GLP-1 fusion proteins and uses thereof

Abstract

A method of modulating blood glucose levels and / or treating diabetes is disclosed. The method comprises the step of administering a fusion peptide of a GLP-1 peptide and an Fc region. The Fc region is a hybrid Fc region comprising a hinge region, a CH2 domain and a CH3 domain in a direction from N-terminus to C-terminus wherein the hinge region comprises a human IgD hinge region, the CH2 domain comprises part of amino acid residues of the CH2 domain of human IgD and human IgG4, and the CH3 domain comprises part of amino acid residues of the CH3 domain of human IgG4 and is glycosylated in the IgD hinge region. The fusion peptides exhibit reduced side effects, such as emesis, nausea and / or increased heart rate.

Description

[0001] This application is a divisional application with application date of March 9, 2020, application number 202080019785.0, and invention name “GLP-1 fusion protein and its use”. Technical Field

[0002] This application claims priority to U.S. Provisional Application No. 62 / 815,486, filed on March 8, 2019, the contents of which are incorporated by reference in their entirety.

[0003] Disclosed is a use of a fusion protein using a glucagon-like peptide and an Fc region in regulating blood sugar levels. Background Art

[0004] Diabetes is associated with higher cardiovascular morbidity and mortality. Hypertension, hyperlipidemia, and diabetes are independently associated with an increased risk of cardiovascular disease. Patients with type 2 diabetes have a two- to four-fold increased risk of cardiovascular disease compared with those without diabetes.

[0005] Glucagon-like peptide-1 (GLP-1) is known as a pleiotropic peptide with metabolic and cardiovascular benefits. It is derived from preproglucagon, a 158 amino acid precursor polypeptide that is processed in different tissues to form many different proglucagon-derived peptides. Proglucagon-derived peptides include glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM), which are involved in a variety of physiological functions including glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, and regulation of food intake.

[0006] GLP-1 is a 37-amino acid peptide corresponding to amino acids 72 to 108 of proglucagon (92 to 128 of proglucagon). The major biologically active form is a 30-amino acid peptide hormone (GLP-1(7-37) acid) that is produced in the intestine after a meal and rapidly degraded by the abundant endogenous protease DPP4.

[0007] Many GLP-1 analogs and derivatives are known. These GLP-1 analogs include exendins, which are peptides found in the venom of the Gila monster. Exendins have sequence homology with native GLP-1 and can bind to the GLP-1 receptor and initiate the signal transduction cascade responsible for many of the activities attributed to GLP-1 (7-37) OH. These GLP-1 analogs and derivatives are referred to herein as "GLP-1 peptides," "GLP-1 compounds," or "GLP-1RAs," and these terms are used interchangeably throughout the application.

[0008] GLP-1 peptides show the most promise in treating non-insulin-dependent diabetes. Unlike insulin, which can cause hypoglycemia when administered, GLP-1 is controlled by blood glucose levels, and there is no risk of hypoglycemia associated with GLP-1 peptide therapy.

[0009] Various long-acting GLP-1 peptides (such as GLP-1 fusion proteins) have longer half-lives while maintaining effects on β-cell function, insulin sensitivity, body weight, and cardiovascular system. 1-4 multiple beneficial effects and lack of life-threatening adverse events such as hypoglycemia 5 , the various long-acting GLP-1 peptides mentioned above have been intensively studied in the past few decades.

[0010] However, despite its appeal as an antidiabetic drug, some studies suggest that side effects of GLP-1 therapy, such as nausea, vomiting, and increased heart rate, may interfere with the continued growth of GLP-1RAs. 6-8 .

[0011] According to a cross-sectional survey by Sikirica et al., nausea / vomiting was the most important factor affecting physicians and patients in discontinuing GLP-1 peptides, accounting for approximately 46% and 64%, respectively. In addition, approximately half of the patients reported that nausea / vomiting-related factors were the most troublesome problem associated with GLP-1RAs. 8 Another potential disadvantage of GLP-1 peptides is an increase in heart rate, which has been reported in almost all clinical trials of GLP-1 peptides. 10,11 This may be a direct effect of peripherally administered GLP-1 peptide on cardiomyocytes 12,13 Long-acting GLP-1 peptides are more significant and sustained than short-acting GLP-1 peptides 6 The increase in heart rate caused by long-acting GLP-1 peptides was small, but the increase in heart rate may represent a safety issue as it is one of the risk factors for cardiovascular disease in diabetic patients with advanced heart failure. Overall, these side effects may weaken the efficacy of GLP-1 peptides in real-world treatment, so this suggests the need to develop safer GLP-1 peptides to ultimately improve treatment efficacy. Summary of the invention

[0012] Technical issues

[0013] The present disclosure relates to the use of Fc-fused GLP-1 peptides (hereinafter sometimes referred to as "GLP-1-gFc" or simply "fusion protein" or "fusion peptide") having unique binding affinity properties to its receptor, which is designed to improve in vivo stability and safety. The GLP-1-gFc described herein exhibits good pharmacokinetic (PK) and pharmacodynamic (PD) properties as a long-acting GLP-1RA, and has safer properties compared to commercial GLP-1 analogs (such as dulaglutide).

[0014] Technical Solution

[0015] In one aspect, a method of treating diabetes is provided, comprising administering a fusion protein to a subject in need thereof. In one embodiment, the diabetes is insulin-dependent. In another embodiment, the diabetes is non-insulin-dependent.

[0016] Another aspect includes a method of controlling or regulating glucose levels in a subject, the method comprising administering to a subject in need thereof a fusion protein described herein.

[0017] In one embodiment, the subject has type 2 diabetes.

[0018] In another embodiment, the subject may suffer from metabolic syndrome.

[0019] The fusion protein comprises IgFc and a GLP-1 peptide linked to the Fc. In one embodiment, the Fc is a hybrid comprising an IgG4 CH2 / CH3 portion, an IgD CH2 portion, and an IgD hinge portion, wherein the IgD hinge portion is glycosylated.

[0020] In another embodiment, the GLP-1 peptide may have no more than 6 amino acids that are different from the corresponding amino acids in GLP-1 (7-37) (SEQ ID NO: 1), GLP-1 (7-36) (SEQ ID NO: 11), or Exendin-4 (SEQ ID NO: 10). Even more preferably, the GLP-1 peptide has no more than 5 amino acids that are different from the corresponding amino acids in GLP-1 (7-37) having the sequence SEQ ID NO: 1, GLP-1 (7-36) (SEQ ID NO: 11), or Exendin-4 having the sequence SEQ ID NO: 10. Preferably, the GLP-1 peptide has no more than 4, 3 or 2 amino acids that are different from the corresponding amino acids in GLP-1 (7-37), GLP-1 (7-36), or Exendin-4. In a specific embodiment, the GLP-1 peptide as part of the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 and 11-34. In one embodiment, the IgD hinge portion can have an amino acid sequence selected from the group consisting of SEQ ID NOS: 35-38.

[0021] The following exemplary embodiments are disclosed.

[0022] Embodiment 1. A method for regulating blood glucose level in a subject in need thereof, comprising administering to the subject an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region,

[0023] Among them, the immunoglobulin Fc region (b) contains

[0024] (i) an isolated IgD hinge region consisting of 35 to 49 consecutive amino acid residues from the C-terminus of SEQ ID NO: 35; and

[0025] (ii) CH2 domain and CH3 domain of immunoglobulin Fc polypeptide.

[0026] Embodiment 2. The method of embodiment 1, wherein the effective amount is from about 0.01 mg / kg to about 1 mg / kg body weight.

[0027] Embodiment 3. The method of any of the preceding embodiments, wherein the fusion peptide is administered parenterally at intervals of one week or longer.

[0028] Embodiment 4. The method of any of the preceding embodiments, wherein the subject suffers from diabetes, glucose intolerance and / or insulin resistance.

[0029] Embodiment 5. The method of any of the preceding embodiments, wherein the GLP-1 peptide (a) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NOS: 10 to 34.

[0030] Embodiment 6. The method of any of the preceding embodiments, wherein the isolated IgD hinge region (i) comprises the amino acid sequence of SEQ ID NO:36, 37 or 38.

[0031] Embodiment 7. The method of any of the preceding embodiments, wherein the immunoglobulin Fc region (b) comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 4 to 8.

[0032] Embodiment 8. The method of any of the preceding embodiments, wherein the fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 40 to 42 or 54.

[0033] Embodiment 9. The method of any of the preceding embodiments, wherein the fusion peptide is administered at a dose of 0.01 mg / kg to 0.2 mg / kg at weekly intervals or once a week.

[0034] Embodiment 10. The method of any of the preceding embodiments, wherein the fusion peptide is administered at a dose of 0.2 mg / kg to 0.5 mg / kg at intervals of two weeks or every other week.

[0035] Embodiment 11. The method of any of the preceding embodiments, wherein the subject suffers from diabetes.

[0036] Embodiment 12. The method of any of the preceding embodiments, wherein the diabetes is type II diabetes.

[0037] Embodiment 13. The method of any of the preceding embodiments, wherein the fusion peptide is administered subcutaneously.

[0038] Embodiment 14. The method of any of the preceding embodiments, wherein the fusion peptide is a dimer comprising two peptides linked together by a sulfide bond, wherein each peptide comprises an Fc region (b) having a sequence number of SEQ ID NO: 4, 5, 6, 7 or 8.

[0039] Embodiment 15. A method for preventing and / or treating diabetes in a subject in need thereof, comprising the step of administering to the subject an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region,

[0040] Among them, the immunoglobulin Fc region (b) contains

[0041] (i) an isolated IgD hinge region consisting of 35 to 49 consecutive amino acid residues from the C-terminus of SEQ ID NO: 35; and

[0042] (ii) CH2 domain and CH3 domain of immunoglobulin Fc polypeptide.

