inclusion bodies.

[0129] The obtained inclusion bodies were purified by washing and elution, followed by a protein refolding process. Specifically, the obtained inclusion bodies were washed 2 to 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 remove bacterial proteins, and 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 are completely denatured, the protein refolding process was performed as follows.

[0130] First, 8M urea buffer was gradually diluted with 20mM glycine buffer (pH 9.0) to remove urea, and CuSO4 was added from a concentration of 2M to 80μM to induce stable protein folding. The refolded protein was resuspended in PBS buffer (pH 7.4) and filtered through a 0.22μm filter to remove impurities, then loaded into a protein A affinity column. The column was washed with 1X PBS buffer (pH 7.4) and then eluted with 100mM glycine buffer (pH 3.0) to prepare the DFD6 (E. coli) fusion protein.

[0131] In the case of RGE (Amgen) fusion protein, the protein that has completed the refolding process is suspended in 50 mM Tris buffer (pH 8.0), the suspension is 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 (pH 8.0), followed by elution of the RGE (Amgen) fusion protein with a concentration gradient of 50 mM Tris buffer (pH 8.0). The RGE (Amgen) fusion protein obtained by anion exchange resin was mixed with ammonium sulfate to a concentration of 1 M and then purified using a hydrophobic interaction column (Phenylesepharose FF, GE Healthcare). Specifically, the column was washed with 50 mM Tris buffer (pH 8.0) containing 1 M ammonium sulfate, followed by elution with a concentration gradient of 50 mM Tris buffer (pH 8.0), and the eluted fractions were analyzed by 10% Tris-glycine gel electrophoresis. The gel was stained with Coomassie Brilliant Blue R with gentle agitation, and the fraction containing high-purity FGF21 mutant fusion protein was collected and dialyzed overnight at 4°C with the final buffer solution (1X PBS, 1 mM EDTA, pH 7.4). After dialysis, the obtained protein stock solution was concentrated at 3,000 rpm using a 30,000 MW cutoff centrifuge filter at 4°C. The concentration of the FGF21 mutant fusion protein was measured by BCA quantitative analysis.

[0132] Preparation Examples 1-5. Purification of FGF21 mutant fusion protein

[0133] The protein A affinity column (GE Healthcare) was equilibrated with 1X PBS buffer (pH 7.4). Culture supernatant containing each FGF21 mutant fusion protein obtained in Preparation Examples 1-3 was filtered through a 0.2 μm filter and then loaded into the protein A affinity column. The column was washed with 1X PBS buffer (pH 7.4) and then eluted with 100 mM glycine buffer (pH 3.0). An anion exchange resin column (POROS) was used. @ The fusion protein obtained by affinity chromatography was purified using a 50 μm HQ (Thermo Fisher Scientific) column. The anion exchange resin column was equilibrated with 50 mM Tris buffer (pH 8.0) before eluting the FGF21 mutant fusion protein from the column. Specifically, after washing the column with 50 mM Tris buffer (pH 8.0), 50 mM Tris buffer (pH 8.0) was partitioned along a concentration gradient, and the eluted fractions were analyzed. Each eluted fraction was analyzed using size exclusion chromatography (SEC-HPLC), and the fraction containing high-purity FGF21 mutant fusion protein was collected. Concentration and quantification were performed according to the methods described in Preparation Examples 1-4.

[0134] Experimental Example 1. In vitro activity of fusion proteins

[0135] Experimental Example 1-1. Effect of FGF21 mutation on protein activity

[0136] The in vitro activities of the fusion proteins DFD4, DFD5, DFD6, DFD6 (E. coli), DFD7, DFD9, DFD13, DFD18, DFD72, DFD73 and DFD74 prepared in Preparation Example 1 were measured.

[0137] Specifically, the in vitro FGF21 activity of the fusion protein was evaluated using the HEK293 cell line (Yuhan Corporation, Korea), which was modified to overexpress human β-klotho (a co-receptor of FGF21). To evaluate activity, the concentrates containing the fusion protein prepared in Preparation Examples 1-4 and 1-5 were serially diluted 3-fold at a concentration of 3 μM. After culturing for 5 hours in a serum-deficient state, the cell lines overexpressing human β-klotho were treated with the diluted fusion protein for 20 minutes, followed by lysis at room temperature with stirring at 60 rpm for 30 minutes by adding cell lysis buffer (Cisbio / catalog number 64ERKPEG). The cell lysate solution was mixed with an antibody (Cisbio / catalog number 64ERKPEG) capable of detecting extracellular signal-regulated kinase (ERK) and phosphorylated ERK, and the mixture was incubated at room temperature for 2 hours. Fluorescence was detected using a fluorescence detector (TECAN / GENiosPro). The activity was assessed by comparing the EC50 values. 50 The value is used to measure the activity of the fusion protein.

[0138] like Figures 1a to 1c As shown, the in vitro activity of the fusion proteins prepared by introducing mutant sequences into the wild-type FGF21 protein was not inhibited, and the activities of each fusion protein were similar to those of the others. It was also confirmed that the in vitro activity of the fusion proteins prepared by introducing N-glycosylation mutations into the wild-type FGF21 protein was not inhibited, using DFD6 samples expressed in *E. coli* and DFD6 samples expressed in animal cells.

[0139] Experimental Example 1-2. The Role of Adaptor Sequences in Protein Activity

[0140] The in vitro activities of the fusion proteins DFD1, DFD3, DFD4 and DFD13 prepared in Preparation Example 1 were measured.

