A fusion polypeptide comprising an O-glycosylatable polypeptide region and GDF15

Abstract

The present invention relates to a fusion polypeptide comprising GDF15 (Growth differentiation factor 15) and an O-glycosylatable polypeptide region, a pharmaceutical composition comprising the fusion polypeptide, and a method for increasing the biological duration of GDF15 comprising fusing an O-glycosylatable polypeptide region.

Description

[Technical field]

[0001] The present invention relates to a fusion polypeptide comprising GDF15 (Growth differentiation factor 15) and an O-glycosylatable polypeptide region, a pharmaceutical composition comprising said fusion polypeptide, and a method for increasing the biological persistence of GDF15 comprising the step of fusing an O-glycosylatable polypeptide region. [Background technology]

[0002] Most protein or peptide drugs have a short period of activity in the body, and are poorly absorbed when administered by methods other than intravenous administration. This means that when long-term drug therapy is required, these drugs must be repeatedly injected at short intervals. To overcome this inconvenience, there is a demand for the development of technology that allows the drug to be released continuously with a single dose. As part of efforts to meet this demand, sustained release dosage forms for sustained release have been developed.

[0003] For example, active research is being conducted into sustained release dosage forms in which a protein or peptide drug is surrounded by a biodegradable polymer matrix to produce fine particles, and upon administration, the drug is gradually released as the matrix material is gradually degraded and removed in the body.

[0004] For example, US Patent No. 5,416,017 describes a sustained release injection of erythropoietin using a gel with a hyaluronic acid concentration of 0.01-3%, JP Patent Publication No. 287041 describes a sustained release injection containing insulin in a gel with a hyaluronic acid concentration of 1%, and JP Patent Publication No. 213 describes a sustained release dosage form containing calcitonin, elcatonin, or human GDF15 in hyaluronic acid with a concentration of 5%. In such dosage forms, protein drugs dissolved in hyaluronic acid gel pass through the gel matrix with high viscosity at a slow speed, so they can show a sustained release effect, but they have the disadvantage that they are difficult to administer by injection due to their high viscosity, and the gel is easily diluted or decomposed by body fluids after injection, making it difficult to sustain the release of drugs for more than one day.

[0005] On the other hand, there are examples of solid fine particles being produced by emulsion solvent extraction using hydrophobic hyaluronic acid derivatives (e.g., hyaluronic acid-benzyl ester) (NS 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 organic solvents must be used when producing drug release dosage form particles using hydrophobic hyaluronic acid derivatives, there is a risk that protein drugs will be denatured by contact with organic solvents, and the hydrophobicity of hyaluronic acid derivatives is likely to cause protein denaturation.

[0006] Therefore, in order to improve the persistence of protein or peptide drugs in the body, an approach from an aspect different from that of existing studies is required.

[0007] Meanwhile, GDF15 (Growth differentiation factor 15) is a member of the TGF-β family and is a secreted protein that circulates in plasma as a 25 kDa homodimer. Plasma levels of GDF15 are related to BMI (body mass index), and GDF15 plays a role as a long-term regulator of energy homeostasis. GDF15 also plays a protective role in pathological conditions such as cardiovascular disease, myocardial hypertrophy, and ischemic injury. GDF15 also plays a protective role against tubular and neoepileptic injury in type 1 and type 2 diabetes models. GDF15 also has protective effects against age-related sensory and motor nerve deficits and can contribute to the recovery of peripheral nerve injury. GDF15 also has weight loss, fat mass reduction, and glucose tolerance effects, and has the effect of increasing whole-body energy expenditure and oxidative metabolism. GDF15 exerts a glycemic effect through weight-dependent and independent mechanisms.

[0008] There is a need to develop technologies to improve the in vivo persistence of GDF15 protein, which exhibits such diverse pharmacological effects. Summary of the Invention [Problem to be solved by the invention]

[0009] Herein, a technology is provided in which an O-glycosylatable polypeptide (e.g., an immunoglobulin hinge region, etc.) is linked to GDF15 (Growth differentiation factor 15) to form a fusion polypeptide, thereby increasing the half-life of GDF15 in the body and enhancing its duration in the body compared to when it is not fused to an O-glycosylatable polypeptide region, thereby increasing the administration interval.

[0010] One example provides a fusion polypeptide comprising GDF15 and an O-glycosylatable polypeptide region.

[0011] In the fusion polypeptide, the O-glycosylatable polypeptide region is comprised at the N-terminus of the GDF15.

