FUSION POLYPEPTIDE COMPRISING Fc REGION OF IMMUNOGLOBULIN AND GDF15

Abstract

Provided is a fusion polypeptide comprising GDF15 (Growth/differentiation factor 15) and an Fc region of immunoglobulin, a pharmaceutical composition comprising the fusion polypeptide, and a method of increasing in vivo duration of GDF15 comprising fusing with an Fc region of immunoglobulin.

Claims

1. A fusion polypeptide, comprising GDF15 (Growth/differentiation factor 15) or its functional variant, and Fc region of an immunoglobulin, wherein, the Fc region of the immunoglobulin is a single chain of IgG1 Fc region or IgG4 Fc region, and is linked to the N-terminus of the GDF15 or its functional variant, via a flexible peptide linker, the functional variant of GDF15 is a deletion variant in which at least one of 14 amino acids at positions 1 to 14 of the amino acid sequence of SEQ ID NO: 1 is deleted, and the flexible peptide linker is represented by (GGGGS)n (n is 1, 2, 3, 4, or 5).

2. The fusion polypeptide according to claim 1, wherein the Fc region of the immunoglobulin is human IgG4 Fc region.

3. The fusion polypeptide according to claim 2, wherein the human IgG4 Fc region (1) comprises the amino acid sequence of SEQ ID NO: 5, or (2) further comprises the amino acid sequence of SEQ ID NO: 10 at the N-terminus of the amino acid sequence of SEQ ID NO: 5.

4. The fusion polypeptide according to claim 3, wherein the human IgG4 Fc region (1) comprises an amino acid sequence selected from SEQ ID NOs: 6 to 9, or (2) further comprises an amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 at the N-terminus of the amino acid sequence selected from SEQ ID NOs: 6 to 9.

5. The fusion polypeptide according to claim 1, wherein the functional variant of GDF15 comprises the amino acid sequence of SEQ ID NO: 2.

6. The fusion polypeptide according to claim 1, wherein the GDF15 or its functional variant linked to the Fc region of the immunoglobulin in the fusion polypeptide has at least 1.5 times increased in vivo half-life, compared to GDF15 or its functional variant which is not linked to Fc region of the immunoglobulin.

7. A fusion polypeptide dimer, comprising 2 fusion polypeptides of claim 1.

8. A nucleic acid molecule encoding the fusion polypeptide of claim 1.

9. A recombinant vector comprising the nucleic acid molecule of claim 8.

10. A recombinant cell comprising the recombinant vector of claim 9.

11. A method of preparation of the fusion polypeptide of claim 1, comprising culturing a recombinant cell comprising a recombinant vector that comprises a nucleic acid molecule encoding the fusion polypeptide of claim 114

12. A method of enhancing in vivo stability of GDF15 or its functional variant, comprising linking an Fc region of the immunoglobulin to the N-terminus of GDF15 or its functional variant via a flexible peptide linker, wherein the Fc region of the immunoglobulin is a single chain of IgG1 Fc region or IgG4 Fc region the functional variant of GDF15 is a deletion variant in which at least one of 14 amino acids from 1 to 14 of the amino acid sequence of SEQ ID NO: 1 are deleted, and the flexible peptide linker is represented by (GGGGS)n ((SEQ ID NO: 13)n, wherein n is 1, 2, 3, 4, or 5).

13. The method for enhancing in vivo stability of GDF15 or its functional variant according to claim 12, wherein the Fc region of the immunoglobulin is human IgG4 Fc region.

14. The method for enhancing in vivo stability of GDF15 or its functional variant according to claim 13, wherein the human IgG4 Fc region (1) comprises the amino acid sequence of SEQ ID NO: 5, or (2) further comprises the amino acid sequence of SEQ ID NO: 10 at the N-terminus of the amino acid sequence of SEQ ID NO: 5.

15. The method for enhancing in vivo stability of GDF15 or its functional variant according to claim 14, wherein the human IgG4 Fc region (1) comprises an amino acid sequence selected from SEQ ID NOs: 6 to 9, or (2) further comprises an amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 at the N-terminus of the amino acid sequence selected from SEQ ID NOs: 6 to 9.

16. The method for enhancing in vivo stability of GDF15 or its functional variant according to claim 12, wherein the functional variant of GDF15 comprises the amino acid sequence of SEQ ID NO: 2.

17. The method for enhancing in vivo stability of GDF15 or its functional variant according to claim 12, wherein the GDF15 or its functional variant linked to the Fc region of the immunoglobulin in the fusion polypeptide has at least 1.5 times increased in vivo half-life, compared to GDF15 or its functional variant which is not linked to Fc region of the immunoglobulin.

18. A composition comprising at least one selected from the group consisting of: the fusion polypeptide of claim 1, a fusion polypeptide dimer comprising two of the fusion polypeptides, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector.

19. A method of weight loss, dietary control, or prevention or treatment of metabolic disease, comprising administering to a subject in need thereof a therapeutically effective amount of at least one selected from the group consisting of: the fusion polypeptide of claim 1, a fusion polypeptide dimer comprising two of the fusion polypeptides, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector.

20. The method of claim 19, wherein the metabolic disease is obesity, diabetes, or nonalcoholic fatty liver disease.

21. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG. 1 is a mimetic diagram of the fusion polypeptide according to one example.

[0075] FIG. 2 shows the amino acid sequence of GDF15 (wild type; mature form; SEQ ID NO: 1) (from N-terminus to C-terminus).

[0076] FIG. 3a and FIG. 3b show the structure of the complex in which Fc-ΔN14GDF15 (3a) or Fc-wtGDF15 (3b) binds to GFRAL.

[0077] FIG. 4 is a mimetic diagram schematically showing the fusion polypeptide gene according to the example.