[0141] Specifically, the FGF21 activity of the fusion protein was measured using the concentrate containing the fusion protein prepared in Preparation Examples 1-5, as described in Experimental Example 1-1. The results are shown in... Figure 2a and 2b middle.

[0142] It was confirmed that the FGF21 mutant fusion protein did not show a significant decrease in activity, but... Figure 2a and 2b As shown, the activity varies slightly depending on the adapter sequence.

[0143] Experimental results of DFD1, RGE (Amgen), and Fc-FGF21 (Lilly) in Experimental Examples 1-3.

[0144] The in vitro activities of the fusion protein DFD1 prepared in Preparation Example 1 and the control proteins RGE (Amgen) and Fc-FGF21 (Lilly) were measured.

[0145] Specifically, the FGF21 activity of the fusion protein was measured using the concentrate containing the fusion protein prepared in Preparation Examples 1-5 and the control protein, according to the method described in Experimental Example 1-1. The results are shown in... Figure 3 middle.

[0146] like Figure 3 As shown, DFD1 and RGE (Amgen) have similar in vitro activities, while Fc-FGF21 (Lilly) has twice the in vitro activity of other proteins.

[0147] Experimental Example 2. Evaluation of the stability of fusion proteins

[0148] Experimental Example 2-1. Experimental Methods for Evaluating Stability

[0149] To measure the amount of protein aggregates in the initial stage of sample preparation, size exclusion chromatography (SEC-HPLC) was used to quantify high molecular weight aggregates (%HMW). Results are shown in... Figure 4 middle.

[0150] Specifically, using TosoHaas type TSK-GEL G3000SW XLThe column was used for SEC-HPLC. The column was equilibrated by flowing buffer solution (1X PBS, 1 mM EDTA, pH 7.4) at a flow rate of 1 mL / min. The DFD4 and DFD13 protein stock solutions prepared in Preparation Examples 1-5 were concentrated to a target concentration of 20 mg / mL or higher at 3000 rpm using a 30,000 MW cutoff centrifuge filter at 4 °C. After determining the concentration of each sample by BCA quantification, the samples were diluted to a final concentration of 20 mg / mL with buffer solution (1X PBS, 1 mM EDTA, pH 7.4). To measure the initial %HMW of DFD4 and DFD13, the 20 mg / mL sample was diluted to a final concentration of 1 mg / mL with buffer solution (1X PBS, 1 mM EDTA, pH 7.4), and each sample in a 100 μL volume was analyzed by SEC-HPLC column.

[0151] For stability evaluation of each sample, the %HMW of the sample was measured using the SEC-HPLC method on days 4, 8, and 14 while the sample was stored at 5°C, 25°C, and 37°C for two weeks.

[0152] like Figure 4 As shown, DFD13 exhibited lower levels of high molecular weight aggregates (HMW%) in the initial phase and up to 2 weeks compared to DFD4. This suggests that the introduction of the EIRP mutation improves the stability of the FGF21 mutant fusion protein, thereby significantly reducing HMW.

[0153] Experimental Example 2-2. Stability Results

[0154] 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. Table 4 below summarizes the analytical results of zero-hour samples (initial stage; day 0) and samples stored for 4, 8, and 14 days for DFD4 and DFD13 (in Table 4, ND indicates "not detected").

[0155] [Table 4]

[0156] Stability of DFD4 and DFD13 at a concentration of 20 mg / mL for 2 weeks (% HMW)

[0157]

[0158] As shown in Table 4, the %HMW level at the initial stage (day 0) was 0.91% for DFD4 and 0.56% for DFD13. After 2 weeks of storage at 25°C, the %HMW level of DFD4 increased to 8.83%, but this was not observed in DFD13. Compared to DFD4, DFD13 showed a lower %HMW rate at both the initial stage and 2 weeks, indicating a significant reduction in the %HMW rate of the FGF21 mutant fusion protein due to the introduction of the EIRP mutation.

[0159] Experimental Example 3. Pharmacokinetic Evaluation of Fusion Proteins

[0160] Experimental Example 3-1. Experimental Methods for Pharmacokinetic Evaluation

[0161] Six-week-old male ICR mice purchased from Orient BIO (Korea) were grouped (n = 3 / blood collection time) to have similar average body weight the day before drug treatment and administered once subcutaneously with each sample at 1 mg / kg (RGE: 2 mg / kg). Blood samples were then collected at 1, 4, 8, 12, 24, 48, 72, and 96 hours post-injection. The concentration of full-length FGF21 protein in blood was measured using a complete human FGF21 ELISA kit (F1231-K01, Eagle Biosciences, USA) that is immunoreactive to the N- and C-termini of the FGF21 protein. The concentrations in blood samples collected up to 96 hours after subcutaneous injection of each fusion protein into the mice were measured, and pharmacokinetic parameters for each sample were calculated.

[0162] Experimental Example 3-2. Pharmacokinetic Activity Assessment

[0163] Based on a graph showing the concentration of each protein in the blood of mice relative to the time following subcutaneous administration of the fusion protein ( Figure 5 The pharmacokinetic parameters were calculated. The data are shown in Table 5 below.

[0164] [Table 5]

[0165]

[0166] The pharmacokinetic profiles of each fusion protein are compared and evaluated based on the area under the curve (AUC), which indicates the level of drug exposure.