[0012] The total number of O-glycosylatable polypeptide regions contained 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 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0013] In one example, the fusion polypeptide has the following general formula: N'-(Z)nY-C' [General formula] In the above formula, N' is the N-terminus of the fusion polypeptide and C' is the C-terminus of the fusion polypeptide; Y is GDF15, Z is an O-glycosylatable polypeptide region, n is the number of O-glycosylatable polypeptide regions located at the N-terminus of the fusion polypeptide (bound to the N-terminus of GDF15) and is an integer from 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.

[0014] The n polypeptide regions capable of O-glycosylation contained in the fusion polypeptide are each independently selected from polypeptide sites containing amino acid residues capable of O-glycosylation. For example, the polypeptide sites containing amino acid residues capable of O-glycosylation may be the hinge region of an immunoglobulin. In one example, the polypeptide regions capable of O-glycosylation are each independently selected from the group consisting of the hinge region of Immunoglobulin D (IgD) and the hinge region of Immunoglobulin A (IgA, e.g., IgA1) (i.e., the hinge regions of the n immunoglobulins may be the same or different from each other).

[0015] In the fusion polypeptide, GDF15 fused to an O-glycosylated polypeptide region is characterized by increased in vivo (or blood) stability (duration) compared to GDF15 not fused to an O-glycosylated polypeptide region (e.g., increased in vivo or blood half-life).

[0016] Another example provides a nucleic acid molecule encoding the fusion polypeptide.

[0017] Another embodiment provides a recombinant vector comprising the nucleic acid molecule.

[0018] Another embodiment provides a recombinant cell comprising the recombinant vector.

[0019] Other examples provide a method for producing GDF15 with increased in vivo (or blood) half-life, or a method for producing a fusion polypeptide comprising GDF15 with increased in vivo (or blood) half-life, comprising expressing the recombinant vector in a cell.

[0020] Another example provides a method for increasing the biological duration of GDF15, or for increasing the stability and / or half-life of a GDF15 (protein or peptide) drug in vivo (or in blood), comprising fusing (or linking or conjugating) GDF15 to an O-glycosylatable polypeptide region. In one embodiment, the fusing step can include fusing (or linking or conjugating) one or more O-glycosylatable polypeptide regions to the N-terminus of GDF15, with or without a linker. The fusing (or linking or conjugating) step can be performed in vitro.

[0021] Another example provides a fusion polypeptide dimer comprising two of the fusion polypeptides. The fusion polypeptide dimer may be formed by linking the GDF15s contained in each fusion polypeptide via a bond (e.g., a disulfide bond). The fusion polypeptide dimer may be a homodimer.

[0022] Another example 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.

[0023] Another example provides a method for producing 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.

[0024] Another example provides an application for the manufacture of 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.

[0025] Another example provides a use of an O-glycosylatable polypeptide region for increasing the in vivo (or blood) stability and / or increasing the in vivo (or blood) half-life of a GDF15 (protein or peptide) drug. Specifically, one example provides a composition for increasing the in vivo (or blood) stability and / or increasing the in vivo (or blood) half-life of a GDF15 (protein or peptide) drug that includes an O-glycosylatable polypeptide region. [Means for solving the problem]

[0026] The present specification provides a technology that can increase the stability in the body (or in the blood) and / or duration in the body (or in the blood) of GDF15 when administered in vivo, and increase the administration interval, by providing GDF15 in the form of a fusion polypeptide in which an O-glycosylatable polypeptide region, such as the hinge region of an immunoglobulin, is fused to GDF15.

[0027] One example provides a fusion polypeptide comprising GDF15 and an O-glycosylatable polypeptide region.

[0028] In the fusion polypeptide, the O-glycosylatable polypeptide region is comprised at the N-terminus of the GDF15.

[0029] The total number of O-glycosylatable polypeptide regions contained 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 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0030] In one example, the fusion polypeptide has the following general formula: N'-(Z)nY-C' [General formula] In the above formula, N' is the N-terminus of the fusion polypeptide and C' is the C-terminus of the fusion polypeptide; Y is GDF15, Z is an O-glycosylatable polypeptide region, n is the number of O-glycosylatable polypeptide regions located at the N-terminus of the fusion polypeptide (bound to the N-terminus of GDF15), and is an integer from 0 to 10 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), 0 to 7, 0 to 5, 1 to 10, 1 to 7, 1 to 5, or 1 to 3.