[0078] FIG. 5 is a graph showing the weight change of the IgG1-GDF (CRL) administration group and HIgG1-GDF (CRL) administration group.

[0079] FIG. 6 is a graph showing the weight change rate at 7 days after administration of the IgG1-GDF (CRL) administration group and HIgG1-GDF (CRL) administration group.

[0080] FIG. 7 is a graph showing the weight change of the IgG4-GDF15 administration group and IgG4-GDF(CRL) administration group.

[0081] FIG. 8 is a graph showing the weight change rate at 7 days (blue bar) and 14 days (red bar) after administration of the IgG4-GDF15 administration group and IgG4-GDF(CRL) administration group.

[0082] FIG. 9 is a graph showing the cumulative feed intake by 7 days after administration of the IgG1-GDF (CRL) administration group and HIgG1-GDF (CRL) administration group.

[0083] FIG. 10 is a graph showing the cumulative feed intake by 7 days, 14 days and 19 days after administration of the IgG4-GDF15 administration group and IgG4-GDF(CRL) administration group.

[0084] FIG. 11 is a graph showing the change in the fusion polypeptide concentration in blood (in serum) according to the elapsed time after administration of the fusion polypeptide.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0085] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are intended to illustrate the present invention only, but the scope of the present invention is not limited by these examples.

EXAMPLE 1

Preparation of Fusion Polypeptide

[0086] 1.1. Cloning and culturing of gene encoding fusion polypeptide IgG1-GDF(CRL), HIgG1-GDF(CRL), IgG4-GDF, HIgG4-GDF, IgG4-GDF(CRL), and HIgG4-GDF(CRL) (See FIG. 1) which were fusion polypeptides fused with Mature GDF15 (represented by GDF or GDF15; SEQ ID NO: 1), or GDF15 variant (represented by GDF(CRL); 14 amino acids at the N-terminus were deleted: SEQ ID NO: 2) that had a target polypeptide of immunoglobulin Fc (IgG1-Fc (not comprising hinge: SEQ ID NO: 3; HIgG1-Fc (comprising hinge; [SEQ ID NO: 4]-[SEQ ID NO: 3]), Mutated IgG4-Fc (not comprising hinge; SEQ ID NO: 7) or Mutated HIgG4-Fc (comprising hinge: [SEQ ID NO: 12]-[SEQ ID NO: 7]) comprising or not comprising hinge, were prepared. The amino acid sequences of each part comprised in the fusion polypeptide were summarized in Table 2, Table 3 and Table 4 below.

TABLE-US-00002 TABLE 2 IgG1-GDF(CRL) (not comprising hinge) & HIgG1-GDF(CRL) (comprising hinge) (direction from N-terminus to C-terminus) SEQ ID Amino acid sequence NO: Signal Peptide MHRPEAMLLL LTLALLGGPT WA 17 IgG1 Fc Hinge DKTHTCPPCP  4 CH2-CH3 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD  3 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG Linker GGGGSGGGGS GGGGSGGGGS 18 GDF(CRL) CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA  2 ANMHAQIKTS LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL LAKDCHCI

TABLE-US-00003 TABLE 3 IgG4-GDF15 (not comprising hinge) & HIgG4-GDF15 (comprising hinge) (direction from N-terminus to C-terminus) SEQ ID Amino acid sequence NO: Signal Peptide MHRPEAMLLL LTLALLGGPT WA 17 Mutated Hinge ESKYGPPCPP CP 12 IgG4 Fc CH2-CH3 APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED  7 PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG Linker GGGGSGGGGS GGGGSGGGGS 18 GDF15 ARNGDHCPLG PGRCCRLHTV RASLEDLGWA DWVLSPREVQ  1 VTMCIGACPS QFRAANMHAQ IKTSLHRLKP DTVPAPCCVP ASYNPMVLIQ KTDTGVSLQT YDDLLAKDCH CI

TABLE-US-00004 TABLE 4 IgG4-GDF(CRL) (not comprising hinge) & HIgG4-GDF(CRL) (comprising hinge) (direction from N-terminus to C-terminus) SEQ ID Amino acid sequence NO: Signal Peptide MHRPEAMLLL LTLALLGGPT WA 17 Mutated Hinge ESKYGPPCPP CP 12 IgG4 Fc CH2-CH3 APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED  7 PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG Linker GGGGSGGGGS GGGGSGGGGS 18 GDF15(CRL) CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA  2 ANMHAQIKTS LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL LAKDCHCI

[0087] 1.1.1 Preparation of Recombinant Expression Vector

[0088] 1.1.1.1. Mature GDF15

[0089] To obtain a gene encoding Mature GDF15, referring to the amino acid of UniprotKB Q99968, a gene encoding Mature GDF15 (SEQ ID NO: 14) was synthesized in Bioneer.

TABLE-US-00005 (339 bp) SEQ ID NO: 14   1 GCCCGGAACG GCGACCACTG CCCCCTGGGG CCCGGACGGT GCTGCCGGCT  51 GCACACCGTG CGGGCCTCCC TGGAGGACCT GGGCTGGGCC GACTGGGTGC 101 TGTCCCCAAG GGAGGTGCAA GTGACCATGT GCATCGGCGC CTGCCCATCT 151 CAGTTCCGGG CCGCCAACAT GCACGCTCAG ATCAAGACCA GCCTGCACCG 201 GCTGAAGCCC GACACCGTGC CCGCCCCCTG CTGCGTGCCC GCCTCCTACA 251 ACCCCATGGT GCTGATTCAG AAGACCGACA CCGGCGTGAG CCTGCAGACC 301 TACGACGACC TGCTGGCCAA GGACTGCCAC TGCATCTAA (The underlined part is GDF(CRL))

[0090] 1.1.1.2. IgG1-Fc

[0091] A gene coding human IgG1 Fc comprising hinge or a gene coding human IgG1 Fc not comprising hinge was obtained using by a plasmid comprising a gene coding core hinge of human IgG1 and IgG1 Fc by PCR.