[0043] Embodiment 16. The method of embodiment 15, wherein the effective amount is about 0.01 mg / kg to about 1 mg / kg body weight.

[0044] Embodiment 17. The method of any one of embodiments 15-16, wherein the fusion peptide is administered parenterally at intervals of one week or longer.

[0045] Embodiment 18. The method of any one of embodiments 15-17, wherein the fusion peptide is administered at a dose of 0.01 mg / kg to 0.2 mg / kg at weekly intervals or once a week.

[0046] Embodiment 19. The method of any one of embodiments 15-18, wherein the fusion peptide is administered at a dose of 0.2 mg / kg to 0.5 mg / kg at intervals of two weeks or every other week.

[0047] Embodiment 20. The method of any one of embodiments 15-19, wherein the diabetes is non-insulin-dependent diabetes or insulin-dependent diabetes.

[0048] Another aspect includes an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein for use in regulating blood glucose levels.

[0049] Yet another aspect includes a composition for regulating blood glucose levels, the composition comprising an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0050] Yet another aspect includes a therapeutic agent for regulating blood glucose levels, the therapeutic agent comprising an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0051] Another aspect includes the use of an effective amount of a fusion peptide for regulating blood glucose levels, the fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region described herein.

[0052] Yet another aspect includes the use of an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein in the preparation of a medicament for regulating blood glucose levels.

[0053] Another aspect includes a fusion peptide for preventing and / or treating diabetes in an effective amount, the fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0054] Another aspect includes a composition for preventing and / or treating diabetes, the composition comprising an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0055] Another aspect includes a therapeutic agent for preventing and / or treating diabetes, the therapeutic agent comprising an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0056] Another aspect includes the use of an effective amount of a fusion peptide for preventing and / or treating diabetes, the fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0057] Another aspect includes the use of an effective amount of a fusion peptide in the preparation of a medicament for preventing and / or treating diabetes, wherein the fusion peptide comprises (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0058] Effects of the Invention

[0059] A method for regulating blood sugar levels and / or treating diabetes is disclosed. The method comprises the step of administering a fusion peptide of a GLP-1 peptide and an Fc region. The fusion peptide exhibits reduced side effects such as vomiting, nausea and / or increased heart rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1a FIG. 4 is a schematic diagram of an embodiment of dulaglutide and the GLP-1-gFc fusion protein of the present invention.

[0061] Figure 1b to Figure 1h GLP-1-gFc has a higher dissociation constant (Kd) and a lower receptor-mediated response. Different concentrations of GLP-1-gFc and dulaglutide were loaded into harvested GLP-1R expressing cells and then treated with Bright-GloTM detection reagent for 2 minutes. Luminescence ( Figure 1b ). In vitro activity in a transgenic cAMP-specific luciferin and GLP-1 receptor (GLP-1R) expressing cell line (GLP1R_cAMP / luc). The binding affinity of each test article was evaluated by SPR (surface plasmon resonance) analysis system ( Figure 1c The sensorgrams and corresponding values ​​(Ka: binding constant, Kd: dissociation constant, KD: equilibrium dissociation constant) are presented. The results are representative of more than two independent experiments. The results of in vitro activity assays and SPR are expressed as mean ± SEM and the average of different concentrations ( Figure 1d ). Figure 1e : Pharmacokinetics of GLP-1 peptide and GLP-1-Fc after IV administration in SD rats (n=4 / group). Figure 1f : Pharmacokinetics of GLP-1-Fc and GLP-1-gFc after SC administration in SD rats (n=4 / group). Figure 1g: IPGTT results of GLP-1 peptide, GLP-1-Fc and GLP-1-gFc in CD-1 mice, which received each test molecule by SC route and then IP challenged with 2g / kg glucose (n=4 / group / day). The AUC of glucose levels that changed daily was calculated and converted into %AUC of the vehicle to plot a graph of %AUC versus time. The results are expressed as mean ± standard deviation for PK and mean ± SEM for other groups. *p<0.05; **p<0.01; ***p<0.001 relative to the vehicle group; #p<0.05 relative to the GLP-1-Fc group. One-way ANOVA followed by Tukey and Dunnett's T3 test as post hoc analysis. T 1 / 2 , half-life; AUC last , serum concentration-time - finally the area under the concentration-time curve can be measured.

[0062] Figure 1h The binding affinity of GLP-1-gFc is shown to be lower than that of dulaglutide determined by the BLI system. The two figures are representative sensorgrams of the binding affinity of GLP-1-gFc and dulaglutide, and the table shows the average values ​​of the affinity parameters. The assay was repeated 3 times, using a new biosensor for each test article. KD, equilibrium dissociation constant; K on , binding constant; K dis , dissociation constant; R 2 , R squared.

[0063] Figure 2a and Figure 2b Results showing the glucose-lowering effects of 0.6 mg / kg body weight of dulaglutide GLP-1-gFc and 0.6 mg / kg body weight and 2.4 mg / kg body weight of GLP-1-gFc. Six-week-old male db / db mice received subcutaneous injections of the designated test articles weekly for six weeks. Blood samples were collected from the tail vein weekly and biweekly for non-fasting glucose and glycosylated hemoglobin (HbA1c), respectively, to monitor the anti-diabetic efficacy of the molecules.

[0064] Results are expressed as mean ± SEM; n = 6-8 / group. Statistical data were evaluated by Student's T-test, where *p < 0.05; **p < 0.01; ***p < 0.001 relative to vehicle.

[0065] Figure 2c The binding structure modeling diagram of GPL-1-Fc / GLP-1 receptor and GLP-1-gFc / GLP-1 receptor made by Pymol software. The left figure is the binding structure modeling between GLP-1 receptor and GLP-1-gFc, and the right figure is the binding structure modeling between GLP-1 receptor and GLP-1-Fc.

[0066] The structures of GLP1-GLP1 receptor complex (PDB 3IOL) and human IgG4 (PDB 4C54) were taken from RCSB PDB (Protein Data Bank). Fc and gFc composed of IgD and IgG4 were obtained from Phyre v2.0 software using human IgG4Fc (PDB 4C54) as template.

[0067] Figure 3a to Figure 3c A comparison of dulaglutide and GLP-1-gFc in glucose lowering and body weight in obese ob / ob mice is shown. Equivalent doses of GLP-1-gFc and dulaglutide were administered subcutaneously weekly to nine-week-old female obese ob / ob mice for four weeks. Food intake and body weight were measured once a week during the treatment period, and HbA1c was measured at the beginning and end of the treatment period (week 0 and week 4). Results are expressed as mean ± SEM; n = 6-8 / group. Statistical data were evaluated by Student's T test, where *p<0.05; **p<0.01; ***p<0.001 relative to vehicle; #p<0.05 for GLP-1-gFc relative to dulaglutide. The results indicate that GLP-1-gFc exhibits similar glucose-lowering effects.

[0068] Figure 4a to Figure 4c Mouse CTA and monkey ECG studies on side effects (nausea and vomiting) and QT prolongation responses of GLP-1-gFc and dulaglutide are shown. To compare the CTA responses of GLP-1-gFc and dulaglutide with the positive control LiCl, blueberry bar consumption was measured before dosing (day 0) (a) and after a 14-day washout period (b) for each molecule.

[0069] The potential effects of GLP-1-gFc and dulaglutide on cardiac electrophysiological signals were evaluated in telemetrically instrumented cynomolgus monkeys (c). Monkeys were administered a single dose of different doses of dulaglutide and GLP-1-gFc by the subcutaneous route. ECG waveforms were recorded from at least 2 hours before injection to approximately 24 hours after administration. In particular, the QT interval of individual monkeys was obtained and converted to QTc (corrected QT). The results are expressed as mean ± SEM; for mouse CTA, n = 10 / group, for monkey ECG studies, n = 2-3 / group. Statistics were evaluated by Mann-Whitney test, where **p<0.01; ***p<0.001 relative to vehicle; ##p<0.01 for GLP-1-gFc relative to dulaglutide.

[0070] Figure 4dConfirmed drug washout assessed by overnight food intake before the second exposure to blueberry bars in the CTA study (n=8-10 / group) is shown. In the GLP-1RA-treated group, overnight food intake on day 1 after injection was significantly reduced. In contrast, there was no difference in overnight food intake between the GLP-1-gFc and dulaglutide groups on the day before the second exposure (day 13), confirming that GLP-1-RA-related food intake suppression was completely eliminated. Results are expressed as mean ± standard error of the mean. ***p<0.001 relative to vehicle; Mann-Whitney U test ns, not significant, #p<0.01 relative to dulaglutide; dulaglutide_0.6, dulaglutide 0.6mg / kg; gFc_2.4, GLP-1-gFc 2.4mg / kg.

[0071] Figure 5a to Figure 5c The pharmacokinetics of GLP-1-gFc (single subcutaneous dose) in healthy human subjects are shown. Six (6) increasing doses of GLP-1-gFc were administered subcutaneously in healthy males. Blood samples collected at the indicated time points were analyzed and plotted versus time after injection (a). The maximum concentration (C max ) and the last measurable time (AUC last ) area under the curve to evaluate the dose dependence of pharmacokinetics (b, c). The results are expressed as mean ± standard deviation; n = 6 / group. The results show that GLP-1-gFc exhibits dose-dependent pharmacokinetics.

[0072] Figure 5d Dose-dependent PK curves are shown. GLP-1-gFc showed a dose-dependent PK curve after a single SC administration in SD rats (n=3 / group) and cynomolgus monkeys (n=3 / sex / dose). The collected serum samples were analyzed using a GLP-1-gFc-specific ELISA method, in which mouse anti-human IgG4 and n-terminal specific GLP-1 antibodies were used as coating and detection antibodies. The results are expressed as mean ± standard deviation. T 1 / 2 ,half life.

