Patent Publication Number: US-2023133672-A1

Title: Fusion polypeptide comprising gdf15 and polypeptide region capable of o-glycosylation

Description:
This application is a 35 U.S.C. 371 National Phase Entry Application from PCT/KR2020/018053 filed on Dec. 10, 2020, which claims priority to and the benefits of Korean Patent Application No. 10-2019-0165052, filed with the Korean Intellectual Property Office on Dec. 11, 2019, the entire contents of which are incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on Dec. 12, 2022, is named 3570-820_ST25.txt and is 538,682 bytes in size. 
     The present invention relates to a fusion polypeptide comprising GDF15 (Growth differentiation factor 15) and a polypeptide region capable of O-glycosylation, a pharmaceutical composition comprising the fusion polypeptide, and a method for increasing in vivo duration of GDF15 comprising the step of fusing a polypeptide region capable of O-glycosylation. 
     BACKGROUND OF THE INVENTION 
     Most protein or peptide drugs have a short duration of activity in the body, and their absorption rate is low when administered by methods other than intravenous administration, and therefore, there is an inconvenience of having to continuously inject these drugs repeatedly at short administration intervals when treatment of long-term drug administration is required. In order to solve such inconvenience, it is required to develop a technology for continuously releasing a drug with single administration. As a part to meet these needs, a sustained-release formulation for sustained release is being developed. 
     For examples, research on a sustained-release formulation in which in which the drug is slowly released while the matrix substance is slowly decomposed in vivo when it is administered, by preparing a microparticle in the form of a protein or peptide drug surrounded by a biodegradable polymer matrix is actively progressed. 
     For example, U.S. Pat. No. 5,416,017 discloses a sustained-release injection of erythropoietin using a gel having a hyaluronic acid concentration of 0.01 to 3%, and Japanese Patent Publication No. 1-287041 discloses a sustained-release injection in which insulin is contained in a gel having a hyaluronic acid concentration of 1%, and Japanese Patent Publication No. 2-213 discloses a sustained-release formulation in which calcitonin, elcatonin or human GDF15 is contained in hyaluronic acid having a concentration of 5%. In such a formulation, the protein drug dissolved in the gel of hyaluronic acid passes through the gel matrix with high viscosity at a slow speed, so it can exhibit a sustained release effect, but there are disadvantages in that it is not easy to administer by injection due to high viscosity, and it is difficult to release the drug for more than 1 day as the gel is easily diluted or decomposed by body fluids after injection. 
     On the other hand, there are examples of preparing solid microparticles by emulsion solvent extraction using a hyaluronic acid derivative having hydrophobicity (for example, hyaluronic acid-benzyl ester) (N. S. Nightlinger, et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 22nd, Paper No. 3205 (1995); L. Ilum, et al., J. Controlled Rel., 29, 133(1994)). Since it is necessary to use an organic solvent in preparation of the drug release formulation particles using a hydrophobic hyaluronic acid derivative, there is a risk of denaturation of the protein drug by contact with the organic solvent, and the possibility of denaturation of the protein due to the hydrophobicity of the hyaluronic acid derivative is high. 
     Therefore, in order to improve in vivo persistence of a protein or peptide drug, an approach different from the conventional studies is required. 
     On the other hand, GDF15 (Growth differentiation factor 15) is a member of the TGF-beta family, and is a 25 kDa homodimer, and is a secretory protein circulating in plasma. The plasma level of GDF15 is related to BMI (body mass index) and GDF15 plays a role as a long-term regulator of energy homeostasis. GDF15 also has protective actions in pathological conditions such as cardiovascular disease, myocardial hypertrophy and ischemic injury. In addition, GDF15 plays a protective role against renal tubular and renal interstitial damage in models of type 1 diabetes and type 2 diabetes. Furthermore, GDF15 has a protective effect against age-related sensory and motor nerve loss, and can contribute to peripheral nerve damage recovery. Moreover, GDF15 has effects of weight loss and body fat reduction and glucose tolerance, and has an effect of increasing systemic energy consumption and oxidative metabolism. GDF15 exhibits an effect of glycemic control through body weight-dependent and non-dependent mechanisms. 
     The development of a technology for improving in vivo persistence of GDF15 protein exhibiting such various pharmacological effects is required. 
     BRIEF SUMMARY OF THE INVENTION 
     In the present description, provided is a technology of increasing an in vivo half-life of GDF15 to enhance the in vivo duration and thereby, increasing the administration interval, by linking a polypeptide capable of O-glycosylation (for example, immunoglobulin hinge region, etc.) to GDF15 (Growth differentiation factor 15) to form a fusion polypeptide, compared to the case where it is not fused with a polypeptide region capable of O-glycosylation. 
     One embodiment provides a fusion polypeptide comprising GDF15 and a polypeptide region capable of O-glycosylation. 
     In the fusion polypeptide, the polypeptide region capable of O-glycosylation may be comprised in the N-terminus of the GDF15. 
     The total number of the polypeptide region capable of O-glycosylation comprised in the fusion polypeptide may be 1 or more, for example, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 to 10, 2 to 8, 2 to 6, 2 to 4 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). 
     In one embodiment, the fusion polypeptide may be represented by the following general formula: 
       N′—(Z) n -Y—C′  [general formula]
 
     in the formula, 
     N′ is the N-terminus of the fusion polypeptide, and C′ is the C-terminus of the fusion polypeptide, and 
     Y is GDF15, and 
     Z is a polypeptide region capable of O-glycosylation, and 
     n is the number of the polypeptide region capable of O-glycosylation positioned at the N-terminus of the fusion polypeptide (bound to the N-terminus of GDF15) and an integer of 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), 1 to 7, 1 to 5, or 1 to 3. 
     The n polypeptide regions capable of O-glycosylation comprised in the fusion polypeptide may be each independently selected among polypeptide regions comprising amino acid residues capable of O-glycosylation. For example, the polypeptide regions comprising amino acid residues capable of O-glycosylation may be immunoglobulin hinge regions. In one embodiment, the polypeptide regions capable of O-glycosylation may be selected from the group consisting of immunoglobulin D (IgD) hinge regions and immunoglobulin A (IgA, for example, IgA1) hinge regions (i.e., n immunoglobulin hinge regions may be same or different each other). 
     In the fusion polypeptide, the GDF15 fused with the polypeptide region capable of O-glycosylation, is characterized by having increased in vivo (or in blood) stability (duration), compared to the GDF15 not fused with the polypeptide region capable of O-glycosylation (for example, in vivo or blood half-life increase). 
     Another embodiment provides a nucleic acid molecule encoding the fusion polypeptide. 
     Another embodiment provides a recombinant vector comprising the nucleic acid molecule. 
     Another embodiment provides a recombinant cell comprising the recombinant vector. 
     Another embodiment provides a method for preparation of GDF15 with an increased in vivo (or in blood) half-life, or a method for preparation of a fusion polypeptide comprising the GDF15 with an increased in vivo (or in blood) half-life, comprising expressing the recombinant vector in a cell. 
     Another embodiment provides a method for increasing in vivo duration of GDF15, or a method for enhancing in vivo (or in blood) stability of a GDF15 (protein or peptide) drug and/or increasing an in vivo (or in blood) half-life, comprising fusing (or linking or binding) GDF15 and a polypeptide region capable of O-glycosylation. In one specific example, the fusing may comprise fusing (or linking or binding) one or more polypeptide regions capable of O-glycosylation at the N-terminus of GDF15 through or not through a linker. The fusing (or linking or binding) may be performed in vitro. 
     Another embodiment provides a fusion polypeptide dimer, comprising two of the fusion polypeptides. The fusion polypeptide dimer may be formed by being linked by a bond (for example, disulfide bond) between GDF15 comprised in each fusion polypeptide. The fusion polypeptide dimer may be a homodimer. 
     Another embodiment provides a pharmaceutical composition comprising one or more selected from the group consisting of the fusion polypeptide, a fusion polypeptide dimer comprising the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule and a recombinant cell comprising the recombinant vector. 
     Another embodiment provides a method for preparing a pharmaceutical composition using one or more selected from the group consisting of the fusion polypeptide, a fusion polypeptide dimer comprising the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule and a recombinant cell comprising the recombinant vector. 
     Another embodiment provides a use of one or more selected from the group consisting of the fusion polypeptide, a fusion polypeptide dimer comprising the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule and a recombinant cell comprising the recombinant vector, for preparing a pharmaceutical composition. 
     Another embodiment provides a use of a polypeptide region capable of O-glycosylation for enhancing in vivo (or in blood) stability and/or increasing an in vivo (or in blood) half-life of a GDF15 (protein or peptide) drug. Specifically, an embodiment provides a composition for enhancing in vivo (or in blood) stability and/or increasing an in vivo (or in blood) half-life of a GDF15 (protein or peptide) drug, the composition comprising a polypeptide region capable of O-glycosylation. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present description provides a technology capable of enhancing in vivo (or in blood) stability and/or in vivo (or in blood) duration in case of in vivo application of GDF15, by providing a fusion polypeptide form in which a polypeptide region capable of O-glycosylation such as an immunoglobulin hinge region is fused to GDF15. 
     One embodiment provides a fusion polypeptide comprising GDF15 and a polypeptide region capable of O-glycosylation. 
     In the fusion polypeptide, the polypeptide region capable of O-glycosylation may be comprised at the N-terminus of the GDF15. 
     The total number of the polypeptide region capable of O-glycosylation comprised in the fusion polypeptide may be 1 or more, for example, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 to 10, 2 to 8, 2 to 6, 2 to 4 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). 
     In one embodiment, the fusion polypeptide may be represented by the following general formula: 
       N′—(Z) n -Y—C′  [general formula]
 
     in the formula, 
     N′ is the N-terminus of the fusion polypeptide, and C′ is the C-terminus of the fusion polypeptide, and 
     Y is GDF15, and 
     Z is a polypeptide region capable of O-glycosylation, and 
     n is the number of the polypeptide region capable of O-glycosylation positioned at the N-terminus of the fusion polypeptide (bound to the N-terminus of GDF15) and an integer of 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), 1 to 7, 1 to 5, or 1 to 3. 
     In one embodiment, in the fusion polypeptide, when the active site of GDF15 is positioned at the C-terminus, the polypeptide region capable of O-glycosylation may be fused to the N-terminus. 
     The n polypeptide regions capable of O-glycosylation comprised in the fusion polypeptide may be each independently selected among polypeptide regions comprising amino acid residues capable of O-glycosylation. For example, the polypeptide regions comprising amino acid residues capable of O-glycosylation may be immunoglobulin hinge regions. In one embodiment, the polypeptide regions capable of O-glycosylation may be selected from the group consisting of immunoglobulin D (IgD) hinge regions and immunoglobulin A (IgA, for example, IgA1) hinge regions (i.e., n immunoglobulin hinge regions may be same or different each other). 
     In one specific embodiment, the polypeptide region capable of O-glycosylation positioned (comprised) at the N-terminus of the fusion polypeptide may be 1 or 2, and in case of 2 or more, each of the polypeptide regions capable of O-glycosylation may be same or different each other. In one specific embodiment, one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptide regions capable of O-glycosylation positioned at the N-terminus may be all IgD hinge regions or IgA (for example, IgA1) hinge regions, or comprise one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) IgD hinge regions and one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) IgA (for example, IgA1) hinge regions in various orders. 
     In other specific embodiment, when all the n polypeptide regions capable of O-glycosylation comprised in the fusion polypeptide are positioned only at the N-terminus of the fusion polypeptide (in other words, when one or more polypeptide regions capable of O-glycosylation are present only at the N-terminus of the fusion polypeptide), the one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptide regions capable of O-glycosylation may be all IgD hinge regions or IgA hinge regions, or comprise one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) IgD hinge regions and one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) IgA hinge regions in various orders. 
     The polypeptide region capable of O-glycosylation (each region when the polypeptide region capable of O-glycosylation is 2 or more) may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more O-glycosylation residues (the upper limit is 100, 50, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8) (for example, 1, 2, 3, 4, 5, 6, 7 or 8). For example, the polypeptide region capable of O-glycosylation (each region when the polypeptide region capable of O-glycosylation is 2 or more) may comprise 1 to 10 or 3 to 10 O-glycosylation residues (amino acid residues capable of O-glycosylation). 
     In one embodiment, the polypeptide region capable of O-glycosylation may be one or more selected from immunoglobulin (for example, human immunoglobulin) hinge regions, and for example, may be IgD hinge regions, IgA hinge regions or a combination thereof. 
     Since hinge regions such as IgD hinge regions (for example, human IgD hinge regions) and/or IgA hinge regions (for example, human hinge regions) among the regions of immunoglobulin (for example, human immunoglobulin) comprise a residue capable of O-glycosylation, the polypeptide region capable of O-glycosylation may necessarily comprise one or more (human) IgD hinge regions and/or one or more (human) IgA hinge regions, or necessarily consist of the hinge regions. In one specific embodiment, the polypeptide region capable of O-glycosylation may not comprise one or more (e.g., 1, 2, or all 3) selected from the group consisting of CH1, CH2, and CH3 of immunoglobulin regions not comprising a residue capable of O-glycosylation (for example, IgD and/or IgA). 
     In addition, considering the number of appropriate residues capable of O-glycosylation in the fusion polypeptide provided in the present description, the polypeptide capable of O-glycosylation may comprise one or more, more specifically, 2 or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10) IgD hinge regions (for example, human IgD hinge regions) and/or IgA hinge regions (for example, human IgA hinge regions). 
     More specifically, the IgD may be human IgD (for example, UniProKB P01880 (invariant domain; SEQ ID NO: 7), etc.), and the hinge region of IgD may be one or more selected from the group consisting of 
     a polypeptide comprising an amino acid sequence of “N′-ESPKAQA SS VP T AQPQAEGSLAKA TT APA TT RNT-C′ (SEQ ID NO: 1); amino acid residues in bold are residues capable of O-glycosylation (7 in total)” or essentially consisting of the amino acid sequence (“IgD hinge”), 
     a polypeptide comprising 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, 22 or more, or 24 or more (the upper limit is 34 or 33) consecutive amino acids comprising one or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more O-glycosylation residues in the amino acid sequence of SEQ ID NO: 1, or essentially consisting of the amino acids (“a part of IgD hinge”; for example, a polypeptide comprising 5 or more continuous amino acids comprising “SSVPT” (SEQ ID NO: 9) in SEQ ID NO: 1 or a polypeptide comprising 7 or more continuous amino acids comprising “TTAPATT” (SEQ ID NO: 10)), and 
     a polypeptide comprising 34 or more or 35 or more continuous amino acids comprising the amino acid sequence (IgD hinge) of SEQ ID NO: 1, in the IgD (for example, SEQ ID NO: 7) or 7 or more, 10 or more, 15 or more, 20 or more, 22 or more or 24 or more continuous amino acids comprising a part of the IgD hinge, or essentially consisting of the amino acids (“extension of IgD hinge”; for example, a polypeptide comprising 34 or more or 35 or more continuous amino acids comprising SEQ ID NO: 1 in “ESPKAQASS VPTAQPQAEG SLAKATTAPA TTRNTGRGGE EKKKEKEKEE QEERETKTP” (SEQ ID NO: 11) in the IgD (SEQ ID NO: 7) or a part of the IgD hinge). 
     The IgA may be human IgA (for example, IgA1 (UniProKB P01876, invariant domain; SEQ ID NO: 8), etc.), and the hinge region of the IgA may be one or more selected from the group consisting of a polypeptide comprising an amino acid sequence of “N′-VP ST PP T P S P S TPP T P S P S -C′ (SEQ ID NO: 2); amino acid residues in bold are residues capable of O-glycosylation (8 in total)” or essentially consisting of the amino acid sequence (“IgA hinge”), 
     a polypeptide comprising 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 17 or more or 18 continuous amino acids comprising 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more or 8 O-glycosylation residues in the amino acid sequence of SEQ ID NO: 2, or essentially consisting of the amino acid sequence (“a part of IgA hinge”; for example, a polypeptide comprising 8 or more or 9 or more amino acids comprising “STPPTPSP” (SEQ ID NO: 12) in SEQ ID NO: 2), and 
     19 or more or 20 or more continuous amino acids comprising the amino acid sequence (IgA (for example, IgA1) hinge), in IgA (for example, IgA1 (SEQ ID NO: 8)), or a polypeptide comprising 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, or 18 continuous amino acids comprising a part of the IgA (for example, IgA1) hinge, or essentially consisting of the amino acid sequence (“extension of IgA hinge”). 
     In other embodiment, the polypeptide region capable of O-glycosylation may be a polypeptide region comprising 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, 20 or more, 22 or more, 25 or more, 27 or more, 30 or more, 32 or more or 35 or more (the upper limit is 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or the total amino acid number of each protein) continuous amino acids comprising 1 or more, 2 or more, 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, 20 or more, or 22 or more (for example, 1 to 10, 3 to 10; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) amino acid residues capable of O-glycosylation in the proteins indicated in the following Table 1 (for example, proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 113) or essentially consisting of the amino acids. In the present description, it is preferred that the polypeptide region capable of O-glycosylation does not affect the function of GDF15. The polypeptide region capable of O-glycosylation of the proteins indicated in Table below may be selected from among regions not involved in the original function of a full-length protein, and thereby, the polypeptide region capable of O-glycosylation may only serve to increase the half-life without affecting the function of GDF15: 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 UniProtK 
                 UniProtK 
                   