TABLE-US-00006 (678 bp) SEQ ID NO: 15   1 GACAAAACTC ACACATGCCC ACCGTGCCCA GCACCTGAAC TCCTGGGGGG  51 ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT 101 CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC 151 CCTGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC 201 CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC CGTGTGGTCA 251 GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGAGTACAAG 301 TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC 351 CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTATACC CTGCCCCCAT 401 CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA 451 GGCTTCTATC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC 501 GGAGAACAAC TACAAGACCA CGCCTCCCGT GCTGGACTCC GACGGCTCCT 551 TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG 601 AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC 651 GCAGAAGAGC CTCTCCCTGT CTCCGGGT (The underlined part is a gene coding IgG1 Core Hinge)

[0092] 1.1.1.3. IgG4-Fc

[0093] A gene coding human IgG4 Fc comprising hinge or a gene coding human IgG4 Fc not comprising hinge was obtained using by a plasmid comprising a gene coding hinge of human IgG4 and IgG4 Fc by PCR.

TABLE-US-00007 (684 bp) SEQ ID NO: 16   1 GAGTCCAAAT ATGGTCCCCC ATGCCCACCC TGCCCAGCAC CTGAGGCCGC  51 CGGGGGACCG TCAGTCTTCC TCTTCCCCCC AAAACCCAAG GACACCCTCA 101 TGATCTCCCG GACCCCTGAG GTCACGTGCG TGGTGGTGGA CGTGTCCCAG 151 GAGGACCCCG AGGTGCAGTT CAACTGGTAC GTGGACGGCG TGGAGGTGCA 201 CAACGCCAAG ACCAAGCCCC GGGAGGAGCA GTTCAACTCC ACCTACCGGG 251 TGGTGTCCGT GCTGACCGTG CTGCACCAGG ACTGGCTGAA CGGCAAGGAG 301 TACAAGTGCA AGGTGTCCAA CAAGGGCCTG CCCTCCTCCA TCGAGAAGAC 351 CATCTCCAAG GCCAAGGGCC AGCCCCGGGA GCCCCAGGTG TACACCCTGC 401 CCCCCTCCCA GGAGGAGATG ACCAAGAACC AGGTGTCCCT GACCTGCCTG 451 GTGAAGGGCT TCTACCCCTC CGACATCGCC GTGGAGTGGG AGTCCAACGG 501 CCAGCCCGAG AACAACTACA AGACCACCCC CCCCGTGCTG GACTCCGACG 551 GCTCCTTCTT CCTGTACTCC CGGCTGACCG TGGACAAGTC CCGGTGGCAG 601 GAGGGCAACG TGTTCTCCTG CTCCGTGATG CACGAGGCCC TGCACAACCA 651 CTACACCCAG AAGTCCCTGT CCCTGTCCCT GGGC (The underlined part is a gene coding IgG4 Hinge)

[0094] 1.1.1.4. Preparation of Expression Vector

[0095] pDHDD-D1G1 (comprising the promoter of KR10-1868139B1) which was a variant of pcDNA3.1(+) (Invitrogen, Cat. No. V790-20) was cut by BamHI and NotI, and the genes (mature GDF15, IgG1-Fc, IgG4-Fc) were combined thereto to insert the gene having the following structure (See FIG. 4), thereby preparing each recombinant vector.

[0096] pHIgG1-GDF(CRL)

[0097] ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG1 Core Hinge (SEQ ID NO: 4)-IgG1 CH2-CH3 (SEQ ID NO: 3)-GS Linker (SEQ ID NO: 18)-GDF (CRL) (SEQ ID NO: 2)- NotI restriction site]-(C-terminus)’

[0098] plgG1-GDF(CRL)

[0099] ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG1 CH2-CH3 (SEQ ID NO: 3)-GS Linker (SEQ ID NO: 18)-GDF (CRL) (SEQ ID NO: 2)-NotI restriction site]-(C-terminus)’

[0100] pH IgG4-G DF15

[0101] ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG4 Hinge (SEQ ID NO: 12)-IgG4 CH2-CH3 (SEQ ID NO: 7)-GS Linker (SEQ ID NO: 18)-GDF15 (SEQ ID NO: 1)- NotI restriction site]-(C-terminus)’

[0102] plgG4-GDF15

[0103] ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG4 CH2-CH3 (SEQ ID NO: 7)-GS Linker (SEQ ID NO: 18)-GDF15 (SEQ ID NO: 1)- NotI restriction site]-(C-terminus)’

[0104] pH IgG4-G DF(CRL)

[0105] ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG4 Hinge (SEQ ID NO: 12)-IgG4 CH2-CH3 (SEQ ID NO: 7)-GS Linker (SEQ ID NO: 18)-GDF(CRL) (SEQ ID NO: 2)- NotI restriction site]-(C-terminus)’

[0106] plgG4-GDF(CRL)

[0107] ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)- IgG4 CH2-CH3 (SEQ ID NO: 7)-GS Linker (SEQ ID NO: 18)-GDF(CRL) (SEQ ID NO: 2)- NotI restriction site]-(C-terminus)’

[0108] 1.1.2. Culturing of Gene Encoding Fusion Polypeptide

[0109] The prepared recombinant expression vectors, pHIgG1-GDF(CRL), plgG1-GDF(CRL), pHIgG4-GDF(CRL), plgG4-GDF(CRL), pHIgG4-GDF and plgG4-GDF were introduced to ExpiCHO-S™ cell (Thermo Fisher Scientific), and were cultured in ExpiCHO Expression Medium (Thermo Fisher Scientific; 400 mL) for 12 days (Fed-Batch Culture; Day 1 & Day 5 Feeding), to express the fusion polypeptides, HIgG1-GDF(CRL), IgG1-GDF(CRL), HIgG4-GDF(CRL), IgG4-GDF(CRL), HIgG4-GDF and IgG4-GDF.