[0073] Figures 6a to 6e The results of evaluating side effects (nausea or vomiting, or heart rate) in an oral glucose tolerance test (OGTT). Blood samples for measuring blood glucose and insulin were collected before and after 0.25, 0.5, 1, 1.5, and 2 hours of ingestion of 75 g of glucose solution. The changes in glucose and insulin were plotted against the time point of blood sampling. The area under the curve of each graph was calculated and plotted against each dose to show the dose-related therapeutic effect of GLP-1-gFc ( Figures 6a to 6cGastrointestinal side effects and vital signs including pulse rate were monitored throughout the study and at follow-up (Day 28). Among the observed gastrointestinal side effects, nausea / vomiting was expressed as the number of patients experiencing each side effect in each dose group ( Figure 6d ). The observed pulse rate data on day 0 were subtracted to show changes after dosing ( Figure 6d ). Pulse rate on days 3 and 5 was plotted against each dose to compare with the treatment effect in the OGTT study, where the effect was assessed at the same time points. Results are expressed as mean ± standard deviation; n = 6 / group. The results indicate that GL-1-gFc exhibits very low side effects. DETAILED DESCRIPTION

[0074] The fusion protein of the embodiment can be represented by the following chemical formula (I):

[0075] GLP-1-gFc formula (I)

[0076] Wherein, GLP-1 is a GLP-1 peptide having a sequence number of SEQ ID NO: 1 or an analog or variant thereof, and gFc is an immunoglobulin Fc region having an IgD hinge region. In one embodiment, GLP-1 may have an amino acid sequence having a sequence number of SEQ ID NO: 1, 10 or 11, an analog or variant thereof, wherein less than 6 amino acids of the sequence number of SEQ ID NO: 1, 10 or 11 are substituted.

[0077] Substitutions may be made that are conservative amino acid substitutions that have no or only minimal effect on the overall protein charge, ie, polarity or hydrophobicity.

[0078] For conservative amino acid substitutions, please refer to Table 1 below.

[0079] Table 1

[0080]

[0081] For each amino acid, additional conservative substitutions include "homologues" of the amino acid. In particular, a "homologue" refers to an amino acid in which a methylene group (CH2) is inserted into the side chain at the β position of the amino acid side chain. Examples of "homologues" may include, but are not limited to, homophenylalanine, homoarginine, homoserine, and the like.

[0082] In one embodiment, the gFc of formula (I) is a modified immunoglobulin or a portion thereof or a variant thereof, wherein the Fc region has an IgD hinge region. The IgD hinge region has one O-glycan.

[0083] In particular, the Fc region of the modified immunoglobulin can be a region in which antibody-dependent cellular toxicity (ADCC) or complement-dependent cytotoxicity (CDC) is weakened due to modification of the binding affinity to Fc receptors and / or complement. The modified immunoglobulin can be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and a combination thereof. Specifically, the Fc region of the modified immunoglobulin can include a hinge region, a CH2 domain and a CH3 domain from N-terminal to C-terminal. In particular, the hinge region may include a human IgD hinge region; the CH2 domain may include a portion of the amino acid residues of human IgD and a portion of the amino acid residues of human IgG4CH2 domain; and the CH3 domain may include a portion of the amino acid residues of human IgG4CH3 domain.

[0084] In addition, the two fusion proteins can form a dimer. For example, the Fc regions can bind to each other to form a dimer. As used herein, the term "Fc region", "Fc fragment" or "Fc" refers to a protein including the constant region 2 (CH2) and the constant region 3 (CH3) of the heavy chain of an immunoglobulin, but excluding the variable region of the heavy chain and the variable region of the light chain and the constant region of the light chain (CL1), which may also include the hinge region of the constant region of the heavy chain.

[0085] In one embodiment, a hybrid Fc or a hybrid Fc fragment thereof may be referred to as "hFc" or "hyFc".

[0086] In addition, as used herein, the term "Fc region variant" refers to a variant prepared by replacing a portion of the amino acids in the Fc region or by combining different types of Fc regions. The Fc region variant can prevent being cut off at the hinge region. Specifically, the 144th amino acid and / or the 145th amino acid of SEQ ID NO: 4 can be modified. Preferably, the variant can be a variant in which the 144th amino acid K is replaced by G or S, and a variant in which the 145th amino acid E is replaced by G or S.

[0087] The modified immunoglobulin Fc region or Fc region variant can be represented by the following formula (II):

[0088] N'-(Z1)pY-Z2-Z3-Z4-C' Formula (II)

[0089] In the above formula (II),

[0090] N' is the N-terminus of the polypeptide, and C' is the C-terminus of the polypeptide;

[0091] p is an integer of 0 or 1;

[0092] Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues from the amino acid residue at position 98 toward the N-terminus among the amino acid residues at positions 90 to 98 of SEQ ID NO: 2;

[0093] Y is an amino acid sequence having 5 to 64 consecutive amino acid residues from the amino acid residue at position 162 toward the N-terminus among the amino acid residues at positions 99 to 162 of SEQ ID NO: 2;

[0094] Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues from the amino acid residue at position 163 toward the C-terminus among the amino acid residues at positions 163 to 199 of SEQ ID NO: 2;

[0095] Z3 is an amino acid sequence having 71 to 106 consecutive amino acid residues from the amino acid residue at position 220 toward the N-terminus among the amino acid residues at positions 115 to 220 of SEQ ID NO: 3; and

[0096] Z4 is an amino acid sequence having 80 to 107 consecutive amino acid residues from the amino acid residue at position 221 toward the C-terminus among the amino acid residues at positions 221 to 327 of SEQ ID NO: 3.

[0097] In addition, the Fc fragment may be in a form having native sugar chains, increased sugar chains, or reduced sugar chains compared to the native form. The immunoglobulin Fc sugar chain may be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms.

[0098] In addition, the Fc region of the modified immunoglobulin may include the amino acid sequence of SEQ ID NO: 4 (hyFc), SEQ ID NO: 5 (hyFcM1), SEQ ID NO: 6 (hyFcM2), SEQ ID NO: 7 (hyFcM3), or SEQ ID NO: 8 (hyFcM4). In addition, the Fc region of the modified immunoglobulin may include the amino acid sequence of SEQ ID NO: 9 (a non-cleaved mouse Fc). The Fc region of the modified immunoglobulin may be as described in U.S. Pat. No. 7,867,491, and the generation of the Fc region of the modified immunoglobulin may be carried out with reference to the disclosure in U.S. Pat. No. 7,867,491, the entire contents of which are incorporated herein by reference. The gFc of formula (I) can be an immunoglobulin region comprising (i) an isolated IgD hinge region consisting of 35 to 49 consecutive amino acid residues from the C-terminus of SEQ ID NO: 35; and (ii) a CH2 domain and a CH3 domain of an immunoglobulin Fc polypeptide. In one embodiment, the IgD hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-38.

[0099] Fusion proteins of formula (I) may be described in US Patent No. 10,538,569, the entire disclosure of which is incorporated herein by reference. The GLP-1 of formula (I) may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 11-34.

[0100] The nucleic acid construct (or genomic construct) comprising the nucleic acid encoding the fusion protein can be used as a part of the gene therapy scheme. In order to rebuild or supplement the function of the desired protein, the expression vector capable of expressing the fusion protein in a specific cell can be administered together with any biologically effective carrier. This can be any preparation or composition capable of effectively delivering the gene encoding the fusion protein to the cell in vivo.

[0101] GLP-1 and gFc can be fused via a peptide linker. The peptide linker can be a peptide of 10 to 20 amino acid residues consisting of Gly and Ser residues.

[0102] In one embodiment, the C-terminus of the GLP-1 peptide can be fused to the N-terminus of the Fc region.

[0103] In one embodiment, the fusion protein of formula (I) has amino acids with sequence numbers SEQ ID NOs: 40, 41, 42 or 54.

[0104] Fusion proteins can be produced by expressing in a suitable host a nucleic acid encoding the fusion protein.

[0105] The nucleic acid molecule may also include a signal sequence or leader sequence.

[0106] As used herein, the term "signal sequence" refers to a fragment that directs the secretion of biologically active molecule drugs and fusion proteins, which is cut off after translation in the host cell. The signal sequence of one embodiment is a polynucleotide encoding an amino acid sequence that initiates the movement of the protein across the endoplasmic reticulum (ER) membrane. Useful signal sequences in one embodiment include antibody light chain signal sequences, such as antibody 14.18 (Gillies et al., Journal of Immunological Methods, 1989, 125: 191-202), antibody heavy chain signal sequences, such as MOPC141 antibody heavy chain signal sequence (Sakano et al., Nature, 1980, 286: 676-683), and other signal sequences known in the art (e.g., see Watson et al., Nucleic Acids Research, 1984, 12: 5145-5164).

[0107] The characteristics of signal peptides are well known in the art, and signal peptides typically have 16 to 30 amino acids, but they may include a greater or lesser number of amino acid residues. Traditional signal peptides consist of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.

[0108] The central hydrophobic region includes 4 to 12 hydrophobic residues, which fix the signal sequence by the membrane lipid bilayer during the translocation of the immature polypeptide. After activation, the signal sequence is often cut by a cellular enzyme called a signal peptidase in the ER cavity. In particular, the signal sequence can be a secretory signal sequence of tissue plasminogen activation (tPa), a signal sequence of herpes simplex virus glycoprotein D (HSV gDs), or a signal sequence of growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells including mammals can be used. In addition, as a secretory signal sequence, a signal sequence contained in GLP-1 can be used, or the signal sequence can be used after a codon substitution with a high expression frequency in a host cell.

[0109] An isolated nucleic acid molecule encoding a fusion protein can be contained in an expression vector.

[0110] As used herein, the term "vector" is understood to be a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell to recombine and insert into the genome of the host cell, or replicate spontaneously as an episome. Vectors can include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and the like. Examples of viral vectors can include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0111] As used herein, the term "gene expression" or "expression" of a target protein is understood to refer to the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of a fusion protein product or fragment thereof.