                   
                   
                   
                 SEQ 
               
               
                 B Entry 
                 B Entry 
                 Protein 
                 Gene 
                   
                   
                 ID 
               
               
                 No. 
                 name 
                 names 
                 names 
                 Length 
                 O-Glycosylation (site) 
                 NO: 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Q96DR8 
                 MUCL1_ 
                 Mucin-like 
                 MUCL1 
                 90 
                 23T, 24T, 30T, 34T, 46T, 
                 23 
               
               
                   
                 HUMAN 
                 protein 1 
                 SBEM 
                   
                 47T, 51T, 52T, 54T, 55T, 
                   
               
               
                   
                   
                   
                 UNQ590/ 
                   
                 59T, 60T, 62T, 63T, 66S, 
                   
               
               
                   
                   
                   
                 PR01160 
                   
                 67T, 68T 
                   
               
               
                   
               
               
                 Q0VAQ4 
                 SMAGP_ 
                 Small cell 
                 SMAGP 
                 97 
                 2T, 3S, 6T, 7T, 9S, 16T, 17T, 
                 24 
               
               
                   
                 HUMAN 
                 adhesion 
                   
                   
                 23T 
                   
               
               
                   
                   
                 glycoprotein 
                   
                   
                   
                   
               
               
                   
               
               
                 P04921 
                 GLPC_ 
                 Glycophorin-C 
                 GYPC 
                 128 
                 3S, 4T, 6S, 9S, 10T, 15S, 
                 25 
               
               
                   
                 HUMAN 
                   
                 GLPC 
                   
                 24S, 26S, 27T, 28T, 31T, 
                   
               
               
                   
                   
                   
                 GPC 
                   
                 32T, 33T, 42S 
                   
               
               
                   
               
               
                 P16860 
                 ANFB_ 
                 Natriuretic 
                 NPPB 
                 134 
                 62T, 63S, 70S, 74T, 79S, 
                 26 
               
               
                   
                 HUMAN 
                 peptides B 
                   
                   
                 84T, 97T 
                   
               
               
                   
               
               
                 P04141 
                 CSF2_ 
                 Granulocyte- 
                 CSF2 
                 144 
                 22S, 24S, 26S,27T 
                 27 
               
               
                   
                 HUMAN 
                 macrophage 
                 GMCS 
                   
                   
                   
               
               
                   
                   
                 colony- 
                 F 
                   
                   
                   
               
               
                   
                   
                 stimulating 
                   
                   
                   
                   
               
               
                   
                   
                 factor 
                   
                   
                   
                   
               
               
                   
               
               
                 P02724 
                 GLPA_ 
                 Glycophorin-A 
                 GYPA 
                 150 
                 21S, 22T, 23T, 29T, 30S, 
                 28 
               
               
                   
                 HUMAN 
                   
                 GPA 
                   
                 31T, 32S, 36T, 38S, 41S, 
                   
               
               
                   
                   
                   
                   
                   
                 44T, 52T, 56T, 63S, 66S, 
                   
               
               
                   
                   
                   
                   
                   
                 69T 
                   
               
               
                   
               
               
                 P10124 
                 SRGN_ 
                 Serglycin 
                 SRGN 
                 158 
                 94S, 96S, 100S, 102S, 104S, 
                 29 
               
               
                   
                 HUMAN 
                   
                 PRG 
                   
                 106S,108S, 110S 
                   
               
               
                   
                   
                   
                 PRG1 
                   
                   
                   
               
               
                   
               
               
                 Q86YL7 
                 PDPN_ 
                 Podoplanin 
                 PDPN 
                 162 
                 25T, 32T, 34T, 35T, 52T, 
                 30 
               
               
                   
                 HUMAN 
                   
                 GP36 
                   
                 55T, 65T, 66T, 76T, 85T, 
                   
               
               
                   
                   
                   
                 PSEC 
                   
                 86S, 88S, 89T, 96S, 98S, 
                   
               
               
                   
                   
                   
                 0003 
                   
                 100T, 102S, 106T, 107S, 
                   
               
               
                   
                   
                   
                 PSEC 
                   
                 109S, 110T, 117T, 119T, 
                   
               
               
                   
                   
                   
                 0025 
                   
                 120T 
                   
               
               
                   
               
               
                 P0DN87 
                 CGB7_ 
                 Choriogonadot 
                 CGB7 
                 165 
                 139S, 141S, 147S, 150S, 
                 31 
               
               
                   
                 HUMAN 
                 ropin subunit 
                   
                   
                 152S, 158S 
                   
               
               
                   
                   
                 beta 7 
                   
                   
                   
                   
               
               
                   
               
               
                 P0DN86 
                 CGB3_ 
                 Choriogonadot 
                 CGB3 
                 165 
                 139S, 141S, 147S, 150S, 
                 32 
               
               
                   
                 HUMAN 
                 ropin subunit 
                 CGB; 
                   
                 152S, 158S 
                   
               
               
                   
                   
                 beta 3 
                 CGB5; 
                   
                   
                   
               
               
                   
                   
                   
                 CGB8 
                   
                   
                   
               
               
                   
               
               
                 P01344 
                 IGF2_ 
                 Insulin-like 
                 IGF2 
                 180 
                 96T, 99T, 163T 
                 33 
               
               
                   
                 HUMAN 
                 growth factor II 
                 PP1446 
                   
                   
                   
               
               
                   
               
               
                 P07498 
                 CASK_ 
                 Kappa-casein 
                 CSN3 
                 182 
                 133T, 143T, 148T, 151T, 
                 34 
               
               
                   
                 HUMAN 
                   
                 CASK 
                   
                 157T, 167T, 169T, 178T 
                   
               
               
                   
                   
                   
                 CSN10 
                   
                   
                   
               
               
                   
                   
                   
                 CSNK 
                   
                   
                   
               
               
                   
               
               
                 P31431 
                 SDC4_ 
                 Syndecan-4 
                 SDC4 
                 198 
                 39S, 61S, 63S 
                 35 
               
               
                   
                 HUMAN 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 P34741 
                 SDC2_ 
                 Syndecan-2 
                 SDC2 
                 201 
                 41S, 55S, 57S, 101T 
                 36 
               
               
                   
                 HUMAN 
                   
                 HSPG1 
                   
                   
                   
               
               
                   
               
               
                 Q99075 
                 HBEGF_ 
                 Proheparin- 
                 HBEGF 
                 208 
                 37T, 38S, 44T, 47T, 75T, 85T 
                 37 
               
               
                   
                 HUMAN 
                 binding EGF- 
                 DTR 
                   
                   
                   
               
               
                   
                   
                 like growth 
                 DTS 
                   
                   
                   
               
               
                   
                   
                 factor 
                 HEGFL 
                   
                   
                   
               
               
                   
               
               
                 P13727 
                 PRG2_ 
                 Bone marrow 
                 PRG2 
                 222 
                 23T, 24S, 25T, 34T, 62S 
                 38 
               
               
                   
                 HUMAN 
                 proteoglycan 
                 MBP 
                   
                   
                   
               
               
                   
                   
                 (BMPG) 
                   
                   
                   
                   
               
               
                   
               
               
                 P24592 
                 IBP6_ 
                 Insulin-like 
                 IGFBP6 
                 240 
                 126T, 144S, 145T, 146T, 
                 39 
               
               
                   
                 MHUAN 
                 growth factor- 
                 IBP6 
                   
                 152S 
                   
               
               
                   
                   
                 binding protein 
                   
                   
                   
                   
               
               
                   
                   
                 6 (IBP-6) 
                   
                   
                   
                   
               
               
                   
               
               
                 Q9UHG2 
                 PCSK1_ 
                 ProSAAS 
                 PCSK1N 
                 260 
                 53T, 228S, 247T 
                 40 
               
               
                   
                 UHMAN 
                 (Proprotein 
                   
                   
                   
                   
               
               
                   
                   
                 convertase 
                   
                   
                   
                   
               
               
                   
                   
                 subtilisin/kexin 
                   
                   
                   
                   
               
               
                   
                   
                 type 1 
                   
                   
                   
                   
               
               
                   
                   
                 inhibitor) 
                   
                   
                   
                   
               
               
                   
               
               
                 P01589 
                 IL2RA_ 
                 lnterleukin-2 
                 IL2RA 
                 272 
                 218T, 224T, 229T, 237T 
                 41 
               
               
                   
                 UHMAN 
                 receptor 
                   
                   
                   
                   
               
               
                   
                   
                 subunit alpha 
                   
                   
                   
                   
               
               
                   
                   
                 (IL-2 receptor 
                   
                   
                   
                   
               
               
                   
                   
                 subunit alpha) 
                   
                   
                   
                   
               
               
                   
               
               
                 P21583 
                 SCF_ 
                 Kit ligand 
                 KITLG 
                 273 
                 167S, 168T, 180T 
                 42 
               
               
                   
                 HUMAN 
                 (Mast cell 
                 MGF 
                   
                   
                   
               
               
                   
                   
                 growth factor) 
                 SCF 
                   
                   
                   
               
               
                   
                   
                 (MGF) 
                   
                   
                   
                   
               
               
                   
               
               
                 A1E959 
                 ODAM_ 
                 Odontogenic 
                 ODAM 
                 279 
                 115T, 119T, 244T, 249S, 
                 43 
               
               
                   
                 HUMAN 
                 ameloblast- 
                 APIN 
                   
                 250T, 251T, 255T, 256S, 
                   
               
               
                   
                   
                 associated 
                   
                   
                 261T, 263T, 273T, 275S 
                   
               
               
                   
                   
                 protein (Apin) 
                   
                   
                   
                   
               
               
                   
               
               
                 P10451 
                 OSTP_ 
                 Osteopontin 
                 SPP1 
                 314 
                 134T, 138T, 143T, 147T, 
                 44 
               
               
                   
                 HUMAN 
                   
                 BNSP 
                   
                 152T 
                   
               
               
                   
                   
                   
                 OPN 
                   
                   
                   
               
               
                   
                   
                   
                 PSEC 
                   
                   
                   
               
               
                   
                   
                   
                 0156 
                   
                   
                   
               
               
                   
               
               
                 P21815 
                 SIAL_ 
                 Bone 
                 IBSP 
                 317 
                 119T, 122T, 227T, 228T, 
                 45 
               
               
                   
                 HUMAN 
                 sialoprotein 2 
                 BNSP 
                   
                 229T, 238T, 239T 
                   
               
               
                   
                   
                 (Bone 
                   
                   
                   
                   
               
               
                   
                   
                 sialoprotein II) 
                   
                   
                   
                   
               
               
                   
                   
                 (BSP II) 
                   
                   
                   
                   
               
               
                   
               
               
                 P02649 
                 APOE_ 
                 Apolipoprotein 
                 APOE 
                 317 
                 26T, 36T, 212T, 307T, 308S, 
                 46 
               
               
                   
                 HUMAN 
                 E(Apo-E) 
                   
                   
                 314S 
                   
               
               
                   
               
               
                 Q99645 
                 EPYC_ 
                 Epiphycan 
                 EPYC 
                 322 
                 60T, 64S, 96S 
                 47 
               
               
                   
                 HUMAN 
                 (Dermatan 
                 DSPG 
                   
                   
                   
               
               
                   
                   
                 sulfate 
                 3 
                   
                   
                   
               
               
                   
                   
                 proteoglycan 
                 PGLB 
                   
                   
                   
               
               
                   
                   
                 3) 
                 SLRR3B 
                   
                   
                   
               
               
                   
               
               
                 Q6UXG3 
                 CLM9_ 
                 CMRF35-like 
                 CD300 
                 332 
                 137T, 143T, 144T, 155T, 
                 48 
               
               
                   
                 HUMAN 
                 molecule 9 
                 LG 
                   
                 161T, 170T, 171T, 177T, 
                   
               
               
                   
                   
                 (CLM-9) 
                 CLM9 
                   
                 187T, 195T, 196S, 199T, 
                   
               
               
                   
                   
                   
                 TREM4 
                   
                 201T, 202S, 207T, 208S, 
                   
               
               
                   
                   
                   
                 UNQ422/ 
                   
                 213S, 214S, 222S, 223T, 
                   
               
               
                   
                   
                   
                 PR0846 
                   
                 224S, 228T, 229S, 237S 
                   
               
               
                   
               
               
                 Q9GZM5 
                 YIPF3_ 
                 Protein YIPF3 
                 YIPF3 
                 350 
                 333T, 334T, 339T, 346T 
                 49 
               
               
                   
                 HUMAN 
                 (Killer lineage 
                 C6orf109 
                   
                   
                   
               
               
                   
                   
                 protein 1) 
                 KLIP1 
                   
                   
                   
               
               
                   
               
               
                 P51681 
                 CCR5_ 
                 C-C 
                 CCR5 
                 352 
                 6S, 7S, 16T, 17S 
                 50 
               
               
                   
                 HUMAN 
                 chemokine 
                 CMKB 
                   
                   
                   
               
               
                   
                   
                 receptor type 5 
                 R5 
                   
                   
                   
               
               
                   
                   
                 (C-C CKR-5) 
                   
                   
                   
                   
               
               
                   
               
               
                 P40225 
                 TPO_ 
                 Thrombopoietin 
                 THPO 
                 353 
                 22S, 58T, 131T, 179T, 180T, 
                 51 
               
               
                   
                 HUMAN 
                 (C-mpl 
                 MGDF 
                   
                 184S, 213T, 265S 
                   
               
               
                   
                   
                 ligand) (ML) 
                   
                   
                   
                   
               
               
                   
               
               
                 P01876 
                 IGHA1_ 
                 Immunoglobulin 
                 IGHA1 
                 353 
                 105S, 106T, 109T, 111S, 
                 8 
               
               
                   
                 HUMAN 
                 heavy 
                   
                   
                 113S, 117T, 119S, 121S 
                   
               
               
                   
                   
                 constant alpha 
                   
                   
                   
                   
               
               
                   
                   
                 1 (Ig alpha-1 
                   
                   
                   
                   
               
               
                   
                   
                 chain C region) 
                   
                   
                   
                   
               
               
                   
               
               
                 P02765 
                 FETUA_ 
                 Alpha-2-HS- 
                 AHSG 
                 367 
                 270T, 280S, 293S, 339T, 
                 52 
               
               
                   
                 HUMAN 
                 glycoprotein 
                 FETUA 
                   
                 341T, 346S 
                   
               
               
                   
                   
                 (Alpha-2-Z- 
                 PR02743 
                   
                   
                   
               
               
                   
                   
                 globulin) 
                   
                   
                   
                   
               
               
                   
               
               
                 P21810 
                 PGS1_ 
                 Biglycan 
                 BGN 
                 368 
                 42S, 47S, 180S, 198S 
                 53 
               
               
                   
                 HUMAN 
                   
                 SLRR1A 
                   
                   
                   
               
               
                   
               
               
                 P01860 
                 IGHG3_ 
                 Immunoglobulin 
                 IGHG3 
                 377 
                 122T, 137T, 152T 
                 54 
               
               
                   
                 HUMAN 
                 heavy 
                   
                   
                   
                   
               
               
                   
                   
                 constant 
                   
                   
                   
                   
               
               
                   
                   
                 gamma 3 
                   
                   
                   
                   
               
               
                   
                   
                 (HDC) 
                   
                   
                   
                   
               
               
                   
               
               
                 P80370 
                 DLK1_ 
                 Protein delta 
                 DLK1 
                 383 
                 94S, 143T, 163S, 214S, 
                 55 
               
               
                   
                 HUMAN 
                 homolog 1 
                 DLK 
                   
                 222T 251S 256T, 260S 
                   
               
               
                   
                   
                 (DLK-1) 
                   
                   
                   
                   
               
               
                   
               
               
                 P01880 
                 IGHD_ 
                 Immunoglobulin 
                 IGHD 
                 384 
                 109S, 110S, 113T, 126T, 
                 7 
               
               
                   
                 MHUAN 
                 heavy 
                   
                   
                 127T, 131T, 132T 
                   
               
               
                   
                   
                 constant delta 
                   
                   
                   
                   
               
               
                   
                   
                 (Ig delta chain 
                   
                   
                   
                   
               
               
                   
                   
                 C region) 
                   
                   
                   
                   
               
               
                   
               
               