[0110] 1.2. Purification of Fusion Polypeptide

[0111] The fusion polypeptide was purified from the cell culture prepared in Example 1.1 using Protein A Affinity Chromatography.

[0112] At first, the culture solution of the cell-removed fusion polypeptide was filter with a 0.22 μm filter. A column in which MabSelect SuRe™ pcc (GE Healthcare Life Sciences) resin was packed was equipped to AKTA™ Pure (GE Healthcare Life Sciences), and Phosphate Buffered Saline (PBS, 10 mM Sodium Phosphate, 150 mM NaCl, pH 7.4) was flowed to equilibrate the column. After injecting the culture solution filtered with the 0.22 μm filter to the equilibrated column, PBS was flowed again to wash the column. After finishing washing of the column, eluting buffer (0.1 M Sodium Citrate pH 3.5) was flowed to the column to elute the target fusion polypeptide. 1M Tris pH 8.5 was added immediately to the eluted solution so as to be neutral pH. Among eluted fractions, fractions with high concentration of the fusion polypeptide and high purity were collected, and were stored frozen.

[0113] For an animal experiment, using Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge, the eluted fraction sample comprising the fusion polypeptide was concentrated with PBS or 20 mM Tris pH 8.0, 150 mM NaCl and buffer exchange was conducted.

[0114] The quantitative analysis of the fusion polypeptide was performed by measuring the absorbance at 280 nm and 340 nm in UV Spectrophotometer (G113A, Agilent Technologies), and calculating the protein concentration by the following equation. As the extinction coefficient of each material, the value theoretically calculated using the amino acid sequence (Table 5) was used.

[00001]$\mathrm{Protein}\mathrm{concentration}\left(\mathrm{mg}/\mathrm{mL}\right)=\frac{\mathrm{Absorbance}\left(A_{280\mathrm{nm}}-A_{340\mathrm{nm}}\right)}{*\mathrm{Extinction}\mathrm{Coefficient}}\times \mathrm{Dilution}\mathrm{Factor}$$\begin{array}{c}{*}^{}\mathrm{Extinction}\mathrm{coefficient}\left(0.1\%\right):a\mathrm{theoretical}\mathrm{absorbance}\mathrm{at}280\mathrm{nm},\\ \mathrm{assuming}\mathrm{that}\mathrm{the}\mathrm{protein}\mathrm{concentration}\mathrm{is}0.1\%\left(1g/L\right),\\ \mathrm{and}\mathrm{all}\mathrm{cysteines}\mathrm{on}\mathrm{the}\mathrm{primary}\mathrm{sequence}\mathrm{are}\mathrm{oxidized}\mathrm{to}\\ \mathrm{form}a\mathrm{disulfide}\mathrm{bond},\mathrm{which}\mathrm{is}\mathrm{calculated}\mathrm{by}\mathrm{ProtParam}\mathrm{tool}\end{array}$

TABLE-US-00008 TABLE 5 Extinction coefficient of fusion polypeptide Extinction coefficient Sample name (0.1%, 1 mg/mL) IgG1-GDF (CRL) 1.317 HIgG1-GDF (CRL) 1.22 IgG4-GDF (CRL) 1.334 IgG4-GDF 1.287

EXAMPLE 2

Pharmacological Effect of Fusion Polypeptide (In Vivo)

[0115] 2.1. Test Process

[0116] The pharmacological effect of the fusion polypeptide produced and purified in Example 1 was tested in mice (C57BL/6J, 6 weeks, male, 100 mice; RaonBio).

[0117] In this example, DIO mouse model (Mouse, C57BL/6J —DIO, male, 100 mice, 14 weeks (8-week feeding of obesity feed) in which the high-fat diet was fed to the C57BL/6J mice for 8 weeks to induce obesity was used. The DIO mouse model exhibits clinical characteristics of type 2 diabetes such as hyperlipidemia, insulin resistance, hyperglycemia, and the like, and therefore it is an animal model widely used for evaluation of improvement of diabetes and insulin resistance, and baseline data comparable for research on metabolic disease such as obesity, diabetes and hyperlipidemia, and the like are accumulated a lot, and therefore it is suitable for the pharmacological effect test of this example, and thus this model was selected.

[0118] The mouse model that was fed the obesity feed for 8 weeks was subjected to a 2-week inspection and purification period, and during this period, the general symptoms were observed once a day to check the health and whether it was suitable for conducting the experiment, to select healthy animals. During the purification period, the individual was marked with a red oil pen on the tail of the animal at the time of acquisition (tail marking), and in the breeding box, a temporary individual identification card (test name, individual number and arrival time) was attached during the inspection and purification period. When separating the group, the individual was marked with a black oil pen on the tail of the animal, and an individual identification card (test name, group information, individual number, gender, arrival time, administration period) was attached to each cage.

[0119] To minimize stress of the experimental animal by subcutaneous administration of the test material (fusion polypeptide), sterile distilled physiological saline was subcutaneously administered at 200 uL/head to all animals using a 1 ml syringe 3 days before administration of the test material, to perform pre-adaptation training for subcutaneous administration.

[0120] For healthy animals with no abnormalities found during the inspection and purification period, the weight and feed intake were measured for all individuals after completion of the purification period.

[0121] The weight and feed intake were measured and group separation was performed so that the mean of two measurements between groups was similar based on the body weight. The administration of the test material was initiated the day after the group separation. Residual animals not selected were excluded from the test system after the end of the group separation.