[0112] As used herein, the term "gene expression" or "expression" of a target protein is understood to refer to the transcription of the DNA sequence, the translation of the mRNA transcript, and the secretion of the Fc fusion protein product or antibody or antibody fragment thereof.

[0113] Useful expression vectors may be RcCMV (Invitrogen, Carlsbad) or variants thereof. The expression vector may include a human cytomegalovirus (CMV) for promoting continuous transcription of the target gene in mammalian cells and a polyadenylation signal sequence of bovine growth hormone for increasing RNA stability after transcription. In an exemplary embodiment, the expression vector is pAD15, which is a modified form of RcCMV.

[0114] By transducing or transfecting a DNA sequence of an embodiment, the expression vector can be included in a suitable host cell suitable for expression and / or secretion of the target protein.

[0115] As used herein, the term "host cell" or "host" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, the terms "transduced," "transformed," and "transfected" refer to the introduction of a nucleic acid (e.g., a vector) into a cell using techniques known in the art.

[0116] Examples of suitable host cells may include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells), TM4, W138, Hep G2, MMT060562, or COS cells.

[0117] Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera litura Sp, Spodoptera litura H5, etc., and plant cells.

[0118] Nucleic acid molecules encoding GLP-1 peptides can be prepared by known methods, including cloning methods, such as those described above, and chemically synthesized DNA. In view of the short length of the encoded peptide, chemical synthesis can be used. The amino acid sequence of GLP-1 and the sequence of the preglucagon gene have been published. [Lopez et al., (1983) Proceedings of the National Academy of Sciences of the United States of America, 80:5485-5489; Bell et al. (1983) Nature, 302:716-718; Heinrich, G. et al. (1984) Endocrinology, 115:2176-2181; Ghiglione, M. et al. 91984) Diabetology 27:599-600]. Therefore, primers can be designed based on the natural sequence to generate DNA encoding the GLP-1 peptide.

[0119] The gene encoding the fusion protein can then be constructed by in-frame ligating the nucleic acid encoding the GLP-1 peptide to the nucleic acid encoding the Fc region described herein. The DNA encoding the wild-type GLP-1 and IgG4 Fc fragment can be mutated prior to ligation or in the context of the cDNA encoding the entire fusion protein by using known mutagenesis techniques. The gene encoding the GLP-1 peptide and the gene encoding the Fc region (e.g., the gene encoding hyFc of SEQ ID NO: 4) can also be ligated in-frame directly or via a DNA encoding a G-rich linker peptide.

[0120] Various forms of fusion proteins can be recovered from culture medium or host cell lysate. If membrane-bound, suitable detergent solutions (e.g., Triton-X 100) or enzyme cleavage can be used to release from the membrane. Cells used in fusion protein expression can be broken by various physical or chemical means such as freeze-thaw cycles, ultrasonic treatment, mechanical disruption or cell lysing agents.

[0121] Once the fusion protein is expressed in a suitable host cell, the fusion protein can be isolated and purified. The following procedures are examples of suitable purification procedures: fractionation on carboxymethylcellulose; gel filtration such as Sephadex G-75; anion exchange resins such as DEAE or Mono-Q; cation exchange such as CM or Mono-S; metal chelate columns to bind epitope-tagged forms of the polypeptide; reversed-phase high performance liquid chromatography; chromatofocusing; silica gel; ethanol precipitation; and ammonium sulfate precipitation.

[0122] A variety of protein purification methods can be used, and such methods are known in the art and described in, for example, Deutscher, Methods in Enzymology 182:83-9 (1990) and Scope, Protein Purification: Principles and Practice, Springer, New York (1982). The purification step selected will depend on the nature of the production process used and the specific fusion protein produced. For example, protein A or protein G affinity matrix can be used to effectively purify the fusion protein containing the Fc fragment. Low or high pH buffer can be used to elute the fusion protein from the affinity matrix. Gentle elution conditions will help prevent the irreversible denaturation of the fusion protein.

[0123] The fusion protein can be formulated with one or more pharmaceutically acceptable carriers or excipients. The fusion protein can be combined with a pharmaceutically acceptable buffer, a pH value adjusted to provide acceptable stability, and an acceptable pH value for administration such as parenteral administration. Optionally, one or more pharmaceutically acceptable antimicrobial agents can be added. Metacresol and phenol are preferred pharmaceutically acceptable microbial agents. One or more pharmaceutically acceptable salts can be added to adjust ionic strength or tension. One or more excipients can be added to further adjust the isotonicity of the preparation.

[0124] Glycerol is an example of an isotonicity adjusting excipient. Pharmaceutically acceptable means is suitable for administration to humans or other animals and therefore does not contain toxic elements or undesirable contaminants and does not interfere with the activity of the active compounds therein.

[0125] The fusion protein can be formulated as a solution formulation or a lyophilized powder that can be reconstituted with a suitable diluent. A lyophilized dosage form is a fusion protein-stable dosage form with or without buffering capacity to maintain the pH value of the solution within the expected shelf life of the reconstituted product. Preferably, the solution containing the heterologous fusion protein discussed herein is substantially isotonic before lyophilization to be able to form an isotonic solution after reconstitution.

[0126] Pharmaceutically acceptable salt forms of the fusion protein are also within the scope of the present invention. Acids commonly used to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid. Preferred acid addition salts are salts formed from inorganic acids such as hydrochloric acid and hydrobromic acid.

[0127] Base addition salts include salts derived from inorganic bases such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.

[0128] The fusion protein of the present invention has biological activity. Biological activity refers to the ability of the fusion protein to bind to and activate the GLP-1 receptor in vivo and induce a reaction. Reactions include but are not limited to insulin secretion, glucagon inhibition, appetite suppression, weight loss, satiety induction, apoptosis inhibition, pancreatic β cell proliferation induction and pancreatic β cell differentiation. A representative number of GLP-1 fusion proteins were tested for in vitro and in vivo activity.

[0129] Fusion protein can be administered by any effective approach known to ordinary physicians. Peripheral injection is a method of this. Parenteral administration is generally understood in medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device (such as an infusion pump). Peripheral parenteral routes can include intravenous, intramuscular, subcutaneous and intraperitoneal routes of administration.

[0130] The fusion protein may also be administered by the oral, rectal, nasal or lower respiratory tract routes.

[0131] The fusion protein can be used to regulate blood sugar in the body or normalize blood sugar in the body.

[0132] The fusion protein exerts its biological effects primarily by acting as a GLP-1 receptor agonist, i.e., binding to a receptor called the GLP-1 receptor. Thus, subjects with diseases and / or conditions that respond favorably to GLP-1 receptor stimulation or to the administration of a GLP-1 compound can be treated with the GLP-1 fusion protein.

[0133] These subjects are said to be "in need of treatment with a GLP-1 compound" or "in need of GLP-1 receptor stimulation". Such subjects may include those suffering from non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus, stroke (see WO 00 / 16797), myocardial infarction (see WO 98 / 08531), obesity (see WO 98 / 19698), post-operative catabolic changes (see U.S. Pat. No. 6,006,753), functional dyspepsia and irritable bowel syndrome (see WO 99 / 64060). Also included are subjects in need of prophylactic treatment with a GLP-1 compound, for example, subjects at risk of developing non-insulin-dependent diabetes mellitus (see WO 00 / 07617). Subjects with impaired glucose tolerance or impaired fasting glucose, subjects whose weight is approximately 25% above normal weight for the subject's height and size, subjects who have undergone partial pancreatectomy, subjects with one or more parents with non-insulin-dependent diabetes mellitus, subjects with gestational diabetes, and subjects with acute or chronic pancreatitis are at risk for developing non-insulin-dependent diabetes mellitus.

[0134] An effective amount of a GLP-1-gFc fusion protein is an amount that produces the desired therapeutic and / or preventive effect without causing unacceptable side effects when administered to a subject requiring GLP-1 receptor stimulation. "Desired therapeutic effect" includes one or more of the following: 1) improvement of symptoms associated with a disease or condition; 2) delayed onset of symptoms associated with a disease or condition; 3) prolonged lifespan compared to no treatment; 4) higher quality of life compared to no treatment. For example, an "effective amount" of a GLP-1-gFc fusion protein for the treatment of diabetes is an amount that better controls blood glucose concentrations than when no treatment is performed, thereby delaying the onset of diabetic complications such as retinopathy, neuropathy, or kidney disease. An "effective amount" of a GLP-1-gFc fusion protein for the prevention of diabetes is an amount that will delay the onset of elevated blood glucose levels that require treatment with antihypoglycemic drugs (such as sulfonylureas, thiazolidinediones, insulin, and / or biguanides) compared to no treatment.

[0135] Compared to commercially available GLP-1 fusion protein drugs, such as dulaglutide, the GLP-1-gFc fusion proteins disclosed herein show lower side effects, such as vomiting, nausea, and / or increased heart rate.

[0136] The dosage of the fusion protein that effectively normalizes blood sugar in patients will depend on many factors, including but not limited to the sex, weight and age of the subject, the severity of being unable to regulate blood sugar, route administration and bioavailability, pharmacokinetic characteristics of the fusion protein, efficacy and formulation. The dosage can be in the range of 0.01mg / kg to 10mg / kg body weight. In one embodiment, the dosage can be in the range of 0.05mg / kg to 5mg / kg body weight. In another embodiment, the dosage can be in the range of 0.01mg / kg to 1mg / kg body weight. In another embodiment, the dosage can be in the range of 0.05mg / kg to 0.5mg / kg body weight. In another embodiment, the dosage can be in the range of 0.05mg / kg to 1mg / kg body weight.

[0137] The fusion protein can be administered at intervals of one week or more.