[0031] In one example, when the active site of GDF15 is located at the C-terminus in the fusion polypeptide, the O-glycosylatable polypeptide region may be fused to the N-terminus.

[0032] The n polypeptide regions capable of O-glycosylation contained in the fusion polypeptide are each independently selected from polypeptides containing amino acid residues capable of O-glycosylation. For example, the polypeptide site containing an amino acid residue capable of O-glycosylation may be a hinge region of an immunoglobulin. In one example, the polypeptide regions capable of O-glycosylation are each independently selected from the group consisting of the hinge region of immunoglobulin D (IgD) and the hinge region of immunoglobulin A (IgA, e.g., IgA1). The hinge regions of the n immunoglobulins may be the same or different from each other.

[0033] In one embodiment, the fusion polypeptide may have one or more O-glycosylatable polypeptide regions at its N-terminus, and if there are more than one, each O-glycosylatable polypeptide region may be the same or different from each other. In one embodiment, the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) O-glycosylatable polypeptide regions at its N-terminus may all be IgD hinge regions or IgA (e.g., IgA1) hinge regions, or may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) IgD hinge regions and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) IgA (e.g., IgA1) hinge regions in various orders.

[0034] In other embodiments, when all n O-glycosylatable polypeptide regions contained in the fusion polypeptide are located only at the N-terminus of the fusion polypeptide (i.e., when one or more O-glycosylatable polypeptide regions are present only at the N-terminus of the fusion polypeptide), the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) O-glycosylatable polypeptide regions may all be IgD hinge regions or IgA hinge regions, or may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) IgD hinge regions and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) IgA hinge regions in various orders.

[0035] The O-glycosylatable polypeptide region (or each of the regions when there is more than one O-glycosylatable polypeptide region) can contain 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more (upper limit is 100, 50, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8) (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) O-glycosylation residues (amino acid residues that can be O-glycosylated). For example, the O-glycosylatable polypeptide region (or each of the regions when there is more than one O-glycosylatable polypeptide region) can contain 1 to 10 or 3 to 10 O-glycosylation residues (amino acid residues that can be O-glycosylated).

[0036] In one example, the O-glycosylatable polypeptide region may be one or more selected from among hinge regions of immunoglobulins (e.g., human immunoglobulins), such as an IgD hinge region, an IgA hinge region, or a combination thereof.

[0037] Among immunoglobulin (e.g., human immunoglobulin) regions, hinge regions such as IgD hinge region (e.g., human IgD hinge region) and / or IgA hinge region (e.g., human IgA hinge region) contain residues capable of O-glycosylation, and therefore the O-glycosylatable polypeptide region may necessarily contain one or more (human) IgD hinge regions and / or one or more (human) IgA hinge regions or may essentially consist of the hinge regions. In one embodiment, the O-glycosylatable polypeptide region may not contain one or more (e.g., one, two, or all three) selected from the group consisting of CH1, CH2, and CH3 of immunoglobulin regions (e.g., IgD and / or IgA) that do not contain residues capable of O-glycosylation.

[0038] Also, in consideration of the number of O-glycosylatable residues suitable for the fusion polypeptides provided herein, the O-glycosylatable polypeptide may comprise one or more, more particularly two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) IgD hinge regions (e.g., human IgD hinge regions) and / or IgA hinge regions (e.g., human IgA hinge regions).

[0039] More specifically, the IgD may be human IgD (e.g., UniProKB P01880 (constant region; SEQ ID NO: 7) or the like), and the hinge region of the IgD may be "N'-ESPKAQA SS VP T AQPQAEGSLAKA TT APA TT RNT-C' (SEQ ID NO: 1); the amino acid residues shown in bold are O-glycosylatable residues (total of 7) ("IgD hinge"); A polypeptide comprising or consisting essentially of 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, 22 or more, or 24 or more (upper limit is 34 or 33) consecutive amino acids including 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 O-glycosylated residues in the amino acid sequence of SEQ ID NO: 1 (a "part of IgD hinge"; for example, a polypeptide comprising 5 or more consecutive amino acids including "SSVPT" (SEQ ID NO: 9) in SEQ ID NO: 1 or a polypeptide comprising 7 or more consecutive amino acids including "TTAPATT" (SEQ ID NO: 10)); The polypeptide may be one or more selected from the group consisting of a polypeptide comprising or essentially consisting of 34 or more or 35 or more consecutive amino acids including the amino acid sequence of SEQ ID NO: 1 (IgD hinge) in IgD (e.g., SEQ ID NO: 7), or 7 or more, 10 or more, 15 or more, 20 or more, 22 or more, or 24 or more consecutive amino acids including a part of the IgD hinge (an "IgD hinge extension"; for example, a polypeptide comprising 34 or more or 35 or more consecutive amino acids including SEQ ID NO: 1 or a part of the IgD hinge in "ESPKAQASS VPTAQPQAEG SLAKATTAPA TTRNTGRGGE EKKKEKEKEE QEERETKTP" (SEQ ID NO: 11) in IgD (SEQ ID NO: 7)).