[0122] The information of the high fat diet (obesity feed; High fat diet (HFD)) fed to the C57BL/6J —DIO was as follows:

[0123] 5.24 kcal/g, fat 60% by weight, protein 20% by weight, and carbohydrate-derived calories 20% by weight; Research Diet Inc., U.S.A.; Product No. High fat diet (Fat 60 kcal %, D12492).

[0124] The feed was fed in a free feeding (feeding during the purification and test period) manner.

[0125] As the drinking manner, the tap water was filtered with a filter water flowing sterilizer and then irradiated with ultraviolet light, and it was freely consumed using a polycarbonate drinking water bottle (250 mL).

[0126] 2.1.1. HIgG1-GDF (CRL) and IgG1-GDF(CRL)

[0127] The administration of the test materials (HIgG1-GDF(CRL) and IgG1-GDF(CRL)) and control material GDF15 (R&D Systems) was conducted the day after the group separation, and the administration time was 9 AM every day. All the control material and test materials were administered subcutaneously. The administration route of the control material and test materials was selected as a subcutaneous route depending on the clinical intended route of administration.

[0128] The dose of all the control material and test materials was 5 mL/kg, and the dose by individual was calculated on the basis of the recently measured weight, and they were administered subcutaneously once at the test start day using a disposable syringe (1 mL). The test materials were administered only once at the test start day. For comparison, the control group in which the control material GDF15 was prepared, and for the comparison group in which GDF15 was administered, they were administered once a day for 5 days, 5 times in total, and all administrations were progressed from 9 AM.

[0129] The test group composition and administration dose, and the like were summarized in the following Table 6:

TABLE-US-00009 TABLE 6 IgG1-GDF (CRL) fusion polypeptide administration group composition Adminis- Adminis- High tration tration Fat Administration dose volume Animal Diet Test material route (nmol/kg) (mL/kg) number X Vehicle Subcutaneous — 5 5 O Vehicle, qd Subcutaneous — 5 5 O Vehicle, qw Subcutaneous — 5 5 O GDF15 Subcutaneous 2.86 5 3 O IgG1-GDF Subcutaneous 1 5 5 (CRL) O IgG1-GDF Subcutaneous 10 5 5 (CRL) O HIgG1-GDF Subcutaneous 1 5 5 (CRL) O HIgG1-GDF Subcutaneous 10 5 5 (CRL)

[0130] The observation, measurement and examination schedule for the test group was set Day 0 for the start of administration, and 7 days from the start of administration was set as 1 week of administration.

[0131] The examination schedule was summarized in Table 7:

TABLE-US-00010 TABLE 7 Examination schedule Purification period (week) Period (day) Observation item 1 2 0 1 2 3 4 5 6 7 High fat feed .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. feeding Oral and .circle-solid. subcutaneous administration a daptation Administration .circle-solid. Weight .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. measurement Feed intake .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. .circle-solid. measurement

[0132] For all animals, general clinical symptoms were observed once a day, and the presence of dying and dead animals was confirmed twice a day, and this observation was conducted from 1 day of administration to the end of administration. During the observation, only the case that there was an abnormal symptom was recorded on a recording sheet.

[0133] The weight of each mouse was measured at the day of the start of administration of the test materials (before administration), and then the weight was measured every day (measured by 9 days at maximum). The dose of the test materials was determined on the basis of the most recently measured weight.

[0134] In addition, after administering the test materials to mice, the feed intake was measured every day, and the amount of feeding was measured using an electronic scale by each breeding box, and then the residual amount was measured to calculate the feed intake a day. Individuals who severely ate the feed were excluded from the measurement.

[0135] All the experimental results obtained in this example were represented by mean±standard deviation and were examined using Prism5 (version 5.01). For all the data, one-way analysis of variance (ANOVA) was performed, and when the significance was observed, Dunnett's test was conducted to find the test group with a significant difference with the control group (significance level: both sides 5% and 1%, 0.1%).

[0136] 2.1.2. IgG4-GDF and IgG4-GDF (CRL)

[0137] The test materials (IgG4-GDF GDF IgG4-GDF(CRL)) and control material

[0138] Semaglutide (Bachem) were subcutaneously administered. The dose of all the control material and test materials was 5 mL/kg, and the dose by individual was calculated on the basis of the recently measured weight, and they were administered subcutaneously once at the test start day using a disposable syringe (1 mL). The test materials were administered only once at the test start day, and for comparison, the control group which is administered with the control material Semaglutide was prepared. For a comparison group in which Semaglutide was administered, Semaglutide were administered every day once a day. All administrations were progressed from 9 AM.

[0139] The test group composition and administration dose, and the like were summarized in the following Table 8:

TABLE-US-00011 TABLE 8 IgG4-GDF15 fusion polypeptide administration group composition Adminis- Adminis- High tration tration Fat Administration dose volume Animal Diet Test material route (nmol/kg) (mL/kg) number O Vehicle, qw Subcutaneous — 5 5 O Semaglutide, Subcutaneous  3 5 5 qd O IgG4-GDF Subcutaneous  1 5 5 O IgG4-GDF Subcutaneous 10 5 5 O IgG4-GDF Subcutaneous  1 5 5 (CRL) O IgG4-GDF Subcutaneous 10 5 5 (CRL)

[0140] The observation, measurement and examination schedule for the test groups was set Day 0 for the start of administration, and they were conducted as same as 2.1.1.

[0141] The weight of each mouse was measured at the day of the start of administration of the test materials (before administration), and then the weight was measured every day (measured by 19 days at maximum). The dose of the test materials was determined on the basis of the most recently measured weight.