[0167] As shown in Table 5, after comparing DFD4 with DFD13 and DFD6 with DFD73, it was determined that the introduction of the EIRP sequence increased the AUC value by approximately 10 to 20%. By comparing DFD9 with DFD4, the introduction of TGLEAV increased the AUC value by approximately 6 times.

[0168] Furthermore, the mutations TGLEAN, G170N, and G174N were engineered to prolong the half-life by introducing N-glycosylation into the C-terminus of FGF21, which is known to be proteasically hydrolyzed in vivo. The increased AUC due to the introduction of N-glycosylation was determined by comparing the mutants with each control. To confirm the AUC increase due to the introduction of N-glycosylation, the AUC values ​​of DFD6 (from *E. coli*) without glycosylation were compared with those of DFD6 from human cell lines. DFD6 from human cell lines showed a 3-fold or greater increase in AUC compared to DFD6 (from *E. coli*), demonstrating an improvement in the pharmacokinetic profile due to glycosylation.

[0169] A180E is a mutation disclosed in WO 2009 / 149171 owned by Amgen Inc. When the A180E mutation was further introduced into mutants DFD13 or DFD73 containing the TGLEAV or G170N mutations, respectively, the resulting mutants DFD18 or DFD74 showed an additional 2 to 3-fold increase in AUC values.

[0170] In summary, the pharmacokinetic parameters were improved by introducing different mutations and their combinations compared to DFD9 (wild-type FGF21 fusion protein). The fusion protein showing the greatest improvement in AUC was DFD74, which contained EIRP, G170N, and A180E mutations, showing an approximately 45-fold increase in AUC compared to DFD9. Furthermore, considering that the dose of RGE (Amgen) is 2 mg / kg body weight, DFD74 may have a higher level of drug exposure compared to RGE. The overall pharmacokinetic improvements due to mutations are summarized in Table 6 below.

[0171] [Table 6]

[0172]

[0173] Experiment Example 4. Activity evaluation of the fusion protein in ob / ob mice

[0174] Experimental Example 4-1. Experimental Methods for Evaluating Activity in ob / ob Mice

[0175] ob / ob mice, characterized by hyperglycemia, insulin resistance, hyperphagia, fatty liver, and obesity due to a leptin genetic defect, are widely used in type 2 diabetes research. Male ob / ob mice (Harlan, USA) were purchased from Raonbio (Korea). These mice were 5–6 weeks old upon arrival and 8–9 weeks old after 3 weeks of acclimatization before drug treatment. Mice were grouped (n = 8 / group) to have similar body weight and tail blood glucose levels the day before drug treatment (day 0), and samples were administered subcutaneously once according to the appropriate dose for each sample. Treatment was administered Dulbecco phosphate-buffered saline (DPBS, Gibco, USA) as a carrier, and blood glucose concentrations were measured using a GlucoDr glucose meter (All Medicus, Korea). Non-fasting blood glucose levels and body weight were measured daily until day 14 post-treatment. Glycated hemoglobin levels were also measured in each group before administration and after testing. Glycated hemoglobin levels were calculated using the DCA 2000 HbA1c kit (Siemens, 5035C).

[0176] Example 4-2. Evaluation of activity in ob / ob mice

[0177] Changes in non-fasting blood glucose levels and body weight were observed in male ob / ob mice after a single subcutaneous injection of 30 or 100 nmol / kg DFD18 and DFD72, or 10, 30 or 100 nmol / kg DFD74.

[0178] It was confirmed that DFD18, DFD72, and DFD74 all have a dose-dependent effect in lowering blood glucose levels. Comparing the three agents at a high dose of 100 nmol / kg, DFD72 and DFD74 showed a more significant effect in lowering blood glucose levels compared to DFD18. Figure 6 In addition, Fc-FGF21 (Lilly), which was used as a control in the test, was less effective in lowering blood glucose levels compared to DFD18, DFD72, and DFD74 at the same dose level (30 nmol / kg).

[0179] Regarding their effects on weight loss, comparing the three agents at a high dose of 100 nmol / kg, DFD72 was the most effective in ob / ob mice, resulting in a weight loss of approximately 6%, followed by DFD18, and then DFD74. Figure 7 ).

[0180] After the test, glycated hemoglobin levels, which indicate the mean blood glucose level, were measured, and changes in mean blood glucose were analyzed in each test group. Except for the control group treated with the control protein Fc-FGF21 (Lilly), all treatment groups showed negative differences between before and after administration, confirming the effectiveness of the test protein in lowering blood glucose compared to the control. Figure 8 ).

[0181] Experimental Example 5. Activity Evaluation of Fusion Protein in HFD / STZ Mice

[0182] Experimental Example 5-1. Experimental Methods for Evaluating Activity in HFD / STZ Mice

[0183] The effects of the FGF21 mutant fusion protein on reducing blood glucose and body weight were compared and evaluated in another diabetic model, the HFD / STZ mouse model. A conventional diet-induced obese mouse model (induced by feeding C57BL / 6 mice a 60 kcal% high-fat diet for 8 weeks or longer) still exhibits weak hyperglycemia and diabetes characteristics despite inducing insulin resistance. HFD / STZ mice, which compensate for the defects in the conventional diet-induced obese mouse model, are able to generate dysfunctional β-cells in the pancreas and exhibit reduced insulin secretion due to the high-fat diet (HFD) and administration of low levels of streptozotocin (STZ), and are therefore suitable for pharmacological studies of type 2 diabetes.