                 P15529 
                 MCP_ 
                 Membrane 
                 CD46 
                 392 
                 290S, 291S, 292T, 298S, 
                 56 
               
               
                   
                 HUMAN 
                 cofactor 
                 MCP 
                   
                 300S, 302S, 303T, 304S, 
                   
               
               
                   
                   
                 protein (TLX) 
                 MIC10 
                   
                 305S, 306T, 307T, 309S, 
                   
               
               
                   
                   
                   
                   
                   
                 312S, 313S, 315S, 320T, 
                   
               
               
                   
                   
                   
                   
                   
                 326S 
                   
               
               
                   
               
               
                 P04280 
                 PRP1_ 
                 Basic salivary 
                 PRB1 
                 392 
                 40S, 87S, 150S, 330S 
                 57 
               
               
                   
                 HUMAN 
                 proline-rich 
                   
                   
                   
                   
               
               
                   
                   
                 protein 1 
                   
                   
                   
                   
               
               
                   
               
               
                 P78423 
                 X3CL1_ 
                 Fractalkine (C- 
                 CX3CL1 
                 397 
                 183T, 253S, 329T 
                 58 
               
               
                   
                 HUMAN 
                 X3-C motif 
                 FKN 
                   
                   
                   
               
               
                   
                   
                 chemokine 1) 
                 NTT 
                   
                   
                   
               
               
                   
                   
                   
                 SCYD1 
                   
                   
                   
               
               
                   
                   
                   
                 A- 
                   
                   
                   
               
               
                   
                   
                   
                 152E5.2 
                   
                   
                   
               
               
                   
               
               
                 P16150 
                 LEUK_ 
                 Leukosialin 
                 SPN 
                 400 
                 21T, 22T, 26T, 28T, 29S, 
                 59 
               
               
                   
                 HUMAN 
                 (GPL115) 
                 CD43 
                   
                 35S ,36T, 37S, 41S, 42S, 
                   
               
               
                   
                   
                   
                   
                   
                 46T, 47T, 48S, 50T, 58T, 
                   
               
               
                   
                   
                   
                   
                   
                 69T, 99S, 103S, 109T, 113T, 
                   
               
               
                   
                   
                   
                   
                   
                 114S, 136T, 137T, 173T, 
                   
               
               
                   
                   
                   
                   
                   
                 178T 
                   
               
               
                   
               
               
                 P13473 
                 LAMP2_ 
                 Lysosome- 
                 LAMP2 
                 410 
                 195S, 196T, 200T, 203T, 
                 60 
               
               
                   
                 HUMAN 
                 associated 
                   
                   
                 204T, 207S, 209T, 21OT, 
                   
               
               
                   
                   
                 membrane 
                   
                   
                 211T, 213T 
                   
               
               
                   
                   
                 glycoprotein 2 
                   
                   
                   
                   
               
               
                   
                   
                 (LAMP-2) 
                   
                   
                   
                   
               
               
                   
               
               
                 P11279 
                 LAMP1_ 
                 Lysosome- 
                 LAMP1 
                 417 
                 197S, 199T, 200T, 207S, 
                 61 
               
               
                   
                 HUMAN 
                 associated 
                   
                   
                 209S, 211S, 
                   
               
               
                   
                   
                 membrane 
                   
                   
                   
                   
               
               
                   
                   
                 glycoprotein 1 
                   
                   
                   
                   
               
               
                   
                   
                 (LAMP-1) 
                   
                   
                   
                   
               
               
                   
               
               
                 P21754 
                 ZP3_ 
                 Zona pellucida 
                 ZP3 
                 424 
                 156T, 162T, 163T 
                 62 
               
               
                   
                 HUMAN 
                 sperm-binding 
                 ZP3A 
                   
                   
                   
               
               
                   
                   
                 protein 3 
                 ZP3B 
                   
                   
                   
               
               
                   
                   
                 (Sperm 
                 ZPC 
                   
                   
                   
               
               
                   
                   
                 receptor) 
                   
                   
                   
                   
               
               
                   
               
               
                 P05783 
                 K1C18_ 
                 Keratin, type I 
                 KRT18 
                 430 
                 30S, 31S, 49S 
                 63 
               
               
                   
                 HUMAN 
                 cytoskeletal 18 
                 CYK18 
                   
                   
                   
               
               
                   
                   
                   
                 PIG46 
                   
                   
                   
               
               
                   
               
               
                 Q08629 
                 TICN1_ 
                 Testican-1 
                 SPOC 
                 439 
                 228T, 383S, 388S 
                 64 
               
               
                   
                 HUMAN 
                 (Protein 
                 K1 
                   
                   
                   
               
               
                   
                   
                 SPOCK) 
                 SPOC 
                   
                   
                   
               
               
                   
                   
                   
                 KTIC1 
                   
                   
                   
               
               
                   
                   
                   
                 TICN1 
                   
                   
                   
               
               
                   
               
               
                 O75056 
                 SDC3_ 
                 Syndecan-3 
                 SDC3 
                 442 
                 80S, 82S, 84S, 91S, 314S, 
                 65 
               
               
                   
                 HUMAN 
                 (SYND3) 
                 KIAA0468 
                   
                 367S 
                   
               
               
                   
               
               
                 P10645 
                 CMGA_ 
                 Chromogranin- 
                 CHGA 
                 457 
                 181T, 183T, 251T 
                 66 
               
               
                   
                 HUMAN 
                 A (CgA) 
                   
                   
                   
                   
               
               
                   
               
               
                 P15169 
                 CBPN_ 
                 Carbo xypeptidase 
                 CPN1 
                 458 
                 400T, 402T, 409T 
                 67 
               
               
                   
                 HUMAN 
                 N catalytic 
                 ACBP 
                   
                   
                   
               
               
                   
                   
                 chain (CPN) 
                   
                   
                   
                   
               
               
                   
               
               
                 P00740 
                 FA9_ 
                 Coagulation 
                 F9 
                 461 
                 85T, 99S,107S 
                 68 
               
               
                   
                 HUMAN 
                 factor IX (EC 
                   
                   
                   
                   
               
               
                   
                   
                 3.4.21.22) 
                   
                   
                   
                   
               
               
                   
               
               
                 P20333 
                 TNR1B_ 
                 Tumor 
                 TNFR 
                 461 
                 30T, 206T, 221S, 222T, 
                 69 
               
               
                   
                 HUMAN 
                 necrosis factor 
                 SF1B 
                   
                 224S, 230T, 234S, 235T, 
                   
               
               
                   
                   
                 receptor 
                 TNFBR 
                   
                 239T, 240S, 248S 
                   
               
               
                   
                   
                 superfamily 
                 TNFR2 
                   
                   
                   
               
               
                   
                   
                 member 1B 
                   
                   
                   
                   
               
               
                   
               
               
                 P08670 
                 VIME_ 
                 Vimentin 
                 VIM 
                 466 
                 7S, 33T, 34S 
                 70 
               
               
                   
                 HUMAN 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Q8WXD2 
                 SCG3_ 
                 Secretogranin- 
                 SCG3 
                 468 
                 216T, 231T, 359S 
                 71 
               
               
                   
                 HUMAN 
                 3 
                 UNQ2 
                   
                   
                   
               
               
                   
                   
                 (Secretogranin 
                 502/ 
                   
                   
                   
               
               
                   
                   
                 III) (SgIII) 
                 PR05990 
                   
                   
                   
               
               
                   
               
               
                 Q16566 
                 KCC4_ 
                 Calcium/ 
                 CAMK4 
                 473 
                 57T, 58S, 137S, 189S, 344S, 
                 72 
               
               
                   
                 HUMAN 
                 calmodulin- 
                 CAMK 
                   
                 345S, 356S 
                   
               
               
                   
                   
                 dependent 
                 CAMK- 
                   
                   
                   
               
               
                   
                   
                 protein kinase 
                 GR 
                   
                   
                   
               
               
                   
                   
                 type IV (CaMK 
                 CAMKI 
                   
                   
                   
               
               
                   
                   
                 IV) (EC 
                 V 
                   
                   
                   
               
               
                   
                   
                 2.7.11.17) 
                   
                   
                   
                   
               
               
                   
               
               
                 P31749 
                 AKT1_ 
                 RAC-alpha 
                 AKT1 
                 480 
                 126S, 129S, 305T, 312T, 
                 73 
               
               
                   
                 HUMAN 
                 serine/threonine- 
                 PKB 
                   
                 473S 
                   
               
               
                   
                   
                 protein 
                 RAC 
                   
                   
                   
               
               
                   
                   
                 kinase (EC 
                   
                   
                   
                   
               
               
                   
                   
                 2.7.11.1) 
                   
                   
                   
                   
               
               
                   
               
               
                 P31751 
                 AKT2_ 
                 RAC-beta 
                 AKT2 
                 481 
                 128S, 131S, 306T, 313T 
                 74 
               
               
                   
                 HUMAN 
                 serine/threonine- 
                   
                   
                   
                   
               
               
                   
                   
                 protein 
                   
                   
                   
                   
               
               
                   
                   
                 kinase (EC 
                   
                   
                   
                   
               
               
                   
                   
                 2.7.11.1) 
                   
                   
                   
                   
               
               
                   
               
               
                 O60883 
                 G37L1_ 
                 G-protein 
                 GPR37L1 
                 481 
                 79T, 85T, 86S, 95T, 107T 
                 75 
               
               
                   
                 HUMAN 
                 coupled 
                 ETBRLP2 
                   
                   
                   
               
               
                   
                   
                 receptor 37- 
                   
                   
                   
                   
               
               
                   
                   
                 like 1 
                   
                   
                   
                   
               
               
                   
               
               
                 Q9BXF9 
                 TEKT3_ 
                 Tektin-3 
                 TEKT3 
                 490 
                 7T, 9T, 10T 
                 76 
               
               
                   
                 HUMAN 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 P05155 
                 IC1_ 
                 Plasma 
                 SERPING1 
                 500 
                 47T, 48T, 64S, 71T, 83T, 
                 77 
               
               
                   
                 HUMAN 
                 protease C1 
                 C1IN 
                   
                 88T, 92T, 96T 
                   
               
               
                   
                   
                 inhibitor (C1 
                 C1NH 
                   
                   
                   
               
               
                   
                   
                 Inh) 
                   
                   
                   
                   
               
               
                   
               
               
                 P11831 
                 SRF_ 
                 Serum 
                 SRF 
                 508 
                 277S, 307S, 309S, 316S, 
                 78 
               
               
                   
                 HUMAN 
                 response 
                   
                   
                 383S 
                   
               
               
                   
                   
                 factor (SRF) 
                   
                   
                   
                   
               
               
                   
               
               
                 P0DOX3 
                 IGD_ 
                 Immunoglobulin 
                   
                 512 
                 238S, 255T, 256T, 260T, 
                 79 
               
               
                   
                 HUMAN 
                 delta heavy 
                   
                   
                 261T, 
                   
               
               
                   
                   
                 chain 
                   
                   
                   
                   
               
               
                   
               
               
                 O75487 
                 GPC4_ 
                 Glypican-4 (K- 
                 GPC4 
                 556 
                 494S, 498S, 500S 
                 80 
               
               
                   
                 HUMAN 
                 glypican) 
                 UNQ474/ 
                   
                   
                   
               
               
                   
                   
                   
                 PR0937 
                   
                   
                   
               
               
                   
               
               
                 P35052 
                 GPC1_ 
                 Glypican-1 
                 GPC1 
                 558 
                 486S, 488S, 490S 
                 81 
               
               
                   
                 HUMAN 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 P78333 
                 GPC5_ 
                 Glypican-5 
                 GPC5 
                 572 
                 441S, 486S, 495S, 507S, 
                 82 
               
               
                   
                 HUMAN 
                   
                   
                   
                 509S 
                   
               
               
                   
               
               
                 Q8N158 
                 GPC2_ 
                 Glypican-2 
                 GPC2 
                 579 
                 55S, 92S, 155S, 500S, 502S 
                 83 
               
               
                   
                 HUMAN 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 P00748 
                 FA12_ 
                 Coagulation 
                 F12 
                 615 
                 109T, 299T, 305T, 308S, 
                 84 
               
               
                   
                 HUMAN 
                 factor XII (EC 
                   
                   
                 328T, 329T, 337T 
                   
               
               
                   
                   
                 3.4.21.38) 
                   
                   
                   
                   
               
               
                   
               
               
                 P01042 
                 KNG1_ 
                 Kininogen-1 
                 KNG1 
                 644 
                 401T, 533T, 542T, 546T, 
                 85 
               
               
                   
                 HUMAN 
                 (Alpha-2-thiol 
                 BDK 
                   
                 557T, 571T, 577S, 628T 
                   
               
               
                   
                   
                 proteinase 
                 KNG 
                   
                   
                   
               
               
                   
                   
                 inhibitor) 
                   
                   
                   
                   
               
               
                   
               
               
                 P51693 
                 APLP1_ 
                 Amyloid-like 
                 APLP1 
                 650 
                 215T, 227S, 228T 
                 86 
               
               
                   
                 HUMAN 
                 protein 1 
                   
                   
                   
                   
               
               
                   
                   
                 (APLP) (APLP- 
                   
                   
                   
                   
               
               
                   
                   
                 1) 
                   
                   
                   
                   
               
               
                   
               
               
                 Q9NQ79 
                 CRAC1_ 
                 Cartilage 
                 CRTAC1 
                 661 
                 608T, 618T, 619T, 621T, 
                 87 
               
               
                   
                 HUMAN 
                 acidic protein 1 
                 ASPIC1 
                   
                 626T 
                   
               
               
                   
                   
                 (68 kDa 
                 CEP68 
                   
                   
                   
               
               
                   
                   
                 chondrocyte- 
                   
                   
                   
                   
               
               
                   
                   
                 expressed 
                   
                   
                   
                   
               
               
                   
                   
                 protein) (CEP- 
                   
                   
                   
                   
               
               
                   
                   
                 68) (ASPIC) 
                   
                   
                   
                   
               
               
                   
               
               
                 Q14515 
                 SPRL1_ 
                 SPARC-like 
                 SPARCL1 
                 664 
                 31T, 40T, 44S, 116T 
                 88 
               
               
                   
                 HUMAN 
                 protein 1 (High 
                   
                   
                   
                   
               
               
                   
                   
                 endothelial 
                   
                   
                   
                   
               
               
                   
                   
                 venule protein) 
                   
                   
                   
                   
               
               
                   
                   
                 (Hevin) (MAST 
                   
                   
                   
                   
               
               
                   
                   
                 9) 
                   
                   
                   
                   
               
               
                   
               
               
                 Q16820 
                 MEP1B_ 
                 Meprin A 
                 MEP1B 
                 701 
                 593S, 594T, 599T, 603S 
                 89 
               
               
                   
                 HUMAN 
                 subunit beta 
                   
                   
                   
                   
               
               
                   
                   
                 (EC 3.4.24.63) 
                   
                   
                   
                   
               
               
                   
               
               
                 P17600 
                 SYN1_ 
                 Synapsin-1 
                 SYN1 
                 705 
                 55S, 87T, 96S, 103S, 261S. 
                 90 
               
               
                   
                 HUMAN 
                 (Brain protein 
                   
                   
                 432S, 526T, 564T, 578S 
                   
               
               
                   
                   
                 4.1) (Synapsin 
                   
                   
                   
                   
               
               
                   
                   
                 I) 
                   
                   
                   
                   
               
               
                   
               
               
                 P19835 
                 CEL_ 
                 Bile salt- 
                 CEL 
                 753 
                 558T, 569T, 579T, 607T, 
                 91 
               
               
                   
                 HUMAN 
                 activated 
                 BAL 
                   
                 618T, 629T, 640T, 651T, 
                   
               
               
                   
                   
                 lipase (BAL) 
                   
                   
                 662T, 673T 
                   
               
               
                   
                   
                 (EC 3.1.1.13) 
                   
                   
                   
                   
               
               
                   
                   
                 (EC 3.1.1.3) 
                   
                   
                   
                   
               
               
                   
               
               
                 Q9HCU0 
                 CD248_ 
                 Endosialin 
                 CD248 
                 757 
                 60T, 401T, 428T, 448T, 
                 92 
               
               
                   
                 HUMAN 
                 (Tumor 
                 CD164 
                   
                 456T, 459T, 472T, 519T, 
                   
               
               
                   
                   
                 endothelial 
                 L1 
                   
                 541T, 543T, 544T, 545T, 
                   
               
               
                   
                   
                 marker 1) (CD 
                 TEM1 
                   
                 587T, 593T, 594T, 595T, 
                   
               
               
                   
                   
                 antigen 
                   
                   
                 598S, 601S, 612T, 619T, 
                   
               
               
                   
                   
                 CD248) 
                   
                   
                 623S, 625S, 627T, 630T, 
                   
               
               
                   
                   
                   
                   
                   
                 631S. 636T, 640S, 
                   
               
               