[0138] Depending on the disease being treated, it may be necessary to administer the fusion protein more frequently than weekly, such as two to three times per week.

[0139] For example, according to an embodiment, the dosage can be administered at intervals of one week or longer. In one embodiment, the dosage can be administered at intervals of two weeks or longer. In another embodiment, the dosage can be administered at intervals of three weeks or longer. In yet another embodiment, the dosage can be administered at intervals of 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 30 days, 40 days or longer. In another embodiment, the above dosage can be administered once a week, twice a week, once every other week, twice a month, three times a month, etc.

[0140] In one aspect, a method for reducing the glucose level of a subject without or with reduced side effects is provided, wherein the fusion protein is administered. In one embodiment, the side effects are one or more of nausea, vomiting, and increased heart rate. In one embodiment, the subject suffers from diabetes. On the one hand, the subject suffers from type II diabetes.

[0141] Thus, in one aspect, a method of treating diabetes in a subject by administering a fusion protein is provided.

[0142] In one aspect, the method comprises about 0.01 mg / kg to about 10 mg / kg, about 0.02 mg / kg to about 10 mg / kg, about 0.03 mg / kg to about 10 mg / kg, about 0.04 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.06 mg / kg to about 10 mg / kg, about 0.07 mg / kg to about 10 mg / kg, about 0.08 mg / kg to about 10 mg / kg, about 0.09 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.15 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 10 mg / kg, about 0.25 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 10 mg / kg, about 0.35 mg / kg to about 10 mg / kg, about 0.4 mg / kg to about 10 mg / kg, about 0.45 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 10 mg / kg, from about 0.55 mg / kg to about 10 mg / kg, about 0.6 mg / kg to about 10 mg / kg, about 0.65 mg / kg to about 10 mg / kg, about 0.7 mg / kg to about 10 mg / kg, from about 0.75 mg / kg to about 10 mg / kg, about 0.8 mg / kg to about 10 mg / kg, about 0.85 mg / kg to about 10 mg / kg, about 0.9 mg / kg to about 10 mg / kg, about 0.95 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1.1 mg / kg to about 10 mg / kg, about 1.2 mg / kg to about 10 mg / kg, about 1.3 mg / kg to about 10 mg / kg, about 1.4 mg / kg to about 10 mg / kg, about 1.5 mg / kg to about 10 mg / kg, about 1.6 mg / kg to about 10 mg / kg, about 1.7 mg / kg to about 10 mg / kg, about 1.8 mg / kg to about 10 mg / kg, about 1.9 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg , from about 2.1 mg / kg to about 10 mg / kg, about 2.2 mg / kg to about 10 mg / kg, about 2.3 mg / kg to about 10 mg / kg, about 2.4 mg / kg to about 10 mg / kg, about 2.5 mg / kg to about 10 mg / kg, about 2.6 mg / kg to about 1.0 mg / kg, about 2.7 mg / kg to about 10 mg / kg, about 2.8 mg / kg to about 10 mg / kg, about 2.9 mg / kg to about 10 mg / kg, about 3.1 mg / kg to about 10 mg / kg, about 3.2 mg / kg to about 10 mg / kg, about 3.3 mg / kg to about 10 mg / kg, about 3.4 mg / kg to about 10 mg / kg, about 3.6 mg / kg to about 10 mg / kg, about 3.7 mg / kg to about 10 mg / kg, about 3.9 mg / kg to about 10 mg / kg, about 3.1 mg / kg to about 10 mg / kg, about 3.The fusion protein may be administered at intervals of one week or longer, two weeks or longer, three weeks or longer, or four weeks or longer. In an embodiment, the upper limit of the above range may be about 5 mg / kg. In another embodiment, the dose may be administered at intervals of 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 30 days, 40 days or longer. In another embodiment, the dose may be administered once a week, twice a week, once every other week, twice a month, three times a month, etc.

[0143] In another embodiment, the dosage may be about 0.01 mg / kg to about 1 mg / kg, about 0.02 mg / kg to about 1 mg / kg, about 0.03 mg / kg to about 1 mg / kg, about 0.04 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 1 mg / kg, about 0.06 mg / kg to about 1 mg / kg, about 0.07 mg / kg to about 1 mg / kg, about 0.08 mg / kg to about 1 mg / kg, about 0.09 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.16 mg / kg to about 1 mg / kg, about 0.2 mg / kg to about 1 mg / kg, about 0.24 mg / kg to about 1 mg / kg, about 0.3 mg / kg to about 1 mg / kg, about 0. The fusion protein can be administered at intervals of one week or more, two weeks or more, three weeks or more, or four weeks or more. In yet another embodiment, the dosage can be administered at intervals of 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 30 days, 40 days or longer. In another embodiment, the dosage can be administered once a week, twice a week, once every other week, twice a month, three times a month, etc.

[0144] In another aspect, the method comprises administering the drug at a dosage of about 0.1 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 5 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.4 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.6 mg / kg to about 5 mg / kg, about 0.7 mg / kg to about 5 mg / kg, about 0.8 mg / kg to about 5 mg / kg, about 0.9 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1.1 mg / kg to about 5 mg / kg. g / kg to about 5 mg / kg, about 1.2 mg / kg to about 5 mg / kg, about 1.3 mg / kg to about 5 mg / kg, about 1.4 mg / kg to about 5 mg / kg, about 1.5 mg / kg to about 5 mg / kg, about 1.6 mg / kg to about 5 mg / kg, about 1.7 mg / kg to about 5 mg / kg, about 1.8 mg / kg to about 5 mg / kg, about 1.9 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 2.1 mg / kg to about 5 mg / kg, about 2.2 mg / kg to about 5 mg / kg. g / kg to about 5 mg / kg, about 2.3 mg / kg to about 5 mg / kg, about 2.4 mg / kg to about 5 mg / kg, about 2.5 mg / kg to about 5 mg / kg, about 2.6 mg / kg to about 5 mg / kg, about 2.7 mg / kg to about 5 mg / kg, about 2.8 mg / kg to about 5 mg / kg, about 2.9 mg / kg to about 5 mg / kg, about 3 mg / kg to about 5 mg / kg, about 3.1 mg / kg to about 5 mg / kg, about 3.2 mg / kg to about 5 mg / kg, about 3.3 mg / kg to about 5 mg / kg, about 3.4 mg / kg to about 5 mg / kg, about 3.6 mg / kg to about 5 mg / kg, about 3.7 mg / kg to about 5 mg / kg, about 3.9 mg / kg to about 5 mg / kg, about 3.1 mg / kg to about 5 mg / kg, about 3.2 mg / kg to about 5 mg / kg, about 3.3 mg / kg to about 5 mg / kg, about 3.6 mg / kg to about 5 mg / kg, about 3.7 mg / kg to about 5 mg / kg, about 3.8 mg / kg to about 5 mg / kg, about 3.9 mg / kg to about 5 mg / kg, about 3. The dosage of about 5 mg / kg to about 5 mg / kg, about 3.4 mg / kg to about 5 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 3.6 mg / kg to about 5 mg / kg, about 3.7 mg / kg to about 5 mg / kg, about 3.8 mg / kg to about 5 mg / kg, about 3.9 mg / kg to about 5 mg / kg, or about 4 mg / kg to about 5 mg / kg, with 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 30 days, 40 days or longer intervals of fusion protein. In another embodiment, the dosage can be administered at a frequency of once a week, twice a week, once every two weeks, once a month, twice a month, three times a month, etc.

[0145] In an exemplary embodiment, with the dosage of 0.05mg / kg, 0.06mg / kg, 0.07mg / kg, 0.08mg / kg, 0.09mg / kg, 0.1mg / kg, 0.11mg / kg, 0.12mg / kg, 0.13mg / kg, 0.14mg / kg, 0.15mg / kg, 0.16mg / kg, 0.17mg / kg, 0.18mg / kg, 0.19mg / kg, 0.2mg / kg, 0.21mg / kg, 0.22mg / kg, 0.23mg / kg, 0.24mg / kg, 0.25mg / kg, 0.26mg / kg, 0.27mg / kg, 0.28mg / kg, 0.29mg / kg or 3mg / kg, fusion protein is used at intervals of one week or two weeks. It should be understood that the two-week interval time scheme can be replaced with a frequency every other week.

[0146] In another exemplary embodiment, the fusion protein is administered at a dose of 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.11 mg / kg, 0.12 mg / kg, 0.13 mg / kg, 0.14 mg / kg, 0.15 mg / kg, 0.16 mg / kg, 0.17 mg / kg, 0.18 mg / kg, 0.19 mg / kg, or 0.2 mg / kg at intervals of one week or 10 days.

[0147] In another exemplary embodiment, the fusion protein is administered at a dose of 0.1 mg / kg, 0.11 mg / kg, 0.12 mg / kg, 0.13 mg / kg, 0.14 mg / kg, 0.15 mg / kg, 0.16 mg / kg, 0.17 mg / kg, 0.18 mg / kg, 0.19 mg / kg, 0.2 mg / kg, 0.21 mg / kg, 0.22 mg / kg, 0.23 mg / kg, 0.24 mg / kg, 0.25 mg / kg, 0.26 mg / kg, 0.27 mg / kg, 0.28 mg / kg, 0.29 mg / kg or 3 mg / kg, every two weeks, or at a frequency of once every other week, twice a month or three times a month.

[0148] In embodiments, administration may be parenteral, such as subcutaneous administration.

[0149] In another aspect, provided is an effective amount of a fusion peptide for regulating blood glucose levels, the fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0150] In another aspect, a composition for regulating blood sugar level is provided, comprising an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0151] In another aspect, a therapeutic agent for regulating blood sugar level is provided, comprising an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0152] In another aspect, provided is a use of an effective amount of a fusion peptide for regulating blood glucose levels, the fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0153] In another aspect, provided is a use of an effective amount of a fusion peptide in the preparation of a medicament for regulating blood glucose levels, the fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0154] In another aspect, provided is an effective amount of a fusion peptide for preventing and / or treating diabetes, the fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) the immunoglobulin Fc region described herein.