[0184] Specifically, to induce the HFD / STZ mouse model, C57BL / 6 mice (Japan, SLC) were fed a high-fat diet of 60 kcal% for 4 weeks, followed by intraperitoneal administration of 50 mg / kg STZ (Sigma, 85882) daily for 3 days to induce pancreatic β-cell dysfunction. After an additional 2 weeks of high-fat feeding, mice with non-fasting blood glucose levels of 200 mg / dL or higher were used for testing. Mice were grouped (n = 6 / group) based on the average body weight and tail blood glucose levels of similar individuals the day before drug treatment (day 0), and samples were administered subcutaneously once according to the appropriate dose for each sample. Dulbecco phosphate-buffered saline (DPBS, Gibco, USA) was administered as a carrier, and blood glucose concentrations were measured using a GlucoDr glucose meter (All Medicus, Korea). Non-fasting blood glucose levels and body weight were measured daily until day 14 post-administration. Glycated hemoglobin levels were also measured in each group before administration and after testing. Glycated hemoglobin levels were calculated using the DCA 2000 HbA1c kit (Siemens, 5035C).

[0185] Experimental Example 5-2. Activity Evaluation in HFD / STZ Mice

[0186] Changes in non-fasting blood glucose levels and body weight over time were observed in male HFD / STZ mice after a single subcutaneous injection of 10 nmol / kg DFD72 or DFD74.

[0187] Regarding changes in non-fasting blood glucose levels, DFD72 and DFD74 were confirmed to have similar effects in lowering blood glucose levels, and the blood glucose-lowering effect was maintained until day 10 after administration, and then lost due to drug metabolism after day 10. Figure 9 DFD72 showed a longer-lasting effect than DFD74 on changes in non-fasting blood glucose levels after 10 days of administration.

[0188] Regarding the effect of FGF21 mutant protein on weight loss, both DFD72 and DFD74 were shown to have similar effects in reducing weight by approximately 5%, and the effect disappeared after 10 days post-administration. Figure 10 ).

[0189] After the test, glycated hemoglobin levels, which indicate the mean blood glucose level, were measured, and changes in mean blood glucose were analyzed in each test group. While the glycated hemoglobin level increased by 0.25 in the carrier group, it increased by 0.1 in the DFD74-treated group and decreased by 0.27 in the DFD72-treated group. Figure 11 ).

[0190] Experimental Example 6. Activity of the fusion protein in diet-induced obese mice

[0191] Experimental Example 6-1. Experimental Methods for Evaluating Activity in Diet-Induced Obese Mice

[0192] The weight-reduction effect of DFD18 (an FGF21 mutant fusion protein) was evaluated in diet-induced obese mice. For a diet-induced obesity model, C57BL / 6J mice were purchased from Central Lab.Animal Inc. and fed a high-fat diet (investigation diet) containing 60 kcal% fat for 8 to 12 weeks. Mice were grouped (n = 8 / group) to have similar mean body weights the day before drug treatment (day 0), followed by a single subcutaneous administration of 30 nmol / kg sample. Changes in body weight were compared with those treated with a carrier (PBS).

[0193] Experimental Example 6-2. Protein Activity in Diet-Induced Obese Mice

[0194] In a diet-induced obese mouse model following a single administration of 30 nmol / kg DFD18, the study confirmed that the weight loss effect persisted up to day 10 after administration, with the maximum weight loss (approximately 18%) occurring on day 11 and maintained until day 14. Figure 12).

[0195] Preparation Example 2. Preparation and purification of bifunctional proteins

[0196] Preparation Example 2-1. Preparation of expression vectors for expressing bifunctional proteins

[0197] To identify the effects of the sequence of the GLP-1 mutant protein and the sequence of its fused Fc hinge on its in vitro activity, pharmacokinetic profile, and pharmacological potency, multiple sequences of the Fc-fused GLP-1 mutant protein were designed. The sequences of the GLP-1 mutant protein are listed in Table 7 below, and the sequences of the Fc-fused GLP-1 mutant are listed in Table 8.

[0198] [Table 7]

[0199] 43 GLP-1(A2G) 44 GLP-1(GE) 45 GLP-1 (GG) 46 GLP-1 (GEG)

[0200] [Table 8]

[0201] 49 DFD52: GLP1(A2G)-HyFc5 50 DFD53: GLP1(A2G)-HyFc40 51 DFD54: GLP1(GE)-HyFc5 52 DFD55: GLP1(GE)-HyFc40 53 DFD56: GLP1(GG)-HyFc5 54 DFD57: GLP1(GG)-HyFc40 55 DFD58: GLP1(GEG)-HyFc5 56 DFD59: GLP1(GEG)-HyFc40

[0202] In Table 8, HyFc5 refers to SEQ ID NO: 47, and HyFc40 refers to SEQ ID NO: 48.

[0203] To investigate the effects of the sequences of GLP-1 mutant and FGF21 mutant proteins, the sequence of the Fc hinge fused to the GLP-1 mutant, and the sequence of the linker connecting the FGF21 mutant and the Fc region on their in vitro activity, pharmacokinetic profile, and pharmacological potency, multiple sequences of bifunctional proteins were designed. The sequences of bifunctional proteins containing both GLP-1 mutant and FGF21 mutant proteins are listed in Table 9 below. Each bifunctional protein contains, from the N-terminus to the C-terminus, the GLP-1 mutant protein, the Fc region of the immunoglobulin, the linker, and the FGF21 mutant protein.