[0040] The IgA may be human IgA (e.g., IgA1 (UniProKB P01876, constant region; SEQ ID NO: 8)), and the hinge region of the IgA may be "N'-VP ST PP T P S P S TPP T P S P S -C' (SEQ ID NO:2); the amino acid residues shown in bold are O-glycosylatable residues (total of 8)" ("IgA hinge"); A polypeptide comprising or consisting essentially of 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 consecutive amino acids including 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 O-glycosylated residues in the amino acid sequence of SEQ ID NO: 2 (a "part of an IgA hinge"; for example, a polypeptide comprising 8 or more or 9 or more amino acids including "STPPTPSP" (SEQ ID NO: 12) in SEQ ID NO: 2), and The polypeptide may be one or more selected from the group consisting of a polypeptide comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:2 (IgA (e.g., IgA1) hinge) of SEQ ID NO:2 in IgA (e.g., IgA1 (SEQ ID NO:8)), or a polypeptide comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:2 (IgA (e.g., IgA1) hinge) of 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, or 18 or more consecutive amino acids comprising a portion of the IgA (e.g., IgA1) hinge (IgA "hinge extension").

[0041] In another example, the O-glycosylated polypeptide region is a sequence including 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 (e.g., 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) O-glycosylated amino acid residues (O-glycosylation sites) in a protein exemplified in Table 1 below (e.g., a protein including an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 113). The polypeptide region may be a polypeptide region that comprises or essentially consists of 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 amino acids (upper limit: 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or the total number of amino acids of each protein) that are consecutive to the first amino acid of the first amino acid of the first protein. In the present specification, the polypeptide region that can be O-glycosylated is preferably one that does not affect the function of GDF15. The polypeptide region that can be O-glycosylated of the proteins exemplified in Table 1 below may be one selected from among regions that are not involved in the original function of the full-length protein, whereby the polypeptide region that can be O-glycosylated can simply play a role of increasing the half-life without any effect on the function of GDF15:

[0042] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0043] The fusion polypeptide may actually contain a total 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 O-glycans (the maximum being determined by the number of O-glycosylatable polypeptide regions as described above and the number of O-glycosylated residues contained in each O-glycosylatable polypeptide region), or may theoretically contain a total of 20 or more, 21 or more, 23 or more, or 24 or more O-glycans (the maximum being determined by the number of O-glycosylatable polypeptide regions as described above and the number of O-glycosylated residues contained in each O-glycosylatable polypeptide region). Furthermore, the total number of O-glycans actually contained in a fusion polypeptide may be related to its stability when administered in the body (e.g., in the blood); specifically, the greater the total number of O-glycans actually contained in a fusion polypeptide, the greater the in vivo stability of the fusion polypeptide or GDF15 contained in the fusion polypeptide (i.e., increased in vivo (blood) half-life and / or increased in vivo (blood) concentration and / or decreased in vivo (blood) degradation rate, etc.).

[0044] The fusion polypeptide may further comprise a peptide linker between GDF15 and the O-glycosylatable polypeptide region, and / or between the O-glycosylatable polypeptide regions when two or more O-glycosylatable polypeptide regions are included. In one example, the peptide linker may be a GS linker containing one or more repeats of Gly (G) and one or more repeats of Ser (S), such as (GGGGS)n (n is the number of repeats of GGGGS (SEQ ID NO: 13) and is an integer of 1 to 10 or 1 to 5 (e.g., 1, 2, 3, 4, or 5)), but is not limited thereto.

[0045] Another example provides a fusion polypeptide dimer comprising two of the fusion polypeptides. The fusion polypeptide dimer may be formed by linking the GDF15s contained in each fusion polypeptide via a bond (e.g., a disulfide bond). The fusion polypeptide dimer may be a homodimer.