[0155] In another aspect, a composition for preventing and / or treating diabetes is provided, comprising an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) the immunoglobulin Fc region described herein.

[0156] In another aspect, provided is a therapeutic agent for preventing and / or treating diabetes, comprising an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) the immunoglobulin Fc described herein.

[0157] In another aspect, provided is a use of an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0158] In another aspect, provided is a use of an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region as described herein.

[0159] Embodiments of the present invention

[0160] Various aspects will now be described, by way of non-limiting example only, with reference to the following examples.

[0161] Preparation Example 1: Preparation of GLP-1-hyFc5, GLP-1-hyFc9, GLP-1-hyFc8 and GLP-1-hyFc11

[0162] By following the method described in Example 1-1 of U.S. Patent No. 10,538,569, the contents of which are incorporated herein by reference, GLP-1-hyFc5 fusion polypeptide (SEQ ID NO: 54), GLP-1-hyFc9 (SEQ ID NO: 41), GLP-1-hyFc8 (SEQ ID NO: 40) and GLP-1-hyFc11 (SEQ ID NO: 42) fusion polypeptides.

[0163] Monoclonal selection of transfected cells and purification of secreted proteins were performed in a similar manner as previously described for other hybrid Fc fusion recombinant proteins. 17,18 . Dulaglutide purchased from Eli Lilly and Company, and lithium chloride used in CTA studies was purchased from Sigma-Aldrich (USA).

[0164] Example 1

[0165] Material

[0166] GLP-1(A2G)-hyFc9 (SEQ ID NO: 41) obtained in Preparation Example 1 was used as GLP-1-gFc of formula (I).

[0167] Cell-based in vitro potency assays

[0168] To evaluate the efficacy of the test article (TA), the extent of GLP-1 specific response to cyclic AMP induction, a transgenic cell line (GLP1R_cAMP / luc) was constructed to express the GLP-1 receptor in a cAMP specific luciferin expressing cell line. After thawing and appropriate maintenance, 2x10 5 Cells / mL were seeded into T-75 flasks with growth medium (90% DMEM / high glucose, 10% FBS, 130ug / mL hygromycin B God, 5ug / mL puromycin) and cultured in a CO2 incubator at 37°C until 70-80% confluence. When the cell confluence reached 70-80%, the cells were washed with PBS and 0.05% TE (trypsin EDTA) was added to separate the cells from the flask. Cells were collected and washed as needed for activity assessment and diluted with 0.5% FBS and DMEM / high glucose medium to 2x10 4Cells were inoculated at 80uL / well. After the cells were cultured in a CO2 incubator at 37°C for about 6 hours, they were treated with 20uL / well of different concentrations of TAs and reacted in a CO2 incubator at 37°C for 5 hours. Bright-GloTM assay reagent (Promega, USA) was treated with 100uL / well and reacted at room temperature for 2 minutes. After the reaction, luminescence was measured using a photometer (Berteng, USA).

[0169] Analysis of binding affinity by SPR (surface plasmon resonance)

[0170] Based on the scheme modified from the general procedure of SPR analysis in the published paper, the binding affinity of each TA was evaluated by SPR (Proteon XPR36, Bio-Rad). Specifically, the protein GLC chip (Bio-Rad, USA) was stabilized with PBST (PBS + 0.01% Tween 20, pH 7.4). The stabilized GLC chip was activated with 150uL sulfo-NHS (0.001M) and EDC (0.04M) (1: 1), and then fixed in 10ug / mL human GLP-1 receptor (Abcam, UK) diluted in acetate buffer (pH 5.0). After recording the fixed level and inactivating by 1M ethanolamine-HCl (pH 8.5), different concentrations of dulaglutide and GLP-1-gFc (0uM, 1.25uM, 2.5uM, 5uM, 10uM) were injected into each channel of the chip. The chip was regenerated by 25mM NaOH and checked for "zero base" before repeating the analysis of the same molecule or other TA. All binding sensorgrams were collected, processed and analyzed using the integrated Proteon Manager software (Bio-Rad, USA). The binding curves were fitted using the Laugmuir model.

[0171] animal

[0172] All animal studies were conducted according to protocols approved by the Institutional Animal Care and Use Committee of Genexine (Korea) or WuXi AppTec (China). Obese (C57BL / 6J-ob / ob) mice and DBA / 2 mice were obtained from SLC (Japan) and Koatech (Korea), respectively. The obtained mice were raised in appropriate numbers per cage at 20±2°C with a 12-hour / 12-hour light-dark cycle. Free feeding with irradiated sterilized solid animal feed (Teklad certified irradiated global 18% protein diet, 2918C, Harlan Co., Ltd., US) and sterile water was performed using appropriate dispensers and bottles.

[0173] Male cynomolgus monkeys were obtained from Hainan Jingang Biotechnology Company (China) and housed individually in stainless steel cages in the animal facility of WuXi AppTec.

[0174] The animals were provided monkey chow twice daily and reverse osmosis purified and chlorinated water was provided ad libitum via an automated system. Monkeys used for ECG studies were equipped with transmitters (DSI TL11M2-D70-PCT) according to Wuxi SOP, and only individuals showing normal ECG parameters were included in the study.

[0175] Dose finding of GLP-1-gFc in db / db mice

[0176] Male diabetic (five-week-old, db / db) mice were adapted to the feeding environment for one week. Blood glucose in the non-fasting state was measured to assign animals to treatment groups (n=8 / group): vehicle, dulaglutide 0.6 mg / kg, GLP-1-gFc 0.6 mg / kg, and GLP-1-gFc 2.4 mg / kg. All TAs were diluted with a dedicated formulation buffer to prepare injectable drug products and analyzed with a GLP-1 ELISA, which detects the active form of GLP-1, in which mouse anti-human IgG4 (Anolan Bio, USA) and biotinylated n-terminal specific GLP-1 antibody (Thermo Fisher Scientific, USA) were used to capture and detect antibodies, respectively. The analyzed TA was administered subcutaneously (SC) weekly for six weeks. Non-fasting blood glucose was measured once a week during the treatment period, and glycated hemoglobin (HbA1c) was measured every two weeks starting from week 0.

[0177] Evaluation of anti-diabetic / obesity effects in ob / ob mice

[0178] Female obese (six-week-old, ob / ob) mice were adapted to the feeding environment and operating procedures, such as injections and grasping, for three weeks. Body weight was measured to assign animals to treatment groups (n=8 / group): vehicle, dulaglutide 0.6 mg / kg, GLP-1-gFc 2.4 mg / kg. All TAs were diluted and analyzed by active GLP-1 ELISA as described above and administered weekly by subcutaneous (SC) route for four weeks. Food intake and body weight were measured once a week during the treatment period, and glycated hemoglobin (HbA1c) was measured at the beginning (week 0) and end (week 4) of the treatment period.

[0179] Conditioned Taste Aversion (CTA) Research

[0180] The CTA study to determine the nausea effect of TA was modified from a previously described protocol. 19. Briefly, acclimated male DBA / 2J (five-week-old) mice were individually housed and given 10 min access to pre-weighed blueberry bars, which were then reweighed to measure consumption. After 10 min of exposure to the blueberry bars, animals were assigned to one of the following treatment groups (n=10 / group): vehicle (sc), 0.3 M lithium chloride (ip), dulaglutide 0.6 mg / kg (sc), and GLP-1-gFc 2.4 mg / kg. Each TA was administered immediately after the first exposure to the blueberry bars to pair the novel taste of blueberries with the disgusting stimulus of the TA. After a 14-day washout period, a second blueberry bar was exposed to the mice to rule out the inhibition of food intake by the GLP-1-derived test article, which could affect the consumption of the second exposed blueberry bars. Therefore, the washout of the TA was assessed by normalization of overnight food intake. The extent of the CTA response was determined by the reduction in bar consumption compared to the vehicle group.

[0181] Assessment of QT interval changes in cynomolgus monkeys

[0182] Telemeter-implanted cynomolgus monkeys were given a single dose of vehicle SC prior to being assigned to a single injection of the following TAs: dulaglutide 0.07 mg / kg (n=3), GLP-1-gFc 0.28 mg / kg (n=2), GLP-1-gFc 1.14 mg / kg (n=2). The dose of dulaglutide (0.07 mg / kg) was determined based on the clinical dose of dulaglutide and the typical dose conversion method using body surface area (1.5 mg / 65 kg x 3.08). 20 . The low dose of GLP-1-gFc (0.28 mg / kg) was multiplied by 4 to become an equivalent dose to dulaglutide, and was multiplied by 4 to obtain a high dose of GLP-1-gFc. Blood pressure and ECG waveforms were recorded from 2 hours before each dose to 24 hours after each dose. ECGs of at least 30 seconds were obtained from all monkeys before each dose (at least 30 minutes apart) and 2, 4, 8, 12, 16 and 24 hours after dosing. The collected ECG data were used to calculate QTc (corrected QT). The QTc averaged per 1 minute on the dosing day was calculated using the equation QTc=QT-βx(RR-500).

[0183] Example 2: Clinical study in healthy subjects

[0184] A first-in-human, phase 1, single ascending dose, randomized, double-blind, placebo-controlled study evaluating the safety, tolerability, and pharmacokinetics of GX-G6 administered subcutaneously to healthy men was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. Subjects provided written informed consent prior to enrollment. 48 healthy male subjects (n=8 / group, 6 groups total, n=6 for active drug and n=2 for placebo) aged between 18 and 40 years with a body mass index (BMI) between 18 kg / m 2 and 29.9kg / m 2 , they were allowed to participate in the study. Exclusion criteria included any clinically severe pancreatic, liver, kidney, gastrointestinal, cardiovascular, respiratory, blood, central nervous system disease or other major disease that may affect the safety of the subjects or the absorption, metabolism, excretion of the active agent under investigation. In addition, subjects with malignant tumors and substance abuse or addition such as alcohol and drugs in the past 3 years were also excluded. According to the decision of the Safety Monitoring Committee (SMC) meeting, 0.01mg / kg, 0.02mg / kg, 0.04mg / kg, 0.08mg / kg, 0.16mg / kg and 0.24mg / kg GLP-1-gFc were administered in sequence.