                   
               
               
                 P05067 
                 A4_ 
                 Amyloid-beta 
                 APP 
                 770 
                 633T, 651T, 652T, 656S, 
                 93 
               
               
                   
                 HUMAN 
                 precursor 
                 A4 
                   
                 659T, 663T, 667S, 
                   
               
               
                   
                   
                 protein (APP) 
                 AD1 
                   
                   
                   
               
               
                   
               
               
                 Q9NR71 
                 ASAH2_ 
                 Neutral 
                 ASAH2 
                 780 
                 62T, 67S, 68T, 70T, 738, 
                 94 
               
               
                   
                 HUMAN 
                 ceramidase 
                 HNAC1 
                   
                 74T, 76T, 78S, 79S, 80T, 
                   
               
               
                   
                   
                 (N-CDase) 
                   
                   
                 82T, 84T 
                   
               
               
                   
                   
                 (NCDase) (EC 
                   
                   
                   
                   
               
               
                   
                   
                 3.5.1.-) (EC 
                   
                   
                   
                   
               
               
                   
                   
                 3.5.1.23) 
                   
                   
                   
                   
               
               
                   
               
               
                 P08047 
                 SP1_ 
                 Transcription 
                 SP1 
                 785 
                 491S, 612S, 640T, 641S, 
                 95 
               
               
                   
                 HUMAN 
                 factor Sp1 
                 TSFP1 
                   
                 698S, 702S 
                   
               
               
                   
               
               
                 Q17R60 
                 IMPG1_ 
                 Interphotorece 
                 IMPG1 
                 797 
                 403T, 421T, 432T, 442T 
                 96 
               
               
                   
                 HUMAN 
                 ptor matrix 
                 IPM150 
                   
                   
                   
               
               
                   
                   
                 proteoglycan 1 
                 SPACR 
                   
                   
                   
               
               
                   
               
               
                 P19634 
                 SL9A1_ 
                 Sodium/hydrogen 
                 SLC9A1 
                 815 
                 42T, 56S, 61T, 62T, 68T 
                 97 
               
               
                   
                 HUMAN 
                 exchanger 
                 APNH1 
                   
                   
                   
               
               
                   
                   
                 1 (APNH) 
                 NHE1 
                   
                   
                   
               
               
                   
               
               
                 P12830 
                 CADH1_ 
                 Cadherin-1 
                 CDH1 
                 882 
                 280S, 285T, 358T, 470T, 
                 98 
               
               
                   
                 HUMAN 
                 (CAM 120/80) 
                 CDHE 
                   
                 472T, 509T, 
                   
               
               
                   
                   
                   
                 UVO 
                   
                 576T, 578T, 580T 
                   
               
               
                   
               
               
                 Q14118 
                 DAG1_ 
                 Dystroglycan 
                 DAG1 
                 895 
                 63T, 317T, 319T, 367T, 
                 99 
               
               
                   
                 HUMAN 
                 (Dystrophin- 
                   
                   
                 369T, 372T, 379T, 388T, 
                   
               
               
                   
                   
                 associated 
                   
                   
                 455T 
                   
               
               
                   
                   
                 glycoprotein 1) 
                   
                   
                   
                   
               
               
                   
               
               
                 Q14624 
                 ITIH4_ 
                 Inter-alpha- 
                 ITIH4 
                 930 
                 719T, 720T, 722T 
                 100 
               
               
                   
                 HUMAN 
                 trypsin inhibitor 
                 IHRP 
                   
                   
                   
               
               
                   
                   
                 heavy chain 
                 ITIHL1 
                   
                   
                   
               
               
                   
                   
                 H4 (ITI heavy 
                 PK120 
                   
                   
                   
               
               
                   
                   
                 chain H4) (ITI- 
                 PRO1 
                   
                   
                   
               
               
                   
                   
                   
                 HC4) 
                 851 
                   
                   
               
               
                   
               
               
                 P19823 
                 ITIH2_ 
                 Inter-alpha- 
                 ITIH2 
                 946 
                 666T, 673S, 675T, 691T 
                 101 
               
               
                   
                 HUMAN 
                 trypsin inhibitor 
                 IGHEP2 
                   
                   
                   
               
               
                   
                   
                 heavy chain 
                   
                   
                   
                   
               
               
                   
                   
                 H2 (ITI heavy 
                   
                   
                   
                   
               
               
                   
                   
                 chain H2) (ITI- 
                   
                   
                   
                   
               
               
                   
                   
                 HC2) 
                   
                   
                   
                   
               
               
                   
               
               
                 Q9UPV9 
                 TRAK1_ 
                 Trafficking 
                 TRAK1 
                 953 
                 447S, 680S, 719S, 935T 
                 102 
               
               
                   
                 HUMAN 
                 kinesin-binding 
                 KIAA1042 
                   
                   
                   
               
               
                   
                   
                 protein 1 
                 OIP106 
                   
                   
                   
               
               
                   
               
               
                 P15941 
                 MUC1 
                 Mucin-1 (MUC- 
                 MUC1 
                 1255 
                 131T, 139T, 140S, 144T 
                 103 
               
               
                   
                 HUMAN 
                 D 
                 PUM 
                   
                   
                   
               
               
                   
               
               
                 Q7Z589 
                 EMSY 
                 BRCA2- 
                 EMSY 
                 1322 
                 228S, 236S, 271T, 501T, 
                 104 
               
               
                   
                 HUMAN 
                 interacting 
                 C11orf30 
                   
                 506T, 557S, 1120T 
                   
               
               
                   
                   
                 transcriptional 
                 GL002 
                   
                   
                   
               
               
                   
                   
                 repressor 
                   
                   
                   
                   
               
               
                   
                   
                 EMSY 
                   
                   
                   
                   
               
               
                   
               
               
                 Q92954 
                 PRG4_ 
                 Proteoglycan 4 
                 PRG4 
                 1404 
                 123S, 136S, 240T, 253T, 
                 105 
               
               
                   
                 HUMAN 
                 (Lubricin) 
                 MSF 
                   
                 277T, 291T, 305T, 306S, 
                   
               
               
                   
                   
                   
                 SZP 
                   
                 31OT, 317S, 324T, 332T, 
                   
               
               
                   
                   
                   
                   
                   
                 338T, 367T, 373S, 376T, 
                   
               
               
                   
                   
                   
                   
                   
                 384T, 385T, 388S, 391T, 
                   
               
               
                   
                   
                   
                   
                   
                 399T, 400T, 407T, 408T, 
                   
               
               
                   
                   
                   
                   
                   
                 415T, 423T, 427S, 430T, 
                   
               
               
                   
                   
                   
                   
                   
                 438T, 439T, 446T, 447T, 
                   
               
               
                   
                   
                   
                   
                   
                 454T, 455T, 477T, 478T, 
                   
               
               
                   
                   
                   
                   
                   
                 485T, 493T, 494T, 501T, 
                   
               
               
                   
                   
                   
                   
                   
                 502T, 509T, 525T, 529S, 
                   
               
               
                   
                   
                   
                   
                   
                 532T, 540T, 541T, 553S, 
                   
               
               
                   
                   
                   
                   
                   
                 555T, 563T, 564T, 571T, 
                   
               
               
                   
                   
                   
                   
                   
                 572T, 579T, 580T, 587T, 
                   
               
               
                   
                   
                   
                   
                   
                 588T, 595T, 603T, 604T, 
                   
               
               
                   
                   
                   
                   
                   
                 611T, 612T, 616T, 619T, 
                   
               
               
                   
                   
                   
                   
                   
                 627T, 676T, 683T, 684T, 
                   
               
               
                   
                   
                   
                   
                   
                 691T, 692T, 699T, 700T, 
                   
               
               
                   
                   
                   
                   
                   
                 704T, 707T, 723T, 724T, 
                   
               
               
                   
                   
                   
                   
                   
                 736T, 768T, 769T, 776T, 
                   
               
               
                   
                   
                   
                   
                   
                 777T, 792T, 793T, 805T, 
                   
               
               
                   
                   
                   
                   
                   
                 812S, 829T, 837T, 838T, 
                   
               
               
                   
                   
                   
                   
                   
                 892S, 900T, 
                   
               
               
                   
                   
                   
                   
                   
                 930T, 931T, 962S, 963T, 
                   
               
               
                   
                   
                   
                   
                   
                 968T, 975T, 978T, 979T, 
                   
               
               
                   
                   
                   
                   
                   
                 980T, 1039T, 1161T 
                   
               
               
                   
               
               
                 Q76LX8 
                 ATS13_ 
                 A disintegrin 
                 ADAMTS13 
                 1427 
                 399S, 698S, 7578,907S, 
                 106 
               
               
                   
                 HUMAN 
                 and 
                 C9orf8 
                   
                 965S, 1027S, 1087S 
                   
               
               
                   
                   
                 metalloprotein 
                 UNQ6102/ 
                   
                   
                   
               
               
                   
                   
                 ase with 
                 PR020085 
                   
                   
                   
               
               
                   
                   
                 thrombospondin 
                   
                   
                   
                   
               
               
                   
                   
                 motifs 13 
                   
                   
                   
                   
               
               
                   
                   
                 (ADAM-TS 13) 
                   
                   
                   
                   
               
               
                   
               
               
                 P49790 
                 NU153_ 
                 Nuclear pore 
                 NUP153 
                 1475 
                 534S, 544S, 908S, 909S, 
                 107 
               
               
                   
                 HUMAN 
                 complex 
                   
                   
                 1113S, 1156T 
                   
               
               
                   
                   
                 protein 
                   
                   
                   
                   
               
               
                   
                   
                 Nup153 (153 
                   
                   
                   
                   
               
               
                   
                   
                 kDa 
                   
                   
                   
                   
               
               
                   
                   
                 nucleoporin) 
                   
                   
                   
                   
               
               
                   
                   
                 (Nucleoporin 
                   
                   
                   
                   
               
               
                   
                   
                 Nup153) 
                   
                   
                   
                   
               
               
                   
               
               
                 P31327 
                 CPSM_ 
                 Carbamoyl- 
                 CPS1 
                 1500 
                 537S, 1331S, 1332T 
                 108 
               
               
                   
                 HUMAN 
                 phosphate 
                   
                   
                   
                   
               
               
                   
                   
                 synthase 
                   
                   
                   
                   
               
               
                   
                   
                 [ammonia], 
                   
                   
                   
                   
               
               
                   
                   
                 mitochondrial 
                   
                   
                   
                   
               
               
                   
                   
                 (EC 6.3.4.16) 
                   
                   
                   
                   
               
               
                   
               
               
                 Q8N6G6 
                 ATL1_ 
                 ADAMTS-like 
                 ADAM 
                 1762 
                 48T, 312T, 391S, 451T 
                 109 
               
               
                   
                 HUMAN 
                 protein 1 
                 TSL1 
                   
                   
                   
               
               
                   
                   
                 (ADAMTSL-1) 
                 ADAM 
                   
                   
                   
               
               
                   
                   
                 (Punctin-1) 
                 TSR1 
                   
                   
                   
               
               
                   
                   
                   
                 C9orf94 
                   
                   
                   
               
               
                   
                   
                   
                 UNQ528/ 
                   
                   
                   
               
               
                   
                   
                   
                 PR01071 
                   
                   
                   
               
               
                   
               
               
                 P46531 
                 NOTC1_ 
                 Neurogenic 
                 NOTCH1 
                 2555 
                 65S, 73T, 116T, 146S, 194T, 
                 110 
               
               
                   
                 HUMAN 
                 locus notch 
                 TAN1 
                   
                 232T, 311T, 341S, 349T, 
                   
               
               
                   
                   
                 homolog 
                   
                   
                 378S, 435S, 458S,466T, 
                   
               
               
                   
                   
                 protein 1 
                   
                   
                 496S, 534S, 609S, 617T, 
                   
               
               
                   
                   
                 (Notch 1) 
                   
                   
                 647S, 692T, 722S, 759S, 
                   
               
               
                   
                   
                 (hN1) 
                   
                   
                 767T, 784S, 797S, 805T, 
                   
               
               
                   
                   
                   
                   
                   
                 921S, 951S, 997T, 1027S, 
                   
               
               
                   
                   
                   
                   
                   
                 1035T, 1065S, 1159T, 
                   
               
               
                   
                   
                   
                   
                   
                 1189S, 1197T, 1273S, 
                   
               
               
                   
                   
                   
                   
                   
                 1362T, 1379T, 1402T, 
                   
               
               
                   
               
               
                 P04275 
                 VWF_ 
                 von Willebrand 
                 VWF 
                 2813 
                 1248T, 1255T, 1256T, 
                 111 
               
               
                   
                 HUMAN 
                 factor (vWF) 
                 F8VW 
                   
                 1263S,1468T, 1477T, 
                   
               
               
                   
                   
                   
                 F 
                   
                 1486S, 1487T, 
                   
               
               
                   
               
               
                 Q9UPA5 
                 BSN_ 
                 Protein 
                 BSN 
                 3926 
                 1343T, 1384T, 2314T, 
                 112 
               
               
                   
                 HUMAN 
                 bassoon (Zinc 
                 KIAA0434 
                   
                 2691T, 2936T 
                   
               
               
                   
                   
                 finger protein 
                 ZNF231 
                   
                   
                   
               
               
                   
                   
                 231) 
                   
                   
                   
                   
               
               
                   
               
               
                 Q86WI1 
                 PKHL1_ 
                 Fibrocystin-L 
                 PKHD1L1 
                 4243 
                 122T, 445T, 1803T, 1839T, 
                 113 
               
               
                   
                 HUMAN 
                 (Polycystic 
                   
                   
                 2320T, 3736T 
                   
               
               
                   
                   
                 kidney and 
                   
                   
                   
                   
               
               
                   
                   
                 hepatic 
                   
                   
                   
                   
               
               
                   
                   
                 disease 1 -like 
                   
                   
                   
                   
               
               
                   
                   
                 protein 1) 
                   
                   
                   
                   
               
               
                   
                   
                 (PKHD1-like 
                   
                   
                   
                   
               
               
                   
                   
                 protein 1) 
               
               
                   
               
            
           
         
       
     