[0142] In addition, after administering the test materials to mice, the feed intake was measured every day, and the amount of feeding was measured using an electronic scale by each breeding box, and then the residual amount was measured to calculate the feed intake a day. Individuals who severely ate the feed were excluded from the measurement.

[0143] 2.2. Weight Loss Test Result

[0144] 2.2.1. HIgG1-GDF (CRL) and IgG1-GDF(CRL)

[0145] The weight change measured in the Example 2.1.1 was shown in FIG. 5 and FIG. 6, and Table 9 (Body Weight (Group, % of initial).

TABLE-US-00012 TABLE 9 Group Day 0 1 2 3 4 5 6 7 8 9 Lean Vehicle Mean 100 102 101 101 101 101 101 101 101 102 Control S.E. 0 1 1 1 1 1 1 1 1 1 (Daily inj.) DIO Vehicle Mean 100 100 100 100 100 100 101 101 101 101 Control S.E. 0 0 0 0 1 1 1 1 0 0 (Daily inj.) DIO Vehicle Mean 100 100 99 100 100 100 101 101 102 102 Control S.E. 0 0 0 0 1 1 1 1 1 1 (Single Inj.) GDF15, Mean 100 99 98 97 96 95 95 95 96 97 2.86 nmol/kg S.E. 0 0 0 1 1 1 1 2 2 2 (Daily Inj.) IgG1-GDF(CRL), Mean 100 99 98 96 96 96 95 95 95 95 1 S.E. 0 1 1 0 1 1 1 1 1 1 (Single Inj.) nmol/kg IgG1-GDF(CRL), Mean 100 98 97 95 94 93 91 90 90 90 10 S.E. 0 0 1 1 1 1 1 1 1 1 (Single Inj.) nmol/kg HIgGl-GDF(CRL), Mean 100 100 98 98 97 96 97 96 96 97 1 S.E. 0 0 0 0 0 0 0 0 0 1 (Single Inj.) nmol/kg HIgGl-GDF(CRL), Mean 100 99 98 97 95 95 94 93 93 93 10 S.E. 0 0 0 0 1 1 1 1 1 1 (Single Inj.) nmol/kg (In the Table 9, Day0: first administration of control material GDF15, single administration og IgG1-GDF (CRL) fusion polypeptide; Lean: group without high fat diet)

[0146] FIG. 5 and Table 9 show the weight change after single administration of fusion protein IgG1-GDF by comparing with the negative control group (vehicle administration group) and positive control group (GDF15 daily administration group). In addition, FIG. 6 is a graph extracting and showing the result at 7 days among the results of FIG. 5.

[0147] As shown in the results, it can be confirmed that there was little weight change in case of the negative control group (vehicle administration group), while the weight loss effect disappeared from Day 6, the first day after stopping administration in case of the GDF15 daily administration group (stopping administration from Day 5). On the other hand, it can be confirmed that the weight loss effect was shown immediately after single administration at Day 0 in case of the fusion polypeptide in which GDF15(CRL) was fused with IgG1 or Fc comprising Hinge, and the weight loss effect was not reduced and was shown consistently throughout the test period (9 days), and as time went by, the concentration weight loss effect was increased, and the weight loss effect was concentration-dependent. This weight loss effect of the fusion polypeptide can be said to be comparable when GDF15 is administered once a day throughout the test period.

[0148] 2.2.2. IgG4-GDF and IgG4-GDF(CRL)

[0149] The weight change measured in Example 2.1.2 was shown in FIG. 7 and Table 10 (Body Weight (Group, % of initial).

TABLE-US-00013 TABLE 10 Group Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 DIO Vehicle Control Mean 100 100 100 99 100 100 100 100 101 101 102 102 103 104 104 104 105 105 105 105 (Single Inj.) S.E. 0 0 1 0 0 0 0 1 0 1 1 1 0 1 1 1 1 1 1 1 Semaglutide, 3 nmol/kg Mean 100 94 91 90 87 87 85 86 83 85 84 82 81 82 81 79 79 78 77 77 (Daily Inj.) S.E. 0 0 1 2 2 3 3 3 3 3 3 3 3 3 3 2 2 2 2 1 IgG4-GDF15, 1 nmol/kg Mean 100 99 97 96 95 93 93 92 92 91 91 91 93 94 94 94 95 96 96 96 (Single Inj.) S.E. 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 2 IgG4-GDF15, 10 Mean 100 98 97 96 94 93 93 92 91 90 90 90 91 92 92 92 92 93 94 94 (Single Inj.) S.E. 0 0 1 1 1 1 1 1 2 2 2 1 2 2 2 2 2 2 2 2 nmol/kg IgG4-GDF(CRL), 1 Mean 100 99 98 97 96 95 94 93 92 92 91 91 91 91 91 91 91 92 92 92 (Single Inj.) S.E. 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 nmol/kg IgG4-GDF(CRL), 10 Mean 100 98 97 95 94 92 91 90 88 87 85 85 84 83 83 82 83 84 84 85 (Single Inj.) S.E. 0 0 0 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 3 2 nmol/kg (In the Table 10, Day0: first administration of control material (Semaglutide) and test materials; The control material was administered every day; The test materials were administered once)

[0150] FIG. 7 and FIG. 8, and Table 10 show the weight change after single administration of fusion protein IgG4-GDF2F IgG4-GDF (CRL) by comparing with the negative control group (vehicle administration group) and positive control group (Semaglutide daily administration group). In addition, FIG. 8 is a graph extracting and showing the result at 7 days and 14 days among the results of FIG. 7.