[0185] The starting dose of 0.01 mg / kg was determined based on the fact that the no observed adverse effect level (NOAEL) in the subchronic toxicity study in cynomolgus monkeys was 30 mg / kg, which resulted in a human equivalent dose (HED) of 9.75 mg / kg. As a very conservative approach, a safety factor of 1000 was applied, resulting in a maximum required starting dose (MRSD) of 0.00975 mg / kg, which is approximately 0.01 mg / kg. Submaximal and maximum doses were used to examine the safety of GLP-1-gFc when administered at an equivalent efficacy dose, which is 4-fold higher than dulaglutide and the same dose used in the clinical trial of dulaglutide. 21 .

[0186] (2-1) Safety (especially pulse rate, adverse events)

[0187] Safety was assessed throughout the study and at multiple follow-up visits by monitoring adverse events, vital signs (blood pressure, pulse rate, temperature), 12-lead ECG, physical examination, and laboratory investigations (including anti-drug antibodies during screening).

[0188] (2-2) Pharmacokinetics (PK)

[0189] Blood samples for PK analysis were collected into serum separator tubes by venipuncture or indwelling intravenous catheter before and at designated time points ranging from 0.25 h to 648 h after dosing. Serum GLP-1-gFc concentrations in blood samples were analyzed using a validated ELISA method that detects both the N-terminal intact GLP-1 and the C-terminal end of gFc. PK parameters were calculated using a compartment-free method using Pharsight The AUC of GLP-1-gFc was plotted. last and C max Plots relative to each dose to assess dose proportionality.

[0190] (2-3) Oral glucose tolerance test (OGTT)

[0191] Blood samples for PK analysis were collected into serum separator tubes by venipuncture or indwelling intravenous catheter before and at designated time points ranging from 0.25 h to 648 h after dosing. Serum GLP-1-gFc concentrations in blood samples were analyzed using a validated ELISA method that detects both the N-terminal intact GLP-1 and the C-terminal end of gFc. PK parameters were calculated using a compartment-free method using Pharsight The AUC of GLP-1-gFc was plotted. last and C max Plots relative to each dose to assess dose proportionality.

[0192] After an overnight fast, the subjects drank 300 mL of a commercially available OGTT drink containing 75 g of glucose within 5 minutes. Blood samples for the determination of blood glucose and insulin were collected before and after 0.25 h, 0.5 h, 1 h, 1.5 h, and 2 h of glucose solution intake. The collected samples were analyzed by spectrophotometry and electrochemiluminescence immunoassay (ECLIA) using Cobas c501 and Cobas e / 601 modules (Roche Diagnostics, Switzerland), respectively, to obtain the kinetics of glucose and insulin changes over time. During the test, the subjects remained seated.

[0193] (2-4) Statistical analysis

[0194] SPSS21 (IBM SPSS, Chicago, IL, USA) was used to exclude outliers and analyze statistical significance. Data for PK and human studies are expressed as mean ± standard deviation, and other data are expressed as mean ± SEM. Statistical significance was determined by Student's t-test or Mann-Whitney U test of nonparametric methods. Differences were considered statistically significant at P < 0.05.

[0195] (2-5) Discussion

[0196] (A) GLP-1-gFc exhibits lower in vitro potency than dulaglutide in GLP-1R overexpressing cell lines because of its rapid dissociation from GLP-1R.

[0197] The GLP-1 of the fusion protein with sequence number SEQ ID NO:41 has a single amino acid substitution at the N-terminus to prevent DPP-4 23 Furthermore, O-glycosylation of the hinge region of IgD is expected to improve in vivo stability without loss of activity. In fact, the introduction of O-glycosylation into the hinge region showed a significant enhancement of pharmacokinetics and pharmacodynamics in rodents without loss of activity. Figure 1a to Figure 1h When the two molecules, GLP-1-gFc and dulaglutide of Preparation Example 1, were analyzed in a cell-based assay using a GLP-1 receptor overexpressing cell line that releases cAMP-dependent luciferin, different response curves were obtained for each molecule when the same molar concentration of the two molecules was incubated with the cell line.

[0198] At the same molar concentration, it showed an EC value 3.5 times lower than that of dulaglutide at 23.33 pM. 50 Compared with the value of 6.66 pM for dulaglutide, GLP-1-gFc showed a relatively lower response ( Figure 1b To determine the reason for the different in vitro activities of these molecules, binding affinity was assessed using SPR by flowing them over a human GLP-1 receptor immobilized chip ( Figure 1c ). GLP-1-gFc and dulaglutide showed a dose-dependent increase in response units (RU), and GLP-1-gFc showed a faster rate of RU decrease than dulaglutide. The dissociation slope of GLP-1-gFc, expressed as a dissociation constant (Kd), was 6.43x10 -2 , which is about 10 times higher than dulaglutide. However, the binding constant (Ka) of GLP-1-gFc is 4.02x10 3 , which differs from dulaglutide by only 1.7-fold. This lower binding affinity of GLP-1-gFc was confirmed in a BLI (biolayer interferometry) system, a different assay format used to identify the binding affinity of molecules. Figure 1h In general, the equilibrium dissociation constants (KD) of GLP-1-gFc and dulaglutide are 1.6x10 -5 and 9.04x 10 -7 , indicating that GLP-1-gFc dissociates faster from the GLP-1 receptor than dulaglutide. These observations suggest that GLP-1-gFc has lower binding affinity and in vitro potency than dulaglutide due to different structural features.

[0199] (B) GLP-1-gFc showed comparable glucose-lowering efficacy in diabetic db / db mice at a dose 4-fold higher than that of dulaglutide

[0200] To find a dose that exhibited comparable antidiabetic effects, 0.6 mg / kg and 2.4 mg / kg of GLP-1-gFc (GLP-1(A2G)-hyFc9) were evaluated at the optimal dose of dulaglutide in b / db mice, which was 0.6 mg / kg. 22,24 ( Figure 2a and Figure 2b GLP-1-gFc and dulaglutide were administered weekly via the SC route for six weeks. By the end of the study, nonfasting blood glucose in the vehicle-treated group increased from 274 mg / dL to 515 mg / dL (Δ glucose: 241 mg / dL).

[0201] All TA treatment groups showed statistically significant reductions in final glucose levels compared to vehicle treatment groups. Dulaglutide significantly prevented the increase in nonfasting blood glucose levels with a final blood glucose level of 348 mg / dL (Δ glucose: 76.3 mg / dL). And GLP-1-gFc showed a dose-dependent effect on delayed glucose increase with final blood glucose levels of 459 mg / dL and 355 mg / dL, an increase of 0.6 mg / kg and 2.4 mg / kg, respectively (Δ glucose 185 mg / dL and 80.1 mg / dL, respectively) ( Figure 2a A similar pattern of efficacy was demonstrated in changes in glycated hemoglobin (HbA1c). Only dulaglutide and high-dose GLP-1-gFc showed meaningful terminal HbA1c (%) reductions after six weeks of dosing, with mean values ​​of 4.26% and 4.34%, respectively ( Figure 2b ). Together, these results suggest that approximately 4-fold higher amounts of GLP-1-gFc may be required to achieve antidiabetic efficacy comparable to that of dulaglutide in vivo.

[0202] (C) GLP-1-gFc showed comparable efficacy in lowering blood glucose, but had weaker effects on food intake and weight loss than dulaglutide

[0203] GLP-1 is a well-known pleiotropic ligand whose receptors are present in various organs such as the pancreas, heart, vagus nerve, brain, etc. 25-27 Reductions in food intake / body weight and insulin secretion are well-known effects of GLP-1 that result from GLP-1 receptor signaling in the vagus nerve / brain and pancreas 28To further investigate and compare the effects of GLP-1-gFc and dulaglutide on GLP-1 receptors in the pancreas and vagus nerve / brain, GLP-1-gFc and dulaglutide were administered subcutaneously (SC) weekly to obese ob / ob mice for four weeks. Both GLP-1-gFc and dulaglutide significantly delayed the increase in HbA1c (%) (ΔHbA1c) compared with vehicle (0.9%, 1.1%, and 2.0% for GLP-1-gFc, dulaglutide, and vehicle, respectively) ( Figure 3a ).

[0204] Compared with vehicle, dulaglutide significantly reduced cumulative food intake and body weight (-17 g / cage and -1.9% relative to vehicle). On the other hand, GLP-1-gFc showed a weaker response for both parameters, with the difference between 2.5 mg / kg GLP-1-gFc and dulaglutide being significant in body weight changes at weeks 2 and 3 ( Figure 3b and Figure 3c ). These findings suggest that dulaglutide and GLP-1-gFc may represent different receptor-mediated responses depending on the organ that expresses the GLP-1 receptor at different levels.

[0205] (D) GLP-1-gFc has less risk of nausea / vomiting and QT prolongation than dulaglutide at equivalent doses

[0206] Conditioned Taste Aversion (CTA) Studies in Mice 7,19 The monkeys were monitored for electrocardiogram (ECG) to further investigate the response of GLP-1-gFc of Preparation Example 1 in extrapancreatic organs compared to dulaglutide. For the CTA study, blueberry bars were exposed to mice (n=10 / group) before administration of vehicle, 0.3M LiCl, dulaglutide 0.6 mg / kg, and GLP-1-gFc 2.4 mg / kg. The second consumption of blueberry bars was recorded to assess the nausea / vomiting response of each test molecule previously paired with the first blueberry bar exposure.