     The fusion polypeptide may have the total number of actually comprised O-glycan of 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, or 21 or more (the maximum value is determined by the number of the disclosed polypeptide region capable of O-glycosylation and the number of O-glycosylation residues comprised in each of the polypeptide region capable of O-glycosylation), or have the total number of theoretically comprised 0-glycan of 20 or more, 21 or more, 23 or 24 or more (the maximum value is determined by the number of the disclosed polypeptide region capable of O-glycosylation and the number of O-glycosylation residues comprised in each of the polypeptide region capable of O-glycosylation). In addition, in the fusion polypeptide, the total number of actually comprised 0-glycan may be related to stability upon administration in the body (for example, in blood), and specifically, in the fusion polypeptide, as the total number of the actually comprised 0-glycan increases, the in vivo stability of the fusion polypeptide or GDF15 comprised in the fusion polypeptide may increases (in other words, in vivo (in blood) half-life increase and/or in vivo (in blood) concentration increase and/or in vivo (in blood) rate of degradation decrease, etc.). 
     The fusion polypeptide may further comprise a peptide linker between GDF15 and a polypeptide region capable of O-glycosylation and/or between polypeptide regions capable of O-glycosylation when 2 or more of the polypeptide regions capable of O-glycosylation are comprised. In one embodiment, the peptide linker may be a GS linker repeatedly comprising one or more Gly(G) and one or more Ser(S), and for example, it may be (GGGGS)n (n is a repetition time of GGGGS (SEQ ID NO: 13) and is an integer of 1 to 10 or 1 to 5 (for example, 1, 2, 3, 4, or 5), but not limited thereto. 
     Other embodiment provides a fusion polypeptide dimer, comprising 2 of the fusion polypeptides. The fusion polypeptide dimer may be formed by being linked by a bond (for example, disulfide bond) between GDF15 comprised in each of the fusion polypeptides. The fusion polypeptide dimer may be a homodimer. 
     In the fusion polypeptide and/or fusion polypeptide dimer, the GDF15 fused with the polypeptide region capable of O-glycosylation is characterized by increased in vivo (or in blood) stability, compared to GDF15 in which the polypeptide region capable of O-glycosylation is not fused (for example, in vivo or in blood half-life increase). 
     Other embodiment provides a nucleic acid molecule encoding the fusion polypeptide. 
     Other embodiment provides a recombinant vector comprising the nucleic acid molecule. 
     Other embodiment provides a recombinant cell comprising the recombinant vector. 
     Other embodiment provides a method for preparation of GGF15 with increased in vivo (or in blood) half-life, or a method for preparation of a fusion polypeptide comprising the GDF15 with increased in vivo (or in blood) half-life, comprising expressing the recombinant vector in a cell. 
     Other embodiment provides a method for increasing in vivo duration of GDF15 comprising fusing (or linking or binding) GDF15 and a polypeptide region capable of O-glycosylation. In one specific embodiment, the fusing may comprise fusing (or linking or binding) one or more polypeptide regions capable of O-glycosylation at the N-terminus, C-terminus or both terminuses of GDF15 through or not through a linker. The fusing (or linking or binding) may be progressed in vitro. 
     Other embodiment provides a pharmaceutical composition comprising one or more selected from the group consisting of the fusion polypeptide, a fusion polypeptide dimer comprising the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule and a recombinant cell comprising the recombinant vector. 
     Other embodiment provides a use for enhancing in vivo (or in blood) stability and/or in vivo (or in blood) of a polypeptide (protein or peptide) drug of a polypeptide region capable of O-glycosylation. Specifically, one embodiment provides a composition for enhancing in vivo (or in blood) stability and/or increasing in vivo (or in blood) half-life of a polypeptide (protein or peptide) drug comprising a polypeptide region capable of O-glycosylation. As used in the present description, enhancing stability and/or increasing half-life mean that the stability is enhanced and/or the half-life is increased, compared to a polypeptide (protein or peptide) not comprising a polypeptide region capable of O-glycosylation. 
     Hereinafter, the present invention will be described in more detail: 
     In the present description, GDF15 (Growth differentiation factor 15) (corresponding to Y in the general formula) is a soluble polypeptide, and consists of amino acids from the 197th (A) to 308th (I) except for a signal peptide and a propeptide in total 308 amino acids (UniProt Q99988) (SEQ ID NO: 3; See  FIG.  1   ; mature form): 
     In the present description, GDF15 means, unless otherwise mentioned, 
     (1) the amino acid sequence from 197th (A) to 308th (I) of the full-length protein (UniProt Q99988) (SEQ ID NO: 3, See  FIG.  1   ; ARNG DHCPLGPGRC CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA ANMHAQIKTS LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL LAKDCHCI); 
     (2) a functional variant of GDF15; and/or 
     (3) a polypeptide essentially comprising the amino acid sequence having the sequence homology of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence of the (1) and/or (2) in a range of maintaining the intrinsic activity and structure. 
     In the present description, the functional variant of GDF15 may be a variant mutated to be advantageous for dimer structure formation, while maintaining the intrinsic activity and structure. In one embodiment, the functional variant of GDF15 may be a N-terminal deletion variant in which one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) (for example, one or more from the N-terminus in order) at the N-terminus of the amino acid sequence of GDF15 of SEQ ID NO: 3 (in other words, 14 amino acid residues in total from the 1st to 14th) in SEQ ID NO: 1), for example, all the 14 amino acid residues are deleted. In one specific embodiment, the functional variant of GDF15 may be a polypeptide essentially comprising the amino acid sequence of SEQ ID NO: 4 (CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA ANMHAQIKTS LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL LAKDCHCI) or the amino acid sequence having the sequence homology of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence in a range of maintaining the intrinsic activity and structure of GDF15. 
     In the fusion polypeptide comprising GDF15 and a polypeptide region capable of O-glycosylation provided in the present description, the GDF15 and polypeptide region capable of O-glycosylation and/or 2 or more of polypeptide regions capable of O-glycosylation may be linked directly (for example, without a linker) or linked through an appropriate linker (for example, peptide linker) covalently or non-covalently. The peptide linker may be a polypeptide consisting of any amino acids of 1 to 20, 1 to 15, 1 to 10, 2 to 20, 2 to 15 or 2 to 10, and the kind of the comprised amino acids is not limited. The peptide linker may comprise, for example, Gly, Asn and/or Ser residues, and may also comprise neutral amino acids such as Thr and/or Ala, but not limited thereto, and the amino acid sequence suitable for a peptide linker is known in the art. In one embodiment, the peptide linker may be a GS linker repeatedly comprising one or more Gly(G) and one or more Ser(S), and for example, may be (GGGGS)n (n is a repetition time of GGGGS (SEQ ID NO: 13) and is an integer of 1 to 10 or 1 to 5 (for example, 1, 2, 3, 4, or 5)), but not limited thereto. 
     In addition, the fusion polypeptide may comprise total 1 or more or total 2 or more (for example, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 or 3) polypeptide regions capable of O-glycosylation. When 2 or more of the polypeptide regions capable of O-glycosylation are comprised, in the fusion polypeptide, 2 or more of the polypeptide regions capable of O-glycosylation are linked to the N-terminus of GDF15 and each of the polypeptide regions capable of O-glycosylation may be same or different each other. Then, between the polypeptide regions capable of O-glycosylation and/or between the polypeptide region capable of O-glycosylation and human GDF15, the aforementioned peptide linker may be further comprised. 
     The fusion polypeptide provided in the present description ma be recombinantly or synthetically produced, and it may not be naturally occurring. 
     The in vivo (in blood) half-life in a mammal of GDF15 comprised in the fusion polypeptide provided in the present description may be increased about 1.1 time or more, about 1.15 times or more, about 1.2 times or more, about 1.5 times or more, about 2 times or more, about 2.5 time or more, about 3 times or more, about 3.5 times or more, about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, or about 10 times or more, compared to the GDF15 in which the polypeptide region capable of O-glycosylation is not fused. Otherwise, the highest blood concentration in case of administration in a mammal body of GDF15 comprised in the fusion polypeptide provided in the present description may be higher about 1.2 times or more, about 1.5 times or more, about 2 times or more, about 2.5 times or more, about 3 times or more, about 3.5 times or more, or about 4 times or more, compared to the not fused GDF15. Otherwise, the time of reaching the highest blood concentration in case of administration in a mammal body of the GDF15 comprised in the fusion polypeptide provided in the present description may be extended about 2 times or more, about 3 times or more, about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, about 11 times or more, about 12 times or more, about 13 times or more, about 14 times or more, about 15 times or more, about 18 times or more, about 20 times or more, or about 22 times or more, compared to the not fused GDF15. Otherwise, the area under the blood concentration-time curve up to the measurable last blood gathering time (AUC last ) and/or the area under the blood concentration-time curve calculated by extrapolating from the measurable last blood gathering time to the infinite time (AUC inf ), in case of administration in a mammal body of the GDF15 comprised in the fusion polypeptide provided in the present description may be increased about 2 times or more, about 2.5 times or more, about 3 times or more, about 3.5 times or more, about 4 times or more, about 4.5 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, about 11 times or more, about 12 times or more, about 13 times or more, about 14 times or more, or about 15 times or more, compared to the GDF15 not fused with the polypeptide region capable of O-glycosylation. 
     As such, due to the increased GDF15 half-life, the GDF15 in a fusion polypeptide form to which a polypeptide region capable of O-glycosylation is linked, has an advantage of having a longer administration interval, compared to the GDF15 in a form to which a polypeptide region capable of O-glycosylation is not linked. 
     The fusion polypeptide comprising GDF15 and a polypeptide region capable of O-glycosylation may be prepared by a common chemical synthesis method or recombinant method. 
     In the present description, the term “vector” means an expression means to express a target gene in a host cell, and for example, may be selected from the group consisting of a plasmid vector, a cosmid vector and a virus vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-related virus vector, and the like. In one embodiment, the vector which can be used for the recombinant vector may be produced on the basis of a plasmid (for example, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phage (for example, λgt4λB, λ-Charon, λΔz1, M13, etc.) or virus (for example, SV40, etc.), but not limited thereto. 
     The nucleic acid molecule encoding the fusion polypeptide in the recombinant vector may be operatively linked to a promoter. The term “operatively linked” means functional binding between a nucleic acid expression regulatory sequence (for example, promoter sequence) and other nucleic acid sequence. The regulatory sequence may regulate transcription and/or translation of other nucleic acid sequence by being “operatively linked”. 
     The recombinant vector may be typically constructed as a vector for cloning or an expression vector for expression. As the expression vector, common ones used for expressing a foreign protein in a plant, animal or microorganism may be used. The recombinant vector may be constructed by various methods known in the art. 
     The recombinant vector may be expressed using a eukaryote as a host. When an eukaryote is to be expressed as a host, the recombinant vector may comprise a replication origin such as f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin and/or BBV replication origin, and the like, but not limited thereto, in addition to a nucleic acid molecule to be expressed and the aforementioned promoter, a ribosome binding site, a secretory signal sequence (See Patent Publication No. 2015-0125402) and/or a transcription/translation termination sequence. In addition, a promoter derived from genome of a mammal cell (for example, metallothionein promoter) or a promoter derived from a mammal virus (for example, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus and tk promoter of HSV) may be used and all secretory signal sequences commonly available as a secretory signal sequence may be used, and for example, the secretory signal sequence disclosed in Patent Publication No. 2015-0125402 may be used, but not limited thereto, and as a transcription termination sequence, a polyadenylation sequence may be comprised. 
     The recombinant cell may be obtained by introducing (transforming or transfecting) the recombinant vector into an appropriate host cell. The host cell may be selected from all eukaryotes which can stably and continuously clone or express the recombinant vector. The eukaryote available as a host includes a yeast ( Saccharomyces cerevisiae ), an insect cell, a plant cell and an animal cell, and the like, and for example, includes mice (for example, COP, L, C127, Sp2/0, NS-0, NS-1, At20, or NIH3T3), rats (for example, PC12, PC12h, GH3, or MtT), hamsters (for example, BHK, CHO, GS genetic defect CHO, or DHFR genetic defect CHO), monkeys (for example, COS (COS1, COS3, COS7, etc.), CV1 or Vero), humans (for example, HeLa, HEK-293, retina-derived PER-C6, cell derived from diploid fibroblast, myeloma cell or HepG2), other animal cells (for example, MDCK, etc.), insect cells (for example, Sf9 cell, Sf21 cell, Tn-368 cell, BTI-TN-5B1-4 cell, etc.), hybridoma, and the like, but not limited thereto. 
     By expressing a nucleic acid molecule encoding the fusion polypeptide provided in the present description in the aforementioned appropriate host cell, GDF15 with enhanced in vivo stability compared to the not fused form and a fusion polypeptide comprising thereof may be prepared. The method for preparation of the fusion polypeptide may comprise culturing a recombinant vector comprising the nucleic acid molecule. The culturing may be performed under a common culturing condition. In addition, the method for preparation may further comprise separating and/or purifying the fusion polypeptide from the culture, after the culturing. 
     For delivery (introduction) of the nucleic acid molecule or recombinant vector comprising the same, a delivery method widely known in the art may be used. As the delivery method, for example, when the host cell is a eukaryote, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection and gene bombardment, and the like may be used, but not limited thereto. 
     The method for selecting the transformed (recombinant vector-introduced) host cell may be easily conducted according to a method widely known in the art, using a phenotype expressed by a selection marker. For example, when the selection marker is a specific antibiotic resistant gene, a recombinant cell in which a recombinant vector is introduced may be easily selected by culturing in a medium containing the antibiotic. 
     The fusion polypeptide may be used in prevention and/or treatment of all diseases which are related to GDF15 deficiency and/or dysfunction or can be treated, alleviated or improved by GDF15 activity. 
     Accordingly, in one embodiment, a pharmaceutical composition comprising one or more selected from the group consisting of the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule and a recombinant cell comprising the recombinant vector is provided. The pharmaceutical composition may be a pharmaceutical composition for prevention and/or treatment of diseases related to deficiency and/or dysfunction of GDF15 comprised in the fusion protein or diseases having a therapeutic and/or preventive effect of the GDF15. 
     Other embodiment provides a method for prevention and/or treatment of diseases related to deficiency and/or dysfunction of GDF15 comprised in the fusion protein or diseases having a therapeutic and/or preventive effect of the GDF15, comprising administering one or more selected from the group consisting of the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule and a recombinant cell comprising the recombinant vector, into a patient in need of prevention and/or treatment of diseases related to deficiency and/or dysfunction of GDF15 comprised in the fusion protein or diseases having a therapeutic and/or preventive effect of the GDF15. The method may further comprise confirming a patient in need of prevention and/or treatment of diseases related to deficiency and/or dysfunction of GDF15 comprised in the fusion protein or diseases having a therapeutic and/or preventive effect of the GDF15, before the administering. 
     The example of the diseases related to deficiency and/or dysfunction of GDF15 comprised in the fusion protein or diseases (or symptoms) having a therapeutic and/or preventive effect of the GDF15 may include obesity, diabetes (type 1 diabetes, type 2 diabetes), cardiovascular disease, myocardial hypertrophy, liver disease (e.g., nonalcoholic steatohepatitis (NASH), etc.), ischemic injury (ischemic brain damage, ischemic retina injury), peripheral nerve injury, age-related sensory and/or motor nerves loss, renal tubular and/or renal epileptic injury, but not limited thereto. 
     In other embodiment, the pharmaceutical composition or method comprising administering the same provided in the present description may have one or more effects selected from the group consisting of body weight loss, diet control (intake reduction), body fat reduction, and giving and/or enhancing glucose tolerance, and in this case, the pharmaceutical composition or method may be applied as a use for reducing body weight, reducing body fat and/or giving and/or enhancing glucose tolerance. 
     Therefore, in one embodiment, it may be a pharmaceutical composition or food composition (health functional food) for reducing a body weight, regulating a diet (reducing an amount of food), reducing body fat, or giving and/or enhancing glucose tolerance, as a composition comprising one or more selected from the group consisting of the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector. 
     Other embodiment provides a method for reducing a body weight, regulating a diet (reducing an amount of food), reducing body fat, or giving and/or enhancing glucose tolerance, administering one or more selected from the group consisting of the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule and a recombinant cell comprising the recombinant vector, into a patient in need of reducing a body weight, regulating a diet (reducing an amount of food), reducing body fat, or giving and/or enhancing glucose tolerance. The method may further comprise confirming the patient in need of reducing a body weight, regulating a diet (reducing an amount of food), reducing body fat, or giving and/or enhancing glucose tolerance, before the administering. 
     The pharmaceutical composition may comprise one or more of active ingredients selected from the group consisting of the fusion polypeptide, a fusion polypeptide dimer, a nucleic acid molecule, a recombinant vector and a recombinant cell comprising the fusion polypeptide in a pharmaceutically effective dose. The pharmaceutically effective dose means a contained amount or a dosage of the active ingredient capable of obtaining a desired effect. The contained amount or dosage of the active ingredient may be variously prescribed by factors such as preparation method, administration method, patient&#39;s age, body weight, gender, morbid condition, food, administration time, administration interval, administration route, excretion rate and reaction sensitivity. For example, the single dosage of the active ingredient may be in a range of 0.001 to 1000 mg/kg, 0.01 to 100 mg/kg, 0.01 to 50 mg/kg, 0.01 to 20 mg/kg, or 0.01 to 1 mg/kg, but not limited thereto. 
     Furthermore, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, in addition the active ingredients. The carrier is one commonly used in preparation of a drug comprising a protein, nucleic acid or cell, and may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like, but not limited thereto. The pharmaceutical composition may also comprise one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, commonly used in preparation of pharmaceutical compositions additionally. 
     The administration subject of the pharmaceutical composition may be a mammal including primates such as humans and monkeys, rodents such as mice and rats, and the like, or a cell, tissue, cell culture or tissue culture derived therefrom. 
     The pharmaceutical composition may be administered by oral administration or parenteral administration, or may be administered by contacting it to a cell, tissue or body fluid. Specifically, in case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration and intrarectal administration, and the like. In case of oral administration, since proteins or peptides are digested, an oral composition should be formulated to coat an active agent or to protect it from degradation in the stomach. 
     In addition, the pharmaceutical composition may be formulated in a form of solution, suspension, syrup or emulsion in an oil or aqueous medium, or in a form of extract, powder, granule, tablet or capsule, or the like, and for formulation, it may further comprise a dispersing agent or stabilizing agent. 
     Advantageous Effects 
     The GDF15 fused with a polypeptide region capable of O-glycosylation provided in the present description has a long duration when administered in vivo, so it is possible to increase the administration interval and thereby reduce the administration dose, and therefore, it has an advantageous effect in terms of administration convenience and/or economics and can be usefully applied to fields requiring GDF15 treatment. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    schematically shows the amino acid sequence of GDF15, SEQ ID NO: 3. 
         FIG.  2   a    schematically shows the fusion polypeptide comprising His Taq according to one example. 
         FIG.  2   b    schematically shows the structure of the fusion polypeptide according to one example. 
         FIG.  3    schematically shows the structures of the various types of fusion polypeptides. 
         FIG.  4    shows the result of analyzing fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 synthesized in one example by SDS-PAGE. 
         FIG.  5    shows the result of analyzing fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 synthesized in one example by Q-TOF Mass Spectrometry. 
         FIG.  6    is a graph showing the body weight change when each of the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 is administered to mice once. 
         FIG.  7    is a graph extracting and showing the result at Day 4 among the result of  FIG.  6   . 
         FIG.  8    is a graph showing the feed intake change of mice administered with the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15. 
         FIG.  9    is a graph showing the body weight change upon repeated administration of each of the fusion polypeptides HT-ID2-GDF15 and HT-ID3-GDF15 to mice. 
         FIG.  10   a    is a graph extracting and showing the result at Day 7 and Day 14 among the result of  FIG.  9   . 
         FIG.  10   b    is a graph extracting and showing the result at Day 21 and Day 28 among the result of  FIG.  9   . 
         FIG.  11    is a graph showing the cumulative feed intake up to Day 7, Day 14, Day 21 and Day 28 when the fusion polypeptides HT-ID2-GDF15 and HT-ID3-GDF15 are repeatedly administered to mice, respectively. 
         FIG.  12    is a graph showing the change in the blood fusion polypeptide concentration with time when the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 are administered to SD Rat. 
     