[0204] [Table 9]

[0205]

[0206] Specifically, based on the amino acid sequence of each protein, the nucleotide sequence encoding each bifunctional protein was synthesized after consulting Bioneer Corporation (Korea). NheI and NotI restriction enzyme sequences were added to the 5′ and 3′ ends of the nucleotide sequence encoding each bifunctional protein, respectively. A start codon for protein translation and a leader sequence (MDAMLRGLCCVLLLCGAVFVSPSHA) for secreting the expressed protein outside the cell were inserted next to the 5′ restriction enzyme sequence. A stop codon was inserted next to the nucleotide sequence encoding each bifunctional protein. The nucleotide sequence encoding each bifunctional protein was cloned into the pTrans-empty expression vector using both NheI and NotI restriction enzymes. The pTrans-empty expression vector, purchased from CEVEC Pharmaceuticals (Germany), has a simple structure containing a CMV promoter, an origin of replication derived from pUC, an origin of replication derived from SV40, and an ampicillin resistance gene.

[0207] Preparation Example 2-2. Construction of plasmid DNA for expressing Fc fusion GLP-1 mutants and bifunctional proteins

[0208] E. coli were transformed with each expression vector constructed in Preparation Example 2-1 to obtain a large quantity of plasmid DNA for expression. E. coli cells with weakened cell walls were transformed with each expression vector by heat shock, and the transformed cells were plated on LB plates to obtain colonies. The colonies thus obtained were inoculated into LB medium and incubated at 37°C for 16 hours, yielding 100 mL of each E. coli culture containing each expression vector. The resulting E. coli were centrifuged to remove the medium, and then P1, P2, P3 solutions (QIAGEN, catalog number: 12963) were added to rupture the cell walls, resulting in a DNA suspension in which the protein and DNA phases were separated. Plasmid DNA was purified from the DNA suspension thus obtained 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 (Thermoscientific, Nanodrop Lite). The DNA thus obtained was used for expression.

[0209] Preparation Example 2-3. Expression of Fc-fused GLP-1 mutant and bifunctional protein in CAP-T cells

[0210] 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) already cultured in PEM medium (Life Technologies) using PEI solution (Polyplus, catalog number: 101-10N). The 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 to 7 days, the culture was centrifuged to remove cells, and a supernatant containing each protein was obtained.

[0211] Preparation Examples 2-4. Fc-fusion GLP-1 mutant and purification of bifunctional protein

[0212] The protein A affinity column (GE Healthcare) was equilibrated with 1X PBS buffer (pH 7.4). Culture supernatant containing each Fc-fusion GLP-1 mutant and bifunctional protein obtained in Preparation Examples 2-3 was filtered through a 0.2 μm filter and then loaded into the protein A affinity column. The column was washed with 1X PBS buffer (pH 7.4) and then eluted with 100 mM glycine buffer (pH 3.0). An anion exchange resin column (POROS) was used. @ The HQ50μn (Thermo Fisher Scientific) was used to purify proteins obtained by affinity chromatography. The anion exchange resin column was equilibrated with 50 mM Tris buffer (pH 8.0) before loading it with the proteins eluted from affinity chromatography.

[0213] After washing the column with 50 mM Tris buffer (pH 8.0), 50 mM Tris buffer (pH 8.0) was partitioned along the concentration gradient, and the eluted fractions were analyzed. Each eluted fraction was analyzed using size exclusion chromatography (SEC-HPLC), and fractions containing high-purity Fc-fusion GLP-1 mutants and bifunctional proteins were collected and dialyzed overnight at 4°C with the final buffer solution (1XPBS, 1 mM EDTA, pH 7.4). After dialysis, the resulting protein stock solution was concentrated at 3,000 rpm at 4°C using a 30,000 MW cutoff centrifuge filter. The concentration of each protein was determined by BCA quantification.

[0214] Experimental Example 7. In vitro activity of bifunctional proteins

[0215] Experimental Example 7-1. Activity of DFD23, DFD24, DFD25, DFD26, DFD27, DFD28 and DFD29

[0216] The in vitro GLP-1 activity of bifunctional 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 bifunctional proteins. To evaluate activity, samples containing the fusion protein (protein stock solutions prepared in Preparation Examples 2-4; hereinafter referred to as "samples") were serially diluted 4-fold at a concentration of 25 nM. Intracellular cAMP (Cisbio, 62AM4PEB) was measured 30 minutes after treatment with the human GLP-1 receptor-overexpressing CHO cell line. The activity was compared with EC... 50 The value is used to evaluate the activity of each protein.

[0217] like Figure 13 As shown, bifunctional proteins containing the GLP-1(A2G) sequence exhibit approximately 2–3 times lower activity than bifunctional proteins containing other GLP-1 mutant sequences. No significant differences in GLP-1 activity were observed among bifunctional proteins containing mutant sequences other than the GLP-1(A2G) sequence.

[0218] Experimental Example 7-2. Activity of DFD59, DFD69, DFD112 and DFD114

[0219] The in vitro GLP-1 activity of the bifunctional proteins DFD69, DFD112, and DFD114, as well as DFD59 (an Fc fusion GLP-1 mutant), prepared in Preparation Example 2, 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 bifunctional proteins. To evaluate activity, samples containing each fusion protein were serially diluted 4-fold at a concentration of 25 nM. Intracellular cAMP (Cisbio, 62AM4PEB) was measured 30 minutes after treatment with the human GLP-1 receptor-overexpressing CHO cell line.