[0151] As shown in the results, it can be confirmed that the weight was slightly increased in case of the negative control group (vehicle administration group), while the weight loss was consistent in case of the positive control group (Semaglutide daily administration group). When IgG4-GDF15 was administered once, regardless of dose, the weight loss effect lasted up to 9 days. On the other hand, it can be confirmed that the weight loss effect was shown consistently up to 10 days when 1 nmol/kg of IgG4-GDF(CRL) was administered once and up to 15 days when 10 nmol/kg was administered once, and as time went by, the concentration weight loss effect was increased, and the weight loss effect was concentration-dependent. In addition, it was confirmed that the GDF (CRL) fusion polypeptide with Fc (Mutated) of IgG4 without Hinge had the excellent weight loss effect at the same dose, compared to the

[0152] GDF (CRL) fusion polypeptide with Fc (Mutated) of IgG4 comprising Hinge. The weight loss effect of this IgG4 Fc fusion polypeptide can be said to be comparable when Semaglutide is administered once a day throughout the test period.

[0153] 2.3. Diet Intake Test Result

[0154] 2.3.1. HIgG1-GDF (CRL) and IgG1-GDF(CRL)

[0155] The feed intake change measured in Example 2.1.1 was shown in Table 11 and FIG. 9 (cumulative intake by 6 days), respectively.

TABLE-US-00014 TABLE 11 Group Day 0 1 2 3 4 5 6 7 8 9 Lean Vehicle Mean 4.5 4.6 4.6 4.3 4.4 4.6 4.2 4.5 3.9 4.7 Control S.E. 0.2 0.2 0.1 0.2 0.1 0.3 0.2 0.2 0.1 0.3 (Daily inj.) DIO Vehicle Mean 2.8 2.8 3.1 3.0 3.1 2.9 3.0 2.9 2.9 3.3 Control S.E. 0.3 0.2 0.2 0.3 0.3 0.2 0.3 0.2 0.1 0.2 (Daily inj.) DIO Vehicle Mean 3.1 2.9 3.1 3.2 3.3 3.2 3.3 3.2 3.0 3.5 Control S.E. 0.1 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.2 (Single Inj.) GDF15, 2.86 Mean 2.8 2.4 2.7 2.4 2.5 2.6 2.7 3.0 3.3 3.5 nmol/kg S.E. 0.4 0.3 0.2 0.4 0.2 0.3 0.2 0.3 0.2 0.3 (Daily Inj.) IgGl-GDF(CRL), Mean 3.0 2.1 2.2 2.2 2.6 2.6 2.6 2.8 2.7 3.0 1 S.E. 0.2 0.2 0.1 0.1 0.2 0.1 0.0 0.1 0.1 0.3 nmol/kg (Single Inj.) IgGl-GDF(CRL), Mean 2.7 2.0 2.5 2.0 1.9 1.9 2.1 2.1 2.3 2.8 10 S.E. 0.3 0.1 0.1 0.3 0.2 0.2 0.2 0.1 0.1 0.3 (Single Inj.) nmol/kg HIgGl-GDF(CRL), Mean 3.0 2.4 2.4 2.5 2.7 2.6 2.9 2.7 2.9 3.0 1 S.E. 0.2 0.1 0.1 0.0 0.1 0.0 0.1 0.1 0.1 0.1 (Single Inj.) nmol/kg HIgGl-GDF(CRL), Mean 3.8 2.2 2.2 2.3 2.4 2.4 2.3 2.4 2.9 3.3 10 S.E. 0.5 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.3 0.3 (Single Inj.) nmol/kg

[0156] As shown in the result, it can be confirmed that the administration group of the fusion polypeptide in which GDF (CRL) was fused with IgG1 Fc comprising Hinge or Fc of IgG1 not comprising Hinge showed the feed intake reducing effect during the test period (9 days), compared to the negative control (vehicle administration group) administration group, and the feed intake reducing effect was concentration-dependent. This feed intake reducing effect of the fusion polypeptide can be said to be comparable when GDF15 is administered once a day throughout the test period.

[0157] 2.3.2. IgG4-GDF and IgG4-GDF(CRL)

[0158] The feed intake change measured in Example 2.1.2 was shown in Table 12 and FIG. 10 (cumulative intake by 7 days, at 14 days and 19 days).

TABLE-US-00015 TABLE 12 Group Day 0 1 2 3 4 5 6 7 8 9 DIO Vehicle Control Mean 3.3 2.7 3.0 3.0 3.1 3.5 3.0 3.5 3.3 3.4 (Single Inj.) S.E. 0.1 0.1 0.2 0.1 0.1 0.2 0.2 0.1 0.1 0.2 Semaglutide, 3 nmol/kg Mean 3.7 2.1 3.3 2.8 2.3 2.9 2.7 3.2 2.4 3.7 (Daily Inj.) S.E. 0.3 0.7 1.4 0.6 0.4 0.9 0.6 0.3 0.4 0.1 IgG4-GDF15, 1 nmol/kg Mean 3.0 1.9 1.9 2.1 2.0 2.0 2.1 2.4 2.4 2.5 (Single Inj.) S.E. 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.2 0.2 0.2 IgG4-GDF15, 10 Mean 3.0 1.7 2.0 2.0 2.1 2.2 2.4 2.4 2.2 2.7 (Single Inj.) nmol/kg S.E. 0.1 0.1 0.3 0.2 0.2 0.3 0.2 0.2 0.2 0.2 IgG4-GDF(CRL), 1 Mean 3.3 2.0 2.6 2.7 2.4 2.7 2.3 2.5 2.5 2.8 (Single Inj.) nmol/kg S.E. 0.2 0.1 0.2 0.2 0.1 0.1 0.2 0.1 0.2 0.1 IgG4-GDF(CRL), 10 Mean 3.1 1.5 2.3 2.4 2.4 2.3 2.3 2.0 2.3 2.4 (Single Inj.) nmol/kg S.E. 0.2 0.3 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.3 Group Day 10 11 12 13 14 15 16 17 18 19 DIO Vehicle Control Mean 3.0 3.4 3.2 3.5 3.1 3.3 3.1 3.2 3.3 3.3 (Single Inj.) S.E. 0.2 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.1 0.1 Semaglutide, 3 nmol/kg Mean 2.7 2.5 2.4 3.0 2.5 2.1 2.5 2.0 2.2 3.7 (Daily Inj.) S.E. 0.4 0.4 0.3 0.4 0.3 0.3 0.2 0.2 0.2 0.3 IgG4-GDF15, 1 nmol/kg Mean 2.7 3.1 3.2 3.7 3.0 3.1 3.1 3.1 3.1 3.0 (Single Inj.) S.E. 0.1 0.0 0.0 0.1 0.1 0.3 0.2 0.3 0.1 0.1 IgG4-GDF15, 10 Mean 3.1 3.8 3.3 3.7 3.4 3.6 3.1 3.5 3.3 3.0 (Single Inj.) nmol/kg S.E. 0.3 0.7 0.5 0.5 0.4 0.6 0.2 0.1 0.1 0.1 IgG4-GDF(CRL), 1 Mean 2.4 2.8 2.6 3.0 2.8 2.9 3.1 3.3 3.0 3.3 (Single Inj.) nmol/kg S.E. 0.1 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 IgG4-GDF(CRL), 10 Mean 2.1 2.5 2.3 2.5 2.6 2.6 3.0 3.4 3.3 3.1 (Single Inj.) nmol/kg S.E. 0.1 0.2 0.3 0.2 0.4 0.2 0.3 0.3 0.3 0.2