    
    
     Hereinafter, the present invention will be described in more detail by the following examples. However, they 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 
     1.1. Preparation of Fusion Polypeptide Comprising GDF15 
     Fusion polypeptides IgD-GDF15 (ID1-GDF15), IgD-IgD-GDF15 (ID2-GDF15), IgD-IgD-IgD-GDF15 (ID3-GDF15) (See  FIGS.  2   a  and  2   b   ) in which a combination of IgD hinge (ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNT; SEQ ID NO: 1; the underlined parts were sites capable of O-glycosylation) or several (1, 2 or 3) IgD hinges were fused with GDF15 (SEQ ID NO: 3,  FIG.  1   ) were prepared. For convenience of purification, a fusion polypeptide comprising His-tag (SEQ ID NO: 15) and TEV cleavage Site (SEQ ID NO: 16) was also prepared. The amino acid sequences of each part comprised in the fusion polypeptide were summarized in Table 2 below. 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 SEQ ID 
               
               
                   
                 Amino acid sequence (N terminus C terminus) 
                 NO: 
               
               
                   
               
             
            
               
                 Signal Peptide 
                 MHRPEAMLLL LTLALLGGPT WA 
                 14 
               
               
                 (SP7.2) 
                   
                   
               
               
                   
               
               
                 Target 
                 ARNGDHCPLG PGRCCRLHTV RASLEDLGWA 
                  3 
               
               
                 polypeptide 
                 DWVLSPREVQ 
                   
               
               
                 (GDF15) 
                 VTMCIGACPS QFRAANMHAQ IKTSLHRLKP 
                   
               
               
                   
                 DTVPAPCCVP 
                   
               
               
                   
                 ASYNPMVLIQ KTDTGVSLQT YDDLLAKDCH CI 
                   
               
               
                   
               
               
                 Hinge region of 
                 ESPKAQASSV PTAQPQAEGS LAKATTAPAT TRNT 
                  1 
               
               
                 immunoglobulin 
                   
                   
               
               
                 IgD (ID) 
                   
                   
               
               
                   
               
               
                 His-Tag 
                 HHHHHHHH 
                 15 
               
               
                   
               
               
                 TEV Cleavage 
                 ENLYFQG 
                 16 
               
               
                 Site 
                   
                   
               
               
                   
               
               
                 GS Linker 
                 GGGGSGGGGS GGGGSGGGGS 
                 17 
               
               
                   
               
            
           
         
       
     
     1.1.1. Preparation of Recombinant Expression Vector 
     1.1.1.1. Mature GDF15 
     In order to obtain a gene encoding mature GDF15, referring to the amino acid sequence information of UniprotKB Q99968, a gene encoding mature GDF15 (SEQ ID NO: 5) was synthesized (Bioneer). 
     
       
         
           
               
               
            
               
                 (339 bp) 
                   
               
               
                 SEQ ID NO: 5 
                   
               
            
           
           
               
               
               
            
               
                   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 
               
            
           
         
       
     
     1.1.1.2. IgD Hinge (ID) 
     In order to obtain a gene encoding Human IgD Hinge, referring to the amino acid sequence information of UniprotKB P01880, a gene encoding 3 Human IgD Hinges (hereinafter, referred to as ‘ID3’) (SEQ ID NO: 6) was synthesized in Bioneer. 
     
       
         
           
               
               
            
               
                 (306 bp) 
                   
               
               
                 SEQ ID NO: 6 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 
                   GAGAGCCCTA AGGCTCAGGC CTCTAGCGTG CCAACAGCTC AGCCACAAGC 
                 
                   
               
               
                   
               
               
                  51 
                 
                   TGAAGGAAGC CTGGCCAAGG CTACAACCGC CCCTGCCACA ACACGGAATA 
                 
               
               
                   
               
               
                 101 
                 
                   CA 
                   GAGTCCCC CAAGGCCCAG GCTAGCAGCG TGCCTACCGC CCAGCCTCAG 
                 
               
               
                   
               
               
                 151 
                 
                   GCCGAGGGCT CCCTGGCTAA GGCCACAACC GCTCCCGCTA CAACCAGGAA 
                 
               
               
                   
               
               
                 201 
                 
                   CACCG 
                   
                     AGTCT CCAAAGGCAC AGGCCTCCTC CGTGCCCACT GCACAACCCC 
                   
                 
               
               
                   
               
               
                 251 
                 
                   
                     AAGCAGAGGG CAGCCTCGCC AAGGCAACCA CAGCCCCAGC CACCACCCGG 
                   
                 
               
               
                   
               
               
                 301 
                 
                   
                     AACACA 
                   
                 
               
            
           
         
       
     
     (1-102 polynucleotide (underlined), 103-204 polynucleotide (bold), and 205-306 polynucleotide (bold+underlined) encode IgD Hinge, respectively) 
     1.1.1.3. Preparation of Expression Vector 
     A variant of pcDNA3.1(+) (Invitrogen, Cat. No. V790-20), pDHDD-D1G1 (comprising the promoter of KR10-1868139B1) was cut with BamHI and NotI, and a gene designed to encode a fusion protein having the structure below (See  FIG.  3   ) by combining the above genes (mature GDF15 encoding gene and ID3 encoding gene) was inserted thereto to prepare each recombinant vector. 
     pGDF15 
     ‘(N-terminus)-[BamHI restriction site (GGATCC)-signal peptide (SEQ ID NO: 14)-Mature GDF15 (SEQ ID NO: 3)-NotI restriction site (GCGGCCGC)]-(C-terminus)’ 
     pHT-GDF15 
     ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-His-Taq (SEQ ID NO: 15)-TEV Cleavage Site (SEQ ID NO: 16)-Mature GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminus)’ 
     pID1-GDF15 
     ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17)-Mature GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminus)’ 
     pHT-ID1-GDF15 
     ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-His-Taq (SEQ ID NO: 15)-TEV Cleavage Site (SEQ ID NO: 16)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17)-GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminus)’ 
     pID2-GDF15 
     ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17)-GDF15 (SEQ ID NO: 3)-NotI restriction peptide]-(C-terminus)’ 
     pHT-ID2-GDF15 
     ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-His-Taq (SEQ ID NO: 15)-TEV Cleavage Site (SEQ ID NO: 16)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17)-GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminus)’ 
     pID3-GDF15 
     ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17)-GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminus)’ 
     pHT-ID3-GDF15 
     ‘(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-His-Taq (SEQ ID NO: 15)-TEV Cleavage Site (SEQ ID NO: 16)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17)-GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminus)’ 
     1.1.2. Expression of Fusion Polypeptide 
     The prepared recombinant expression vectors, pGDF15, pHT-GDF15, pID1-GDF15, pHT-ID1-GDF15, pID2-GDF15, pHT-ID2-GDF15, pID3-GDF15, and pHT-ID3-GDF15 were introduced into ExpiCHO-S™ cell (Thermo Fisher Scientific) and cultured (Fed-Batch Culture; Day 1 &amp; Day 5 Feeding) in ExpiCHO Expression Medium (Thermo Fisher Scientific; 400 mL) for 12 days, to express the fusion polypeptides GDF15, HT-GDF15, ID1-GDF15, HT-ID1-GDF15, ID2-GDF15, HT-ID2-GDF15, ID3-GDF15, and HT-ID3-GDF15. 
     1.1.3. Purification of Fusion Polypeptide 
     The fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 produced through the recombinant expression vector were purified and O-Glycan site Occupancy was analyzed using Sialic Acid content analysis and Q-TOF Mass Spectrometry. 
     Specifically, the fusion polypeptides were purified by continuously performing ultrafiltration/diafiltration, Immobilized Metal Affinity Chromatography (IMAC), and Anion Exchange Chromatography (AEX). At first, the culture solution of the fusion protein in which cells were removed was filtered with a 0.22 μm filter. For the filtered solution, concentration was performed using TFF System and then buffer exchange was conducted with a tromethamine buffer solution. A column in which HiTrap™ Chelating HP (GE Healthcare Life Sciences) resin was packed was equipped and an equilibrium buffer (20 mM Tris pH 8.0, 0.5 M NaCl, 5 mM Imidazole) was flowed to equilibrate the column. The process solution in which the ultrafiltration/diafiltration was completed previously was injected into the column, and then the equilibrium buffer was flowed again to wash the column. After completing the washing operation of the column, the elution buffer (20 mM Tris pH 8.0, 0.5 M NaCl, 0.5 M Imidazole) was flowed into the column to elute a target protein. 
     For the obtained eluted solution, concentration was performed using Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge, and then buffer exchange was conducted with a tromethamine buffer solution. The process solution prepared as such was injected into the equilibrated anion exchange column, and the equilibrium buffer (20 mM Tris pH 8.0) was flowed and the column was washed. After completing the washing operation of the column, the elution buffer (20 mM Tris pH 8.0, 0.5 M NaCl) was flowed into the column under a concentration gradient condition to elute the target protein. Among eluted fractions, fractions with high concentration and high purity of the fusion polypeptide were collected and kept frozen. 
     For an animal experiment, concentration and buffer exchange for samples were performed with Phosphate Buffered Saline (PBS, 10 mM Sodium Phosphate, 150 mM NaCl pH 7.4) using Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge. 
     The quantitative analysis of the fusion polypeptide was conducted by measuring the absorbance at 280 nm and 340 nm in UV Spectrophotometer (G113A, Agilent Technologies) by the following equation. As the extinction coefficient, a value theoretically calculated using the amino acid sequence was used. 
     
       
         
           
             
               Protein 
               ⁢ 
                   
               
                 concentration 
                 ⁢ 
                 
                      
                     
                 
                 ( 
                 
                   mg 
                   / 
                   mL 
                 
                 ) 
               
             
             = 
             
               
                 
                   Absorbance 
                   ⁢ 
                       
                   
                     ( 
                     
                       
                         A 
                         
                           280 
                           ⁢ 
                              
                           nm 
                         
                       
                       - 
                       
                         A 
                         
                           340 
                           ⁢ 
                               
                           nm 
                         
                       
                     
                     ) 
                   
                 
                 
                   
                     * 
                   
                   Extinction 
                   ⁢ 
                       
                   Coefficient 
                 
               
               × 
               Dilution 
               ⁢ 
                   
               Factor 
             
           
         
       
     
     *Extinction coefficient (0.1%): theoretical absorbance at 280 nm, assuming that the protein concentration is 0.1% (1 g/L), and all cysteines in Primary Sequence are oxidized to form disulfide bonds. Calculated via ProtParam tool (https://web.expasy.org/protparam/). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Extinction coefficient of fusion polypeptide 
               
            
           
           
               
               
               
            
               
                   
                 Sample name 
                 Extinction coefficient (0.1%, 1 mg/mL) 
               
               
                   
                   
               
               
                   
                 HT-ID1-GDF15 
                 0.833 
               
               
                   
                 HT-ID2-GDF15 
                 0.706 
               
               
                   
                 HT-ID3-GDF15 
                 0.612 
               
               
                   
                   
               
            
           
         
       
     
     For the purified fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15, after Sialic Acid content analysis and reducing, 0-Glycan site Occupancy was analyzed using Q-TOF Mass Spectrometry. 
     The result of analyzing the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 by SDS-PAGE was shown in  FIG.  4   , and the result of analyzing them by Q-TOF Mass Spectrometry was shown in  FIG.  5    and Table 5. In addition, the sialic acid content was also shown in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Average O-Glycan number and Sialic Acid content 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Theoretical 
                   
                   
                   
               
               
                   
                 O-Glycan 
                 Average 
                 O-Glycan 
                 Sialic Acid 
               
               
                   
                 number 
                 O-Glycan 
                 distribution 
                 content 
               
               
                 Sample 
                 (One Chain) 
                 number 
                 (main) 
                 (mol/mol) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 HT-ID1-GDF15 
                 7 
                 5.4 
                 2-7 
                 (6) 
                 14.4 
               
               
                 HT-ID2-GDF15 
                 14 
                 9.7 
                 3-15* 
                 (10) 
                 16.3 
               
               
                 HT-ID3-GDF15 
                 21 
                 13.5 
                 5-21 
                 (13) 
                 18.9 
               
               
                   
               
               
                 *More than the theoretical O-glycan number is presumed that O-glycan is attached to the GS Linker (Spahr et al., 2014, mAbs, 6: 904) 
               
            
           
         
       
     
     Example 2. Pharmacological Effect of Fusion Polypeptide (In Vivo) 
     2.1. Single Administration 
     2.1.1 Test Process 
     The pharmacological effect of the fusion polypeptides produced and purified in Example 1 above was tested in mice (C57BL/6J, 6-week-old, male, 100 mice; Raonbio). 
     In the present example, DI0 mouse model (Mouse, C57BL/6J-DIO, male, 100 mice, 14-week-old (obesity feed feeding for 8 weeks)) in which obesity was induced by feeding a high-fat diet into the C57BL/6J mice for 8 weeks was used. The DI0 mouse model is an animal model widely used for evaluation of diabetes and insulin improvement efficacy, as it exhibits clinical characteristics of type 2 diabetes such as hyperlipidemia, insulin resistance, and hyperglycemia, and a lot of comparable basic data have been accumulated for the study of metabolic diseases such as obesity, diabetes and hyperlipidemia, and therefore, it was suitable for the pharmacological effect test of the present example, and thus this model was selected. 
     The mouse model fed with the obesity feed for 8 weeks was subjected to a quarantine and acclimatization period of 2 weeks, and during this period, general symptoms were observed once a day, and healthy animals were selected by confirming whether they were healthy and suitable for conducting the experiment. During the acclimatization period, the animal&#39;s tail was marked with a red oil pen at the time of acquisition (tail marking), and temporary individual identification cards (test name, individual number, stocking time) were attached to the breeding box during the quarantine acclimatization period. At the time of group separation, individuals were marked on the tails of animals using a black oil pen and individual identification cards (test name, group information, individual number, gender, stocking time, administration period) were attached to each cage. 
     In order to minimize stress experienced by the experimental animals due to subcutaneous administration of the test substance (fusion polypeptide), 200 uL/head of sterile distilled physiological saline was administered subcutaneously to all animals using a 1 mL syringe from 3 days before the administration of the test substance. Pre-adaptation training for subcutaneous administration was conducted. 
     For healthy animals with no abnormalities found during the quarantine and acclimatization period, the body weight and feed intake were measured for all individuals after the acclimatization period. 
     The body weight and feed intake were measured, and group separation was performed so that the averages of the two measured values were similar between groups based on body weight. Test substance administration was started from the day after group separation. Remaining animals that were not selected were excluded from the test system after group separation was terminated. 
     The information of the high fat diet (obesity feed; HFD) fed to the C57BL/6J-DIO was as follows: 
     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). 
     The feed was fed by a free feeding (feeding during the acclimatization and test period) method. 
     The drinking water method was that tap water was filtered with a filter oil-water sterilizer and then ultraviolet rays were irradiated and it was freely ingested using a polycarbonate drinking water bottle (250 mL). 
     The administration of the test substances HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 and the control substance Semaglutide (Bachem) was conducted from the next day after group separation, and the administration time was performed at 9 AM every day. Subcutaneous administration was conducted for all the control substance and test substances. For the administration route of the control substance and test substances, subcutaneous administration was selected depending on the clinically scheduled administration route. 
     For all the control substance and test substances, the amount of the administration solution was set to 5 mL/kg and the administration liquid by individual was calculated on the basis of the recently measured body weight and it was administered by subcutaneous injection once on the start day of the test using a disposable syringe (1 mL). The test substances were administered only once. For comparison, the control group in which the control substance Semaglutide was administered was prepared, and the comparison group in which Semaglutide was administered was administered once a day, and all the administration was progressed from 9 AM. 
     The composition of the test groups and administration dose were summarized in Table 5 below: 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Fusion polypeptide administration group composition 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Adminis- 
                 Adminis- 
                   
               
               
                   
                   
                 Adminis- 
                 tration 
                 tration 
               
               
                 High 
                 Test 
                 tration 
                 dose 
                 volume 
                 Animal 
               
               
                 Fat Diet 
                 substance 
                 route 
                 (nmol/kg) 
                 (mL/kg) 
                 number 
               