[0220] like Figure 14 As shown, by comparing EC 50 The value is used to evaluate the activity of each protein. Three bifunctional proteins showed similar EC values. 50 The value, while DFD59 (without the FGF21 mutant) showed about 2 times higher activity than the bifunctional protein.

[0221] 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 bifunctional proteins was evaluated using the HEK293 cell line overexpressing human β-klotho (a co-receptor of FGF21). To evaluate activity, samples containing each bifunctional protein were serially diluted 3-fold at a concentration of 3 μM. After culturing for 5 hours in a serum-deficient state, the HEK293 cell line overexpressing human β-klotho was treated for 20 minutes, and then the cells were lysed for 30 minutes at room temperature by adding cell lysis buffer (Cisbio / catalog number 64ERKPEG) with stirring at 60 rpm. The cell lysate solution was mixed with an antibody that could detect ERK and phosphorylated ERK, and the mixture was kept at room temperature for 2 hours. Fluorescence was detected using a fluorescence detector (TECAN / GENiosPro). The activity was compared by comparing their EC50 values. 50 The value is used to measure activity.

[0222] The in vitro activities of the FGF21 moiety of the bifunctional proteins DFD69, DFD112, and DFD114 were confirmed to be similar, such as... Figure 14 As shown.

[0223] Example 8. Pharmacokinetic Evaluation of Bifunctional Proteins

[0224] Experimental Example 8-1. Experimental Methods for Pharmacokinetic Evaluation

[0225] Six-week-old male ICR mice purchased from Orient BIO (Korea) were grouped (n = 3 / blood collection time) to have similar average body weights the day before drug treatment and administered a single subcutaneous dose of 1 mg / kg volume to each sample. Blood samples were collected at 1, 4, 8, 12, 24, 48, 72, 96, 144, 192, and 240 hours post-injection. The concentrations of each bifunctional protein in the blood were measured based on both the FGF21 and GLP-1-Fc moieties. The concentration of the full-length FGF21 moiety of the bifunctional protein in the blood was measured using a full-length human FGF21 ELISA kit (F1231-K01, Eagle Biosciences, USA) that is immunoreactive to the N- and C-termini of the FGF21 protein. Furthermore, the concentration of the active GLP-1-Fc moiety of the bifunctional protein in the blood was measured using antibodies immunoreactive to the N-terminus of GLP-1 and Fc, as determined by ELISA analysis. The concentrations of the FGF21 and GLP-1-Fc fractions of each protein were measured in blood samples collected up to 240 hours after a single subcutaneous injection of each protein into mice, and the pharmacokinetic parameters of each protein were calculated.

[0226] Experimental Example 8-2. Pharmacokinetic Activity Results

[0227] Based on the concentration of each active substance in the blood over time after a single subcutaneous administration of each protein in mice ( Figure 15 The pharmacokinetic parameters of the FGF21 and GLP-1-Fc moieties of the bifunctional protein were calculated. The data are shown in Table 10 below.

[0228] [Table 10]

[0229]

[0230] The pharmacokinetic profiles of each bifunctional protein were compared and evaluated based on the area under the curve (AUC), which indicates the level of drug exposure.

[0231] As shown in Table 10, for the pharmacokinetic parameters of the FGF21 portion, DFD114 showed the highest drug exposure (AUC) and half-life, followed by DFD112 with the second-highest AUC, and then DFD69. Compared to DFD69, DFD114 showed an AUC value that was approximately 2-fold or higher. For the pharmacokinetic parameters of the GLP-1-Fc portion, the four proteins containing the same GLP-1 mutant sequence (DFD59, DFD69, DFD112, and DFD114) showed similar AUC values.

[0232] Example 9. Activity evaluation in db / db mice

[0233] Experimental Example 9-1. Methods for evaluating activity in db / db mice

[0234] Characterized by hyperglycemia, insulin resistance, hyperphagia, fatty liver, and obesity due to a genetic defect in the leptin receptor, and exhibiting more severe hyperglycemia and obesity than ob / ob mice, db / db mice are widely used in type 2 diabetes research. 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 after 3 weeks of acclimatization before drug treatment. Mice were grouped (n = 6 / group) to have similar body weight and tail blood glucose levels as average the day before drug treatment (day 0), and samples were administered subcutaneously once according to the appropriate dose for each sample. Dulbecco phosphate-buffered saline (DPBS, Gibco, USA) was administered as a carrier, and blood glucose concentrations were measured using a GlucoDr glucose meter (All Medicus, Korea). Non-fasting blood glucose levels and body weight were measured daily until day 14 post-administration. Glycated hemoglobin levels were also measured in each group before administration and after testing. Glycated hemoglobin levels were calculated using the DCA2000 HbA1c kit (Siemens, 5035C).

[0235] Experimental Example 9-2. Evaluation of activity in db / db mice

[0236] Following a single subcutaneous injection of 10 or 30 nmol / kg of the bifunctional protein DFD114, a single subcutaneous injection of 30 nmol / kg of the long-acting GLP-1-Fc monofunctional protein DFD59, and a combination of 30 nmol / kg of DFD59 and DFD74 (GLP-1-Fc and Fc-FGF21 monofunctional proteins, respectively), changes in non-fasting blood glucose levels and body weight were observed in male db / db mice to compare the effects of the bifunctional protein DFD114 with the combination of Fc-FGF21 and GLP-1-Fc monofunctional proteins.