[0207] At the first exposure to the blueberry bar, consumption was almost identical between the groups assigned to each test molecule ( Figure 4a ). But in the lithium chloride and dulaglutide paired group, the consumption of blueberry bars after the second exposure was significantly reduced. However, the reduction in bar consumption by GLP-1-gFc was much smaller than that in the LiCl and dulaglutide groups, showing statistical significance with the dulaglutide paired group ( Figure 4b ). On the day before the second blueberry bar exposure, overnight food intake was measured to ensure that the effects of long-acting GLP-1-gFc and dulaglutide on food intake suppression were excluded. Compared with the overnight food intake on the first day after injection, there were no significant differences between the groups.

[0208] like Figure 4d As shown, in the CTA study (n=8-10 / group), overnight food intake on the first day after injection was significantly reduced in the treatment groups receiving dulaglutide or GLP-1-gFc, as confirmed by drug washout assessed by overnight food intake before the second exposure to blueberry bars. In contrast, there was no difference in overnight food intake between the GLP-1-gFc and dulaglutide groups on the day before the second exposure (day 13), confirming that GLP-1-RA-related food intake suppression was completely eliminated. Results are expressed as mean ± standard error of the mean. ***p<0.001 relative to vehicle; #p<0.01 relative to dulaglutide; Mann-Whitney U test ns, not significant; Dula_0.6, dulaglutide 0.6mg / kg; gFc_2.4, GLP-1-gFc 2.4mg / kg.

[0209] This result suggests that the vagus nerve / brain response of GLP-1-gFc is different from that of dulaglutide, which is inconsistent with the trend observed in the pancreas.

[0210] To evaluate and compare the cardiovascular effects of GLP-1-gFc and dulaglutide, a total of 7 male cynomolgus monkeys implanted with Telementry received 0.07 mg / kg dulaglutide (n=3), 0.28 mg / kg GLP-1-gFc (n=2), 1.14 mg / kg GLP-1-gFc (n=2) and 1.2 mg / kg GLP-1-gFc (n=2) by SC route. Figure 4c Monkeys received a single dose of vehicle, followed by a 19-day washout period and administration of GLP-1-gFc or dulaglutide. ECG waveforms, heart rate, and blood pressure were recorded from 2 hours before to 24 hours after dosing.

[0211] Even though there were no treatment-related clinical symptoms after a single dose, a numerically meaningful difference between the corrected QT (QTc) intervals of GLP-1-gFc according to the present disclosure and dulaglutide was determined during ECG monitoring. Dulaglutide increased the QTc interval within a specific time range of 10-20 hours, which is predicted to be T max , while low and high doses of GLP-1-gFc did not increase QTc. However, these differences did not lead to any differences in heart rate and blood pressure.

[0212] Overall, these findings suggest that GLP-1-gFc according to the present disclosure may produce a milder response to GLP-1 receptors on the vagus nerve and heart than on the pancreatic receptors, possibly due to its reduced receptor affinity, and this phenomenon is different from other long-acting GLP-1 analogs with high potency (such as dulaglutide).

[0213] (E) Dose-dependent long-acting pharmacokinetics (PK) of GLP-1-gFc after a single SC administration to healthy subjects.

[0214] The purified GLP-1-gFc of Preparation Example 1 exhibited a long-acting PK profile in SD rats and cynomolgus monkeys, with half-lives of 14.1-15.3 hours and 79.1-113.8 hours, respectively. Moreover, it dose-dependently enhanced insulin secretion and glucose reduction in diabetic db / db mice, as shown in Figure 5d As shown. Based on these results, GLP-1-gFc was administered to healthy subjects to confirm the dose-dependent long-acting pharmacokinetics. Six different doses were administered to healthy subjects in sequence from 0.01 mg / kg to 0.24 mg / kg, and blood collected at designated time points was analyzed using the ELISA method.

[0215] The pharmacokinetics of GLP-1-gFc followed a monoexponential decline, with the median T 1 / 2 The range is 62.5 hours to 108 hours ( Figure 5a and Table 2). Geometric mean serum concentrations reached their respective peaks approximately 36 to 48 hours after administration, with mean C max The doses were 36.4 ng / mL (0.01 mg / kg), 68.2 ng / mL (0.02 mg / kg), 102.6 ng / mL (0.04 mg / kg), 242.4 ng / mL (0.08 mg / kg), 454.4 ng / mL (0.16 mg / kg) and 1087.7 ​​ng / mL (0.24 mg / kg). max and AUC last The AUC of GLP-1-gFc was observed in the figure last and C max R 2 The linearity is 0.9891 and 0.9925.

[0216] (F) Despite its good efficacy in OGTT, GLP-1-gFc was well tolerated with no significant side effects on nausea / vomiting and heart rate.

[0217] The safety and efficacy of GLP-1-gFc were evaluated based on several safety parameters including blood pressure, pulse rate, treatment-emergent adverse events (TEAEs), and OGTT according to a protocol approved by the German Federal Institute for Drugs and Medical Devices (BfArM). Overall, a single SC dose of GLP-1-gFc in the dose range of 0.01 to 0.24 mg / kg was safe and well tolerated, without the development of antibodies against GLP-1-gFc. There were no serious adverse events (SAEs), and all TEAEs were mild to moderate in intensity, which resolved by the end of the study.

[0218] In the OGTT study, GLP-1-gFc reduced gAUC (AUC in the glucose-time graph) in a dose-dependent manner. This reduction was more obvious 3 days after administration than 5 days after administration, which is consistent with the T of GLP-1-gFc in pharmacokinetics. max Consistent for 36-48 hours. On day 3 post-dose, the gAUC inhibition (approximately -65% from baseline) was most significant at the highest dose (0.24 mg / kg), and the changes in gAUC at 0.08 mg / kg and 0.16 mg / kg were also significant, inhibiting 55% and 53% from baseline, respectively. The off-target effects of GLP-1-gFc were assessed by the percentage of subjects experiencing nausea / vomiting during the study and the pulse rate on the same day of the OGTT assessment. Almost no subjects experienced nausea / vomiting before the dose of 0.16 mg / kg, and only one subject experienced nausea at the 0.04 mg / kg dose. At the highest dose, 4 of 6 subjects and 1 of 6 subjects experienced transient nausea and vomiting, respectively. In all groups, there was no significant change in pulse rate compared to baseline on days 3 and 5 after dosing.

[0219] Taken together, these results support that the GLP-1-gFc of the present disclosure exhibits a stronger in vivo glucose-lowering effect at an efficacy dose equivalent to dulaglutide, with significantly reduced side effects of nausea / vomiting and QTc.

[0220] Compare files

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Claims

1. A method for regulating blood glucose level in a subject in need thereof, comprising the step of administering to the subject an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region, in, The immunoglobulin Fc region (b) comprises (i) an isolated IgD hinge region consisting of 35 to 49 consecutive amino acid residues from the C-terminus of SEQ ID NO: 35; and (ii) the CH2 domain and the CH3 domain of the immunoglobulin Fc polypeptide.

2. The method of claim 1, wherein: The effective amount is from about 0.01 mg / kg to about 1 mg / kg body weight.

3. The method of claim 1, wherein: The fusion peptide is administered parenterally at intervals of one week or more.

4. The method of claim 1, wherein: The subject suffers from diabetes, glucose intolerance and / or insulin resistance.

5. The method of claim 1, wherein: The GLP-1 peptide (a) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NOS: 10 to 34.

6. The method of claim 1, wherein: The isolated IgD hinge region (i) comprises the amino acid sequence of SEQ ID NO:36, 37 or 38.

7. The method of claim 1, wherein: The immunoglobulin Fc region (b) comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 4 to 8.

8. The method of claim 1, wherein: The fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 40 to 42 or 54.

9. The method of claim 3, wherein: The fusion peptide is administered at a dose of 0.01 mg / kg to 0.2 mg / kg at weekly intervals or at a frequency of once a week.

10. The method of claim 3, wherein: The fusion peptide is administered at a dose of 0.2 mg / kg to 0.5 mg / kg at a frequency of two-week intervals or every other week.

11. The method of claim 1, wherein: The subject suffers from diabetes.

12. The method of claim 11, wherein: The diabetes is type II diabetes.

13. The method of claim 3, wherein: The fusion peptide is administered subcutaneously.

14. The method of claim 1, wherein: The fusion peptide is a dimer comprising two peptides linked together by a sulfide bond, wherein each peptide comprises an Fc region (b) having a sequence number of SEQ ID NO: 4, 5, 6, 7 or 8.

15. A method for preventing and / or treating diabetes in a subject in need thereof, comprising administering to the subject an effective amount of a fusion peptide comprising (a) a glucagon-like peptide-1 (GLP-1) peptide and (b) an immunoglobulin Fc region, in, The immunoglobulin Fc region (b) comprises (i) an isolated IgD hinge region consisting of 35 to 49 consecutive amino acid residues from the C-terminus of SEQ ID NO: 35; and (ii) the CH2 domain and the CH3 domain of the immunoglobulin Fc polypeptide.

16. The method of claim 15, wherein: The effective amount is from about 0.01 mg / kg to about 1 mg / kg body weight.

17. The method of claim 15, wherein: The fusion peptide is administered parenterally at intervals of one week or more.

18. The method of claim 15, wherein: The fusion peptide is administered at a dose of 0.01 mg / kg to 0.2 mg / kg at weekly intervals or at a frequency of once a week.

19. The method of claim 15, wherein: The fusion peptide is administered at a dose of 0.2 mg / kg to 0.5 mg / kg at a frequency of two-week intervals or every other week.

20. The method of claim 15, wherein: The diabetes mellitus is non-insulin-dependent diabetes mellitus or insulin-dependent diabetes mellitus.

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