[0159] As shown in the result, it can be confirmed that the administration group of the fusion polypeptide in which GDF15 or GDF (CRL) was fused with IgG4 (mutated) not comprising Hinge showed the feed intake reducing effect during the test period (19 days), compared to the negative control (vehicle administration group) administration group. This feed intake reducing effect of the fusion polypeptide can be said to be comparable when Semaglutide is administered once a day throughout the test period. When comparing administration groups of 10 nmol/kg of the fusion polypeptide in which GDF15 or GDF (CRL) was fused with IgG4 Fc (Mutated) not comprising Hinge, in case of the administration group of the full-length GDF15-fused fusion polypeptide, the diet suppression ability was maintained for about 10 days, and in case of the administration group of the GDF(CRL)-fused fusion polypeptide, the diet suppression ability was maintained for about 2 weeks. In other words, it was confirmed that the GDF (CRL) fusion polypeptide had a little excellent weight loss effect at 10 nmol/kg dose, compared to the full-length GDF15 fusion polypeptide.

EXAMPLE 3

Pharmacodynamic Test of Fusion Polypeptide (IgG4-GDF or IgG4-GDF(CRL))

[0160] 3.1. Preparation of Test Group and Control Group Serum

[0161] For evaluation of pharmacodynamic characteristics when each polypeptide was subcutaneously administered to rats, polypeptide IgG4-GDF or IgG4-GDF(CRL) was subcutaneously administered in an amount of 2 mg/kg, respectively, to SD Rat (Koatech, male, 7 weeks, about 250g; n=3 each; test group), and at a fixed time, blood was collected about 200M2 through a caudal vein. Blood collecting was progressed before administration of the fusion polypeptide, in 1, 2, 4, 8, 24, 48, 72, 96, 168, 240 and 336 hours after administration. As a control group for comparison of pharmacodynamic characteristics, GDF15 (R&D Systems) was subcutaneously administered in an amount of 2 mg/kg by the same method as above to prepare GDF15 administration group.

[0162] After administering to SD Rat as above, blood collected by time-point was centrifuged to obtain serum, and ELISA was performed using Human GDF15 Immunoassay (SGD150, R&D Systems), to measure the concentration in serum over time for each polypeptide. Using these data, the values of parameters including AUC (area under the curve) was obtained using a software for PK analysis (WinNonlin (Certara L.P.) et al.).

[0163] 3.2 Pharmacodynamic Test Result

[0164] The obtained pharmacodynamic parameters of the fusion polypeptide were shown in Table 13, and the concentration change of the fusion polypeptide over time was shown in FIG. 11:

TABLE-US-00016 TABLE 13 Group 3 Group 1 Group 2 IgG4-GDF PK Parameter GDF15 IgG4-GDF (CRL) C.sub.max (ug/mL) 0.443 10.6 9.73 T.sub.max (hr) 1 96 48 AUC.sub.last (ug .Math. hr/mL) 4.86 2144 2004 AUC.sub.inf (ug .Math. hr/mL) 4.88 2854 2593 t.sub.1/2 (hr) 19.0 101 114 AUC.sub.extp (%) 0.463 14.6 18.2 (C.sub.max: maximum blood concentration, T.sub.max: Time to reach maximum blood concentration, AUC.sub.inf: area under the blood concentration-time curve calculated by extrapolating from the last measurable blood collection time to infinite time, AUC.sub.last: area under the blood concentration-time curve by the last measurable blood collection time, T.sub.1/2 elimination half-life, AUC.sub.Extp (%): [(AUC.sub.inf − AUC.sub.last)/AUC.sub.inf]*100)

[0165] As shown in the result, it can be confirmed that compared with GDF15 (half-life: 19 hours), in case of IgG4-GDF15 (half-life: 101 hours)and IgG4-GDF (CRL) (half-life: 114 hours) fusion proteins, the stability in blood (serum) was significantly increased (5 times or more).

[0166] From the above description, those skilled in the art to which the present invention belongs will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as illustrative and not restrictive in all aspects. The scope of the present invention should be construed to include all the altered or modified forms derived from the meaning and scope of the claims which will be described later, and their equivalent concepts, rather than the above detailed description.