[0237] The long-acting GLP-1-Fc protein DFD59 caused a sharp decrease in blood glucose levels on day 1 after administration, but the decrease diminished after day 2, and by day 4, blood glucose levels were similar to those in the carrier-treated group. Meanwhile, the group treated with DFD114 showed superior blood glucose-lowering effects on day 3, and the effect of lowering blood glucose levels disappeared more rapidly on day 4 compared to 30 nmol / kg, indicating a dose-dependent difference in the duration of the blood glucose-lowering effect. Compared to other groups, the group treated with each protein in combination showed the most durable blood glucose-lowering effect, indicating that the combination of GLP-1 and FGF21 has excellent glycemic control efficacy. Figure 16 ).

[0238] Regarding the effect on weight loss, the group treated with the combination of DFD59 and DFD74 showed the greatest effect on weight loss, and the group treated with 30 nmol / kg DFD114 also showed a significant effect on weight loss. Figure 17 ).

[0239] After the test, the glycated hemoglobin level, which indicates the average blood glucose level, was measured, and the average blood glucose change in each test group was analyzed. Figure 18 As shown, the groups treated with the carrier exhibited increased glycated hemoglobin levels after the test compared to the untreated groups, and the groups treated with DFD59 showed a similar increase. The group treated with 30 nmol / kg DFD114 showed the largest reduction in glycated hemoglobin levels, followed by the group receiving combination therapy, and then the group treated with 10 nmol / kg DFD114. When the protein was evaluated based on the reduction in glycated hemoglobin levels in each treatment group, the bifunctional protein DFD114 was confirmed to have a stronger glycemic-lowering effect compared to the single-function GLP-1-Fc or Fc-FGF21 proteins alone.

[0240] Experimental Example 10. Activity of fusion protein in HFD / STZ mice

[0241] Experimental Example 10-1. Experimental Methods for Evaluating Activity in HFD / STZ Mice

[0242] The effects of bifunctional proteins on reducing blood glucose and body weight were compared and evaluated in another diabetic model, the HFD / STZ mouse model.

[0243] A conventional diet-induced obese mouse model (induced by feeding C57BL / 6 mice a 60 kcal% high-fat diet for 8 weeks or longer) still exhibits weak hyperglycemia and diabetes characteristics despite inducing insulin resistance. HFD / STZ mice, which compensate for the deficiencies in the conventional diet-induced obese mouse model, can generate dysfunctional β-cells in the pancreas and reduce insulin secretion due to a high-fat diet (HFD) and administration of low levels of streptozotocin (STZ), and can be 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 STZ (Sigma, 85882) daily for 3 days to induce pancreatic β-cell dysfunction. After an additional 2 weeks of high-fat diet feeding, mice with non-fasting blood glucose levels of 200 mg / dL or higher were selected for testing. Mice were grouped (n = 6 / group) to have similar body weight and tail blood glucose levels the day before drug treatment (day 0), and each sample was administered subcutaneously once at the appropriate dose for its respective group. Dulbecco phosphate-buffered saline (DPBS, Gibco, USA) was administered as a carrier, and blood glucose concentrations were measured using a GlucoDr glucose meter (All Medicus, Korea). Non-fasting blood glucose levels and body weight were measured daily until day 14 post-administration. Glycated hemoglobin levels were also measured in each group before administration and after testing. Glycated hemoglobin levels were calculated using the DCA 2000 HbA1c kit (Siemens, 5035C).

[0244] Experimental Example 10-2. Activity in HFD / STZ Mice

[0245] Following a single subcutaneous injection of 3 nmol / kg or 10 nmol / kg of the bifunctional protein DFD114, 10 nmol / kg of the Fc-fused GLP-1 mutant DFD59, or 10 nmol / kg of each of the Fc-fused FGF21 mutants DFD72 and DFD74, changes in non-fasting blood glucose levels and body weight over time were observed in male HFD / STZ mice. DFD59 and DFD74 were also administered subcutaneously once at 10 nmol / kg to compare the effects of combined administration of monofunctional proteins with those of bifunctional proteins.

[0246] like Figure 19 As shown, regarding changes in blood glucose levels up to day 4, administration of DFD72 and DFD74 (long-acting FGF21 monofunctional proteins) resulted in a slower decrease in blood glucose, while administration of DFD114 (a long-acting protein containing GLP-1), DFD59, and the combination of DFD59 and DFD74 showed a more rapid decrease in blood glucose from day 1. Similar to the results in db / db mice, DFD59 showed a sharp decrease in blood glucose in the early stages, but the decrease in blood glucose disappeared 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 groups treated with 10 nmol / kg DFD114, DFD72, DFD74, and the combination of these treatments.

[0247] Regarding the effect on weight loss, the group treated with the combination of DFD59 and DFD74 showed the greatest weight loss (7-8%), and the group treated with 10 nmol / kg DFD114 also showed a significant weight loss (approximately 6%). Figure 20 The group treated with DFD59 showed a 5% weight loss on day 1 after administration, but this effect disappeared after day 2 and became similar to the carrier group after day 7. The groups treated with each long-acting FGF21 monofunctional protein DFD72 and DFD74 showed a slower weight loss of 4–5% until day 7 after administration, and this effect disappeared after day 10.