               
                   
               
               
                 O 
                 Vehicle, qw 
                 Sub- 
                 — 
                 5 
                 5 
               
               
                   
                   
                 cutaneous 
               
               
                 O 
                 Semaglutide, 
                 Sub- 
                 3 
                 5 
                 5 
               
               
                   
                 qd 
                 cutaneous 
               
               
                 O 
                 HT-ID1-GDF15 
                 Sub- 
                 10 
                 5 
                 5 
               
               
                   
                   
                 cutaneous 
               
               
                 O 
                 HT-ID2-GDF15 
                 Sub- 
                 10 
                 5 
                 5 
               
               
                   
                   
                 cutaneous 
               
               
                 0 
                 HT-ID3-GDF15 
                 Sub- 
                 10 
                 5 
                 5 
               
               
                   
                   
                 cutaneous 
               
               
                   
               
            
           
         
       
     
     As for the observation, measurement and test schedule for the test groups, the administration start date was set to Day 0, and 7 days from the administration start date were set to one week of administration. 
     The test schedule was summarized in Table 6: 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Test schedule 
               
            
           
           
               
               
               
            
               
                   
                 Acclimatization 
                   
               
               
                   
                 period 
               
               
                   
                 (week) 
                 Period (day) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Observation item 
                 1 
                 2 
                 0 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
               
               
                   
               
               
                 High fat feed 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
               
               
                 feeding 
               
               
                 Adaptation to 
                   
                 • 
               
               
                 oral and 
               
               
                 subcutaneous 
               
               
                 administration 
               
               
                 Administration 
                   
                   
                 • 
               
               
                 Body weight 
                   
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
               
               
                 measurement 
               
               
                 Feed intake 
                   
                   
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
                 • 
               
               
                 measurement 
               
               
                   
               
            
           
         
       
     
     General clinical symptoms were observed once a day for all animals, and the presence or absence of moribund and dead animals was checked twice a day, and these observations were conducted from the 1st day of administration to the end of administration. Only when there were abnormal symptoms during observation, it was recorded on the recording sheet. 
     The body weight of each mouse was measured on the day of the start of administration of the test substances (before administration), and thereafter, the body weight was measured every day (measured up to 9 days), and the amount of the administration solution of the test substances was determined on the basis of the most recently measured body weight. 
     In addition, after administering the test substances into mice, the daily feed intake was measured, and the feeding amount was measured using an electronic scale for each breeding box, and the remaining amount was measured to calculate the daily feed intake. In case of an individual that gnawed heavily on heed, it was excluded from the measurement. 
     All the experimental results obtained in the present example were expressed as mean±standard error and tested using Prism5 (version 5.01). One-way analysis of variance (ANOVA) was performed on all data, and when significance was observed, Dunnett&#39;s test was performed to find out the test groups with a significant difference from the control group (significance level: two-sided 5% and 1%, 0.1%). 
     2.1.2. Body Weight Loss Test Result 
     The change in the body weight measured in Example 2.1.1 above was shown in  FIG.  6    and  FIG.  7   , and Table 7 (Body Weight (Group, % of initial). 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 Group 
                 Day 
                 0 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 DIO Vehicle 
                 Mean 
                 100 
                 100 
                 100 
                 99 
                 100 
                 100 
                 100 
                 100 
                 101 
                 101 
               
               
                 Control 
                 S.E. 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
               
               
                 (Daily Inj.) 
               
               
                 DIO 
                 Mean 
                 100 
                 94 
                 91 
                 90 
                 87 
                 87 
                 85 
                 86 
                 83 
                 85 
               
               
                 Semaglutide 
                 S.E. 
                 0 
                 0 
                 1 
                 2 
                 2 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 3 nmol/kg 
               
               
                 (Daily Inj.) 
               
               
                 HT-ID1-GDF15 
                 Mean 
                 100 
                 99 
                 98 
                 97 
                 96 
                 95 
                 95 
                 96 
                 96 
                 97 
               
               
                 10 nmol/kg 
                 S.E. 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
               
               
                 (Single Inj.) 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 100 
                 98 
                 97 
                 96 
                 94 
                 94 
                 94 
                 93 
                 93 
                 94 
               
               
                 10 nmol/kg 
                 S.E. 
                 0 
                 0 
                 1 
                 1 
                 1 
                 2 
                 2 
                 2 
                 2 
                 2 
               
               
                 (Single Inj.) 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 100 
                 98 
                 97 
                 96 
                 95 
                 94 
                 95 
                 95 
                 95 
                 96 
               
               
                 10 nmol/kg 
                 S.E. 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
               
               
                 (Single Inj.) 
               
               
                   
               
            
           
         
       
     
       FIG.  6    and Table 7 shows the change in the body weight when the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 were administered once, respectively, compared to the negative control group (vehicle administration group) and positive control group (Semaglutide daily administration group). In addition,  FIG.  7    is a graph showing the result at Day 4 by extracting it among the result of  FIG.  6   . 
     As shown in the above result, it could be confirmed that there was little change in the body weight in case of the negative control group (vehicle administration group), while the body weight loss effect was continuously shown from Day 1 after administration in case of the positive control group (Semaglutide daily administration group). In addition, it could be confirmed that the body weight loss effect was shown immediately after single administration at Day 0 in case of the fusion polypeptide in which GDF15 was fused with IgD Hinge, and the body weight loss effect was not reduced and appeared continuously until 3-4 days. 
     2.1.3. Diet Intake Test Result 
     The change in the feed intake measured in Example 2.1.1 above was shown in Table 8 and  FIG.  8    (cumulative intake up to Day 6), respectively. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 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 
               
               
                 (Daily Inj.) 
                 S.E. 
                 0.1 
                 0.1 
                 0.2 
                 0.1 
                 0.1 
                 0.2 
                 0.2 
                 0.1 
                 0.1 
                 0.2 
               
               
                 DIO Semaglutide 
                 Mean 
                 3.7 
                 2.1 
                 3.3 
                 2.8 
                 2.3 
                 2.9 
                 2.7 
                 3.2 
                 2.4 
                 3.7 
               
               
                 3 nmol/kg(Daily Inj.) 
                 S.E. 
                 0.3 
                 0.7 
                 1.4 
                 0.6 
                 0.4 
                 0.9 
                 0.6 
                 0.3 
                 0.4 
                 0.1 
               
               
                 HT-ID1-GDF15 
                 Mean 
                 3.3 
                 2.2 
                 2.9 
                 2.3 
                 2.8 
                 3.1 
                 3.1 
                 3.7 
                 3.4 
                 3.5 
               
               
                 10 nmol/kg(Single Inj.) 
                 S.E. 
                 0.2 
                 0.3 
                 0.2 
                 0.5 
                 0.2 
                 0.3 
                 0.1 
                 0.3 
                 0.3 
                 0.5 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 2.7 
                 1.7 
                 2.3 
                 2.2 
                 2.6 
                 2.4 
                 2.8 
                 3.1 
                 2.7 
                 3.3 
               
               
                 10 nmol/kg(Single Inj.) 
                 S.E. 
                 0.2 
                 0.2 
                 0.1 
                 0.1 
                 0.3 
                 0.2 
                 0.1 
                 0.1 
                 0.2 
                 0.2 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 3.2 
                 2.0 
                 2.5 
                 2.3 
                 2.3 
                 2.7 
                 2.8 
                 3.3 
                 3.3 
                 3.6 
               
               
                 10 nmol/kg(Single Inj.) 
                 S.E. 
                 0.2 
                 0.2 
                 0.3 
                 0.2 
                 0.2 
                 0.3 
                 0.2 
                 0.3 
                 0.2 
                 0.2 
               
               
                   
               
            
           
         
       
     
     As shown in the above result, in case of the fusion polypeptide administration group in which GDF15 was fused with IgD Hinge, compared to the negative control group (vehicle administration group) administration group, the feed intake reduction effect was shown up to Day 6 at maximum depending on the fusion polypeptide, and this feed intake reduction effect of the fusion polypeptide can be said to be comparable to the case of administering Semaglutide, the positive control group, once a day throughout the test period. 
     2.2. Repeated Administration 
     2.2.1 Test Process 
     Except for the administration dose, animal number and administration cycle, most of the test processes were the same as in Example 2.1.1 above. 
     The test substances were administered twice a week (Days 0, 4, 7, 11, 14, 18, 21, 25) for a total of 8 times. For comparison, the control group in which the control substance Semaglutide was administered every day was prepared, and the comparison group in which Semaglutide was administered every day was administered once a day daily, and all the administration was progressed from 9 AM. 
     The composition of the test groups and administration dose, and the like were summarized in Table 9 below: 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Fusion polypeptide administration group composition 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Administration 
                 Administration 
                   
               
               
                 High Fat 
                   
                 Administration 
                 Administration 
                 dose 
                 volume 
                 Animal 
               
               
                 Diet 
                 Test substance 
                 route 
                 cycle 
                 (nmol/kg) 
                 (mL/kg) 
                 number 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 O 
                 Lean Vehicle Control 
                 Subcutaneous 
                 Once a 
                 — 
                 5 
                 4 
               
               
                   
                   
                   
                 week 
               
               
                 O 
                 DIO Vehicle Control 
                 Subcutaneous 
                 Once a 
                 — 
                 5 
                 8 
               
               
                   
                   
                   
                 week 
               
               
                 O 
                 Semaglutide 
                 Subcutaneous 
                 Once a 
                 3 
                 5 
                 8 
               
               
                   
                   
                   
                 day 
               
               
                 O 
                 HT-ID2-GDF15 
                 Subcutaneous 
                 Twice a 
                 3 
                 5 
                 8 
               
               
                   
                   
                   
                 week 
               
               
                 O 
                 HT-ID2-GDF15 
                 Subcutaneous 
                 Twice a 
                 10 
                 5 
                 8 
               
               
                   
                   
                   
                 week 
               
               
                 O 
                 HT-ID2-GDF15 
                 Subcutaneous 
                 Twice a 
                 30 
                 5 
                 8 
               
               
                   
                   
                   
                 week 
               
               
                 O 
                 HT-ID3-GDF15 
                 Subcutaneous 
                 Twice a 
                 3 
                 5 
                 8 
               
               
                   
                   
                   
                 week 
               
               
                 0 
                 HT-ID3-GDF15 
                 Subcutaneous 
                 Twice a 
                 10 
                 5 
                 8 
               
               
                   
                   
                   
                 week 
               
               
                 0 
                 HT-ID3-GDF15 
                 Subcutaneous 
                 Twice a 
                 30 
                 5 
                 8 
               
               
                   
                   
                   
                 week 
               
               
                   
               
            
           
         
       
     
     2.2.2. Body Weight Loss Test Result 
     The change in the body weight measured in Example 2.2.1 above was shown in  FIG.  9   ,  FIG.  10   a   ,  FIG.  10   b    and Table 10 (Body Weight (Group, % of initial). 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
             
            
               
                 Group 
                 Day 
                 0 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
                 14 
               
               
                   
               
               
                 Lean Vehicle 
                 Mean 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
                 99 
                 100 
                 102 
                 102 
                 101 
                 101 
                 102 
                 101 
               
               
                 Control 
                 S.E. 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 DIO Vehicle 
                 Mean 
                 100.0 
                 99.4 
                 99.1 
                 98.6 
                 98.6 
                 98.3 
                 98.8 
                 97.7 
                 95.8 
                 96.2 
                 97.3 
                 99.0 
                 99.8 
                 100.2 
                 100.5 
               
               
                 Control 
                 S.E. 
                 0.0 
                 0.4 
                 0.4 
                 0.5 
                 0.5 
                 0.6 
                 0.7 
                 1.1 
                 1.5 
                 1.3 
                 1.1 
                 1.1 
                 1.0 
                 1.0 
                 1.0 
               
               
                 DIO Semaglutide 
                 Mean 
                 100.0 
                 93.9 
                 92.6 
                 90.4 
                 88.4 
                 66.2 
                 64.1 
                 82.6 
                 61.9 
                 80.7 
                 80.3 
                 80.3 
                 79.1 
                 76.8 
                 76.7 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.0 
                 0.3 
                 0.6 
                 0.8 
                 1.1 
                 1.5 
                 1.5 
                 1.6 
                 1.6 
                 1.8 
                 2.2 
                 1.9 
                 2.0 
                 2.0 
                 2.0 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 100.0 
                 98.0 
                 96.5 
                 95.0 
                 93.9 
                 91.8 
                 90.8 
                 90.3 
                 89.2 
                 89.2 
                 90.1 
                 92.1 
                 92.5 
                 92.7 
                 93.3 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.0 
                 0.3 
                 0.4 
                 0.4 
                 0.4 
                 0.4 
                 0.4 
                 0.5 
                 0.6 
                 0.6 
                 0.7 
                 0.6 
                 0.6 
                 0.7 
                 0.7 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 100.0 
                 98.3 
                 96.7 
                 95.4 
                 94.6 
                 93.0 
                 91.7 
                 90.8 
                 93.1 
                 90.3 
                 90.6 
                 92.1 
                 91.2 
                 92.0 
                 92.2 
               
               
                 10 nmol/kg 
                 S.E. 
                 0.0 
                 0.3 
                 0.5 
                 0.5 
                 0.4 
                 0.5 
                 0.6 
                 0.7 
                 0.7 
                 0.7 
                 0.7 
                 0.8 
                 0.8 
                 0.9 
                 0.8 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 100.0 
                 98.4 
                 97.3 
                 96.2 
                 95.2 
                 94.0 
                 93.1 
                 92.3 
                 91.5 
                 91.2 
                 91.3 
                 93.2 
                 91.9 
                 92.1 
                 93.0 
               
               
                 30 nmol/kg 
                 S.E. 
                 0.0 
                 0.3 
                 0.5 
                 0.6 
                 0.6 
                 0.8 
                 0.9 
                 1.1 
                 1.2 
                 1.2 
                 1.6 
                 1.8 
                 1.7 
                 1.9 
                 2.1 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 100.0 
                 98.2 
                 96.9 
                 95.6 
                 94.4 
                 92.8 
                 91.9 
                 91.0 
                 90.0 
                 89.6 
                 89.1 
                 91.5 
                 91.5 
                 92.3 
                 92.5 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.0 
                 0.3 
                 0.3 
                 0.3 
                 0.5 
                 0.6 
                 0.6 
                 0.7 
                 0.8 
                 1.0 
                 1.4 
                 1.2 
                 1.6 
                 1.4 
                 1.4 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 100.0 
                 98.5 
                 97.3 
                 95.7 
                 94.6 
                 92.8 
                 91.5 
                 90.7 
                 89.8 
                 89.3 
                 88.7 
                 90.9 
                 89.8 
                 90.1 
                 91.0 
               
               
                 10 nmol/kg 
                 S.E. 
                 0.0 
                 0.3 
                 0.4 
                 0.5 
                 0.5 
                 0.6 
                 0.6 
                 0.7 
                 0.9 
                 0.9 
                 1.0 
                 1.0 
                 1.3 
                 1.4 
                 1.3 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 100.0 
                 98.2 
                 97.1 
                 95.8 
                 94.8 
                 92.8 
                 91.7 
                 90.8 
                 89.8 
                 89.8 
                 89.7 
                 92.0 
                 90.0 
                 90.4 
                 90.9 
               
               
                 30 nmol/kg 
                 S.E. 
                 0.0 
                 0.3 
                 0.3 
                 0.4 
                 0.5 
                 0.4 
                 0.4 
                 0.5 
                 0.6 
                 0.8 
                 1.1 
                 1.3 
                 1.3 
                 1.5 
                 1.5 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Group 
                 Day 
                 15 
                 16 
                 17 
                 18 
                 19 
                 20 
                 21 
                 22 
                 23 
                 24 
                 25 
                 26 
                 27 
                 28 
               
               
                   
               
               
                 Lean Vehicle 
                 Mean 
                 101 
                 102 
                 101 
                 102 
                 102 
                 102 
                 101 
                 101 
                 102 
                 102 
                 101 
                 101 
                 102 
                 101 
               
               
                 Control 
                 S.E. 
                 2 
                 1 
                 1 
                 1 
                 2 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
               
               
                 DIO Vehicle 
                 Mean 
                 100.8 
                 101.3 
                 102.0 
                 102.1 
                 102.4 
                 101.9 
                 101.5 
                 101.8 
                 102.2 
                 103.3 
                 103.3 
                 104.1 
                 104.3 
                 103.9 
               
               
                 Control 
                 S.E. 
                 1.0 
                 1.0 
                 1.1 
                 1.0 
                 1.1 
                 1.1 
                 1.7 
                 1.6 
                 1.7 
                 1.6 
                 1.5 
                 1.5 
                 1.7 
                 1.5 
               