[0248] After the test, the glycated hemoglobin level, which indicates the average blood glucose level, was measured, and the average blood glucose change in each test group was analyzed. Figure 21Compared to pre-treatment levels, the load cell group showed an increase in glycated hemoglobin (HbA1c) levels at the end of the test, and the group treated with DFD59 showed a similar increase. In contrast, the group treated with DFD114 showed a dose-dependent reduction in HbA1c levels, with the group treated with 10 nmol / kg DFD114 showing the greatest effect in terms of the reduction in HbA1c levels (-0.42%). The group treated with a combination of DFD59 and DFD74 showed a similar reduction in HbA1c levels as DFD114 (-0.38%). For the long-acting FGF21 monofunctional protein, DFD72 was observed to be superior to DFD74. Comparing proteins based on the reduction in HbA1c levels in each group confirmed that the bifunctional protein DFD114 was superior to both the GLP-1-Fc and Fc-FGF21 monofunctional proteins.

[0249] Experimental Example 11. Prediction and Evaluation of Immunogenicity

[0250] Experimental Example 11-1. Methods and Results for Predicting Immunogenicity

[0251] To predict the potential immunogenicity of bifunctional proteins, a computer analysis of the immunogenicity of each protein was performed.

[0252] Specifically, by using iTope TM and TCED TM Methods (Prediction of immunogenicity of therapeutic proteins: validity of computational tools, BioDrugs, 2010) were used to rapidly screen for the potential immunogenicity of bifunctional proteins. Based on these two methods, T-cell epitopes can be predicted more accurately than computational analysis methods that rely solely on MHC class II binding assays.

[0253] Experimental Example 11-2. Methods and Results for In Vitro Evaluation of Immunogenicity

[0254] To evaluate the potential immunogenicity of bifunctional proteins, EpiScreen was used. TMAnalysis (Increased brainbio-distribution and chemical stability and decreased immunogenicity of an engineered variant of GDNF, Exp Neurol, 2015). When immunogenicity is detected, the amino acid sequence inducing immunogenicity can be identified by T-cell epitope mapping, and deimmunization mutants with minimized immunogenicity can be designed and prepared using computer prediction to re-evaluate immunogenicity.

Claims

1. A bifunctional protein comprising: a mutant fibroblast growth factor 21 (FGF21) protein; a bioactive protein; and the Fc region of an immunoglobulin. The mutations in the FGF21 mutant protein consist of mutations (1) to (3): (1) The amino acids from the N-terminus of wild-type FGF21 protein, from position 98 to 101, are replaced with the amino acid sequence EIRP (SEQ ID NO: 68). (2) The amino acids from the N-terminus of the wild-type FGF21 protein, from position 170 to 174, are replaced with the amino acid sequence TGLEAN (SEQ ID NO: 70), wherein the amino acid residue N of the FGF21 mutant protein introduced by the mutation is glycosylated; (3) Replace the 180th amino acid from the N-terminus of the wild-type FGF21 protein with amino acid E. The amino acid sequence of the wild-type FGF21 protein is represented by SEQ ID NO:

1. The bifunctional protein comprises, from N-terminus to C-terminus, the bioactive protein, the Fc region of the immunoglobulin, and the FGF21 mutant protein, and, The bioactive protein therein consists of an amino acid sequence selected from any of the amino acid sequences represented by SEQ ID NO: 44 to 46.

2. The bifunctional protein according to claim 1, wherein the amino acid sequence of the FGF21 mutant protein is represented by SEQ ID NO:

21.

3. The bifunctional protein of claim 1, wherein the bifunctional protein further comprises a linker.

4. The bifunctional protein of claim 3, wherein the linker connects the FGF21 mutant protein to the Fc region of the immunoglobulin.

5. The bifunctional protein of claim 4, wherein the linker is connected to the C-terminus of the Fc region of the immunoglobulin and the N-terminus of the FGF21 mutant protein.

6. The bifunctional protein of claim 4, wherein the linker is a peptide consisting of 10 to 30 amino acid residues.

7. The bifunctional protein according to claim 6, wherein the linker has an amino acid sequence represented by any one of SEQ ID NO: 2 to 5.

8. The bifunctional protein of claim 1, wherein the Fc region of the immunoglobulin is any one of the Fc regions of IgG1, IgG2, IgG3, IgG4 and IgD, or a hybrid Fc region containing a combination thereof.

9. The bifunctional protein of claim 8, wherein the hybrid Fc comprises an IgG4 region and an IgD region.

10. The bifunctional protein of claim 1, wherein a linker is further provided between the Fc region of the immunoglobulin and the FGF21 mutant protein.

11. The bifunctional protein of claim 10, wherein the linker is connected to the C-terminus of the Fc region of the immunoglobulin and the N-terminus of the FGF21 mutant protein.

12. The bifunctional protein of claim 10, wherein the linker is a peptide consisting of 10 to 30 amino acid residues.

13. The bifunctional protein of claim 10, wherein the linker has an amino acid sequence represented by any one of SEQ ID NO: 2 to 5.

14. A pharmaceutical composition comprising a bifunctional protein according to any one of claims 1 to 13, for the treatment of diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, or cardiovascular disease.

15. The pharmaceutical composition according to claim 14, for the treatment of non-alcoholic steatohepatitis.

16. An isolated nucleic acid molecule encoding a bifunctional protein according to any one of claims 1 to 13.

17. An expression vector comprising the nucleic acid molecule of claim 16.

18. A host cell comprising the expression vector of claim 17.

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