               
                 DIO Semaglutide 
                 Mean 
                 78.1 
                 78.3 
                 77.2 
                 76.7 
                 77.1 
                 76.1 
                 76.8 
                 75.9 
                 75.6 
                 76.0 
                 76.3 
                 76.5 
                 76.2 
                 75.7 
               
               
                 3 nmol/kg 
                 S.E. 
                 1.8 
                 2.0 
                 2.3 
                 2.2 
                 2.1 
                 2.3 
                 2.3 
                 2.2 
                 2.3 
                 2.4 
                 2.8 
                 2.7 
                 2.4 
                 2.7 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 93.8 
                 94.3 
                 94.7 
                 95.1 
                 95.9 
                 95.7 
                 96.2 
                 96.6 
                 96.8 
                 97.8 
                 98.5 
                 99.3 
                 99.5 
                 99.8 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.9 
                 0.7 
                 0.8 
                 0.9 
                 0.8 
                 0.8 
                 1.0 
                 1.1 
                 1.2 
                 1.2 
                 1.2 
                 1.3 
                 1.3 
                 1.4 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 92.1 
                 92.7 
                 93.6 
                 94.2 
                 94.0 
                 93.7 
                 94.0 
                 93.8 
                 94.3 
                 95.4 
                 95.8 
                 95.8 
                 96.3 
                 96.6 
               
               
                 10 nmol/kg 
                 S.E. 
                 0.9 
                 0.9 
                 0.9 
                 0.9 
                 0.9 
                 1.0 
                 0.9 
                 1.0 
                 0.8 
                 0.9 
                 0.9 
                 0.8 
                 0.8 
                 0.7 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 92.1 
                 92.8 
                 93.4 
                 93.6 
                 92.9 
                 92.5 
                 93.2 
                 92.4 
                 92.6 
                 93.1 
                 93.9 
                 93.6 
                 93.2 
                 93.4 
               
               
                 30 nmol/kg 
                 S.E. 
                 2.2 
                 2.4 
                 2.4 
                 2.4 
                 2.5 
                 2.6 
                 2.6 
                 2.8 
                 2.7 
                 2.7 
                 2.6 
                 2.8 
                 2.7 
                 2.6 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 92.6 
                 93.3 
                 94.0 
                 94.4 
                 95.0 
                 95.2 
                 95.8 
                 96.4 
                 97.1 
                 97.4 
                 97.6 
                 98.5 
                 98.0 
                 98.6 
               
               
                 3 nmol/kg 
                 S.E. 
                 1.3 
                 1.3 
                 1.2 
                 1.2 
                 1.2 
                 1.2 
                 1.3 
                 1.3 
                 1.5 
                 1.4 
                 1.3 
                 1.5 
                 1.7 
                 1.7 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 90.0 
                 91.2 
                 91.8 
                 92.3 
                 91.9 
                 91.2 
                 92.1 
                 92.1 
                 92.5 
                 93.2 
                 93.7 
                 94.2 
                 94.4 
                 94.1 
               
               
                 10 nmol/kg 
                 S.E. 
                 1.5 
                 1.5 
                 1.5 
                 1.5 
                 1.7 
                 1.7 
                 1.7 
                 2.1 
                 2.0 
                 2.0 
                 1.9 
                 2.3 
                 2.3 
                 2.2 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 89.3 
                 89.6 
                 90.4 
                 91.0 
                 90.0 
                 89.6 
                 90.4 
                 90.0 
                 90.0 
                 91.0 
                 91.5 
                 91.5 
                 91.2 
                 91.4 
               
               
                 30 nmol/kg 
                 S.E. 
                 1.4 
                 1.6 
                 1.5 
                 1.7 
                 1.5 
                 1.5 
                 1.4 
                 1.3 
                 1.4 
                 1.5 
                 1.5 
                 1.5 
                 1.7 
                 1.6 
               
               
                   
               
            
           
         
       
     
       FIG.  9    and Table 10 shows the change in the body weight in case of repeated administration of the fusion proteins HT-ID2-GDF15 and HT-ID3-GDF15, respectively (Table 9), compared to the negative control group (vehicle administration group) and positive control group (Semaglutide daily administration group). In addition,  FIG.  10   a    is a graph showing the results at Day 7 and Day 14 by extracting them from the result of  FIG.  9    above and  FIG.  10   b    is a graph showing the results at Day 21 and Day 28 by extracting them from the result of  FIG.  9   . 
     As shown in the above result, it could be confirmed that there was little change in the body weight in case of the negative control group (vehicle administration group), while the body weight loss effect was continuously shown from Day 1 after administration. In addition, it could be confirmed that the body weight loss effect was shown immediately after single administration at Day 0 in case of the fusion polypeptide in which GDF15 was fused with IgD Hinge, and the body weight loss effect was continuously shown without being reduced, and the body weight loss effect was concentration-dependent. 
     2.2.3. Diet Intake Test Result 
     The change in the feed intake measured in Example 2.2.1 above was shown in Table 11 and  FIG.  11    (cumulative intake up to Day 7, Day 14, Day 21 and Day 28), respectively. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
             
            
               
                 Group 
                 Day 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
                 14 
               
               
                   
               
               
                 Lean Vehicle 
                 Mean 
                 4.4 
                 3.9 
                 4.1 
                 4.5 
                 4.4 
                 3.9 
                 4.3 
                 4.1 
                 4.1 
                 4.6 
                 4.5 
                 4.4 
                 4.2 
                 4.3 
               
               
                 Control 
                 S.E. 
                 0.2 
                 0.1 
                 0.1 
                 0.1 
                 0.3 
                 0.1 
                 0.2 
                 0.1 
                 0.3 
                 0.2 
                 0.1 
                 0.2 
                 0.2 
                 0.2 
               
               
                 DIO Vehicle 
                 Mean 
                 2.9 
                 2.6 
                 2.7 
                 3.6 
                 3.3 
                 2.5 
                 3.0 
                 2.7 
                 1.7 
                 2.2 
                 3.0 
                 4.0 
                 3.1 
                 3.4 
               
               
                 Control 
                 S.E. 
                 0.2 
                 0.5 
                 0.3 
                 0.8 
                 0.4 
                 0.2 
                 0.1 
                 0.1 
                 0.4 
                 0.2 
                 0.1 
                 0.2 
                 0.1 
                 0.2 
               
               
                 DIO Semaglutide 
                 Mean 
                 2.9 
                 0.7 
                 1.3 
                 1.4 
                 1.7 
                 1.5 
                 1.6 
                 1.9 
                 2.0 
                 2.3 
                 2.2 
                 3.2 
                 2.2 
                 2.5 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.1 
                 0.1 
                 0.2 
                 0.1 
                 0.1 
                 0.2 
                 0.2 
                 0.4 
                 0.5 
                 0.3 
                 0.3 
                 0.3 
                 0.2 
                 0.2 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 3.0 
                 1.5 
                 2.1 
                 2.1 
                 2.4 
                 1.7 
                 2.3 
                 2.3 
                 2.0 
                 2.8 
                 3.3 
                 4.2 
                 3.7 
                 3.4 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.4 
                 0.1 
                 0.2 
                 0.2 
                 0.1 
                 0.2 
                 0.1 
                 0.2 
                 0.7 
                 0.1 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 2.6 
                 1.5 
                 1.8 
                 2.0 
                 2.0 
                 1.8 
                 2.1 
                 2.1 
                 1.9 
                 2.5 
                 3.1 
                 3.7 
                 2.4 
                 3.0 
               
               
                 10 nmol/kg 
                 S.E. 
                 0.2 
                 0.1 
                 0.2 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 3.0 
                 1.7 
                 2.3 
                 2.3 
                 2.3 
                 2.1 
                 2.4 
                 2.3 
                 2.2 
                 2.6 
                 3.0 
                 3.9 
                 2.3 
                 3.2 
               
               
                 30 nmol/kg 
                 S.E. 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
                 0.3 
                 0.2 
                 0.3 
                 0.2 
                 0.2 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 2.9 
                 1.5 
                 1.9 
                 1.9 
                 2.0 
                 1.6 
                 2.1 
                 2.1 
                 1.9 
                 2.4 
                 2.5 
                 3.9 
                 2.9 
                 3.2 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.2 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.2 
                 0.3 
                 0.2 
                 0.1 
                 0.1 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 2.9 
                 1.7 
                 2.3 
                 2.2 
                 2.1 
                 1.9 
                 2.2 
                 2.1 
                 2.1 
                 2.4 
                 3.0 
                 4.1 
                 2.5 
                 3.3 
               
               
                 10 nmol/kg 
                 S.E. 
                 0.2 
                 0.2 
                 0.2 
                 0.1 
                 0.2 
                 0.1 
                 0.2 
                 0.2 
                 0.1 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 2.5 
                 1.6 
                 2.1 
                 2.2 
                 2.3 
                 1.8 
                 2.2 
                 2.1 
                 2.0 
                 2.5 
                 2.9 
                 4.0 
                 2.1 
                 3.0 
               
               
                 30 nmol/kg 
                 S.E. 
                 0.2 
                 0.1 
                 0.1 
                 0.2 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.2 
                 0.2 
                 0.1 
                 0.1 
                 0.2 
               
               
                   
               
               
                 Group 
                 Day 
                 15 
                 16 
                 17 
                 18 
                 19 
                 20 
                 21 
                 22 
                 23 
                 24 
                 25 
                 26 
                 27 
                 28 
               
               
                   
               
               
                 Lean Vehicle 
                 Mean 
                 4.3 
                 4.1 
                 4.2 
                 4.1 
                 4.9 
                 3.8 
                 4.8 
                 4.2 
                 3.9 
                 4.2 
                 4.2 
                 3.8 
                 3.7 
                 4.3 
               
               
                 Control 
                 S.E. 
                 0.2 
                 0.2 
                 0.1 
                 0.3 
                 0.2 
                 0.2 
                 0.1 
                 0.3 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
                 0.2 
                 0.3 
               
               
                 DIO Vehicle 
                 Mean 
                 3.2 
                 3.2 
                 3.1 
                 3.1 
                 3.1 
                 2.7 
                 3.2 
                 2.7 
                 2.8 
                 3.1 
                 3.4 
                 3.5 
                 2.9 
                 3.2 
               
               
                 Control 
                 S.E. 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.4 
                 0.1 
                 0.2 
                 0.2 
                 0.5 
                 0.1 
                 0.1 
               
               
                 DIO Semaglutide 
                 Mean 
                 2.5 
                 2.2 
                 2.4 
                 2.2 
                 2.6 
                 2.6 
                 2.5 
                 2.9 
                 2.0 
                 2.4 
                 2.9 
                 2.4 
                 2.4 
                 2.7 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.3 
                 0.2 
                 0.1 
                 0.2 
                 0.1 
                 0.3 
                 0.1 
                 0.4 
                 0.2 
                 0.1 
                 0.3 
                 0.2 
                 0.2 
                 0.3 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 3.2 
                 3.4 
                 3.4 
                 3.4 
                 3.7 
                 3.3 
                 3.2 
                 3.4 
                 3.1 
                 3.3 
                 3.4 
                 3.3 
                 3.1 
                 3.4 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.2 
                 0.5 
                 0.4 
                 0.2 
                 0.4 
                 0.5 
                 0.1 
                 0.3 
                 0.1 
                 0.2 
                 0.1 
                 0.2 
                 0.1 
                 0.1 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 3.0 
                 2.4 
                 3.0 
                 3.2 
                 3.3 
                 2.4 
                 2.9 
                 3.1 
                 2.3 
                 3.1 
                 3.3 
                 3.1 
                 2.5 
                 3.0 
               
               
                 10 nmol/kg 
                 S.E 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
               
               
                 HT-ID2-GDF15 
                 Mean 
                 3.3 
                 2.4 
                 3.1 
                 3.2 
                 3.1 
                 2.1 
                 3.2 
                 3.1 
                 2.1 
                 3.0 
                 3.1 
                 3.3 
                 2.1 
                 2.9 
               
               
                 30 nmol/kg 
                 S.E. 
                 0.3 
                 0.2 
                 0.2 
                 0.2 
                 0.1 
                 0.1 
                 0.2 
                 0.1 
                 0.2 
                 0.2 
                 0.1 
                 0.1 
                 0.2 
                 0.2 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 3.1 
                 2.7 
                 3.0 
                 3.1 
                 3.3 
                 2.7 
                 3.0 
                 3.1 
                 2.9 
                 3.1 
                 2.9 
                 3.0 
                 2.8 
                 2.7 
               
               
                 3 nmol/kg 
                 S.E. 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 3.2 
                 2.2 
                 3.3 
                 3.3 
                 3.4 
                 2.4 
                 3.1 
                 3.2 
                 2.7 
                 3.1 
                 3.3 
                 3.3 
                 2.7 
                 3.2 
               
               
                 10 nmol/kg 
                 S.E. 
                 0.2 
                 0.3 
                 0.2 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.2 
                 0.1 
                 0.1 
                 0.1 
                 0.2 
                 0.2 
               
               
                 HT-ID3-GDF15 
                 Mean 
                 3.1 
                 1.9 
                 3.0 
                 3.2 
                 3.3 
                 1.9 
                 3.1 
                 3.4 
                 2.3 
                 3.3 
                 3.2 
                 3.2 
                 2.2 
                 3.2 
               
               
                 30 nmol/kg 
                 S.E. 
                 0.1 
                 0.0 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.1 
                 0.2 
                 0.2 
                 0.2 
                 0.1 
                 0.1 
                 0.1 
                 0.2 
               
               
                   
               
            
           
         
       
     
     As shown in the above result, the administration group of the fusion polypeptide in which GDF15 was fused with IgD Hinge showed the feed intake reduction effect throughout the test period depending on the fusion polypeptide, compared to the negative control group (vehicle administration group) administration group, and showed a concentration-dependent tendency. 
     Example 3. Pharmacokinetic Test of Fusion Polypeptide 
     3.1. Test Group and Control Group Serum Preparation 
     For evaluation of pharmacokinetic characteristics when each fusion polypeptide was subcutaneously administered to rats, the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 were subcutaneously administered into SD rats (Koatech, male, 7-week-old, about 250 g; n=3 each; test group) in an amount of 2 mg/kg, respectively, and about 200 μl of blood was collected through the caudal vein at a predetermined time. The blood collection time was performed before administration, and 1, 2, 4, 8, 24, 48, 72, 96, 168, 240 and 336 hours after administration. As the control group for comparison of pharmacokinetic characteristics, GDF15 (R&amp;D Systems) was subcutaneously administered in an amount of 2 mg/kg by the same method to prepare a GDF15 administration group. 
     After administering into SD Rats as above, blood collected by time-point was centrifuged to obtain serum, and ELISA was performed using Human GDF15 Immunoassay (SGD150, R&amp;D Systems), and the concentration in the serum was measured depending on the time of each polypeptide. Using this data, values of parameters including AUC (area under the curve) were obtained using a software for PK analysis (WinNonlin (Certara L. P.), etc.). 
     3.2 Pharmacokinetic Test Result 
     The obtained pharmacokinetic parameters of the fusion polypeptide were shown in Table 12, and the change in the concentration of the fusion polypeptide with time was shown in  FIG.  12   . 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                   
                   
                 Group 2 
                 Group 3 
                 Group 4 
               
               
                   
                 Group 1 
                 HT-ID1- 
                 HT-ID2- 
                 HT-ID3- 
               
               
                 PK parameter 
                 rhGDF15 
                 GDF15 
                 GDF15 
                 GDF15 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 C max  (ug/mL) 
                 0.443 
                 2.09 
                 0.945 
                 1.11 
               
               
                 T max  (hr) 
                 1 
                 8 
                 24 
                 24 
               
               
                 AUC last (ug*hr/mL) 
                 4.86 
                 81.2 
                 53.5 
                 76.2 
               
               
                 AUC inf (ug*hr/mL) 
                 4.88 
                 81.3 
                 53.5 
                 76.4 
               
               
                 t 1/2  (hr) 
                 19.0 
                 24.7 
                 22.3 
                 26.2 
               
               
                 AUC extp  (%) 
                 0.463 
                 0.0700 
                 0.100 
                 0.287 
               
               
                   
               
               
                 (C max : maximum concentration in blood, T max : reaching time to maximum concentration in blood, AUC inf : area under a blood concentration-time curve by extrapolating from the last measurable blood collecting time to infinity, AUC last : area under a blood concentration-time curve up to the last measurable blood collecting time, T 1/2 : loss half-life, AUC Extp (%): [(AUC inf  − AUC last )/AUC inf ]*100) 
               
            
           
         
       
     
     As shown in the above result, it could be confirmed that the half-life was increased in case of the fusion polypeptide fused with IgD Hinge, compared to GDF15 (half-life: 19 hours), and in particular, in case of AUC last , it was increased 16.7 times at maximum compared to GDF15. 
     From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative and not restrictive in all respects. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalent concepts are included in the scope of the present invention, rather than the above detailed description.