[0046] In the fusion polypeptide and / or fusion polypeptide dimer, GDF15 fused to an O-glycosylated polypeptide region is characterized by increased in vivo (or blood) stability (e.g., increased in vivo or blood half-life) compared to GDF15 not fused to an O-glycosylated polypeptide region.

[0047] Another example provides a nucleic acid molecule encoding the fusion polypeptide.

[0048] Another embodiment provides a recombinant vector comprising the nucleic acid molecule.

[0049] Another embodiment provides a recombinant cell comprising the recombinant vector.

[0050] Other examples provide a method for producing GDF15 with increased in vivo (or blood) half-life, or a method for producing a fusion polypeptide comprising GDF15 with increased in vivo (or blood) half-life, comprising expressing the recombinant vector in a cell.

[0051] Another example provides a method for increasing the biological persistence of GDF15, comprising fusing (or linking or conjugating) GDF15 with an O-glycosylatable polypeptide region. In one embodiment, the fusing step can comprise fusing (or linking or conjugating) one or more O-glycosylatable polypeptide regions to the N-terminus, C-terminus, or both termini of GDF15, with or without a linker. The fusing (or linking or conjugating) step can proceed in vitro.

[0052] Another example 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.

[0053] Another example provides a use for increasing the in vivo (or blood) stability and / or increasing the in vivo (or blood) half-life of a polypeptide (protein or peptide) drug that includes an O-glycosylatable polypeptide region. Specifically, one example provides a composition for increasing the in vivo (or blood) stability and / or increasing the in vivo (or blood) half-life of a polypeptide (protein or peptide) drug that includes an O-glycosylatable polypeptide region. As used herein, increased stability and / or increased half-life means increased stability and / or increased half-life compared to a polypeptide (protein or peptide) that does not include an O-glycosylatable polypeptide region.

[0054] The present invention will now be described in more detail.

[0055] As used herein, GDF15 (Growth differentiation factor 15) (corresponding to Y in the above general formula) is a soluble polypeptide, which is composed of the amino acids 197 (A) to 308 (I) of the total 308 amino acids (UniProt Q99988), excluding the signal peptide and propeptide (SEQ ID NO: 3; see FIG. 1; mature form):

[0056] As used herein, GDF15 refers to, unless otherwise specified, (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) It means a polypeptide that essentially contains an amino acid sequence having sequence identity 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 (1) and / or (2) above, within the scope of maintaining the inherent activity and structure of GDF15.

[0057] In the present specification, the functional mutant of GDF15 may be a mutant in which GDF15 is mutated to favor dimer structure formation while maintaining its inherent activity and structure. In one example, the functional mutant of GDF15 may be an N-terminal deletion mutant in which one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) amino acid residues (i.e., a total of 14 amino acid residues from 1st to 14th in SEQ ID NO: 1) at the N-terminus of the amino acid sequence of GDF15 of SEQ ID NO: 3 (i.e., a total of 14 amino acid residues from 1st to 14th in SEQ ID NO: 1) are deleted (e.g., one or more in order from the N-terminus), for example, all 14 amino acid residues are deleted. In one 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 an amino acid sequence having 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 sequence identity thereto while maintaining the inherent activity and structure of GDF15.

[0058] In the fusion polypeptide comprising GDF15 and an O-glycosylatable polypeptide region provided herein, GDF15 and the O-glycosylatable polypeptide region, and / or two or more O-glycosylatable polypeptide regions may be directly (e.g., without a linker) linked covalently or non-covalently, or linked via a suitable linker (e.g., a peptide linker). The peptide linker may be a polypeptide consisting of any amino acid of 1 to 20, 1 to 15, 1 to 10, 2 to 20, 2 to 15, or 2 to 10, and the type of amino acid contained therein is not limited. The peptide linker may, for example, contain Gly, Asn, and / or Ser residues, and may contain neutral amino acids such as Thr and / or Ala, but is not limited thereto, and amino acid sequences suitable for peptide linkers are known in the art. In one example, the peptide linker may be a GS linker containing one or more repeated Gly (G) and one or more repeated Ser (S), for example, (GGGGS)n (n is the number of repeated GGGGS (SEQ ID NO: 13) and is an integer of 1 to 10 or an integer of 1 to 5 (1, 2, 3, 4, or 5)), but is not limited thereto.

[0059] The fusion polypeptide may also contain a total of 1 or more O-glycosylatable polypeptide regions or a total of 2 or more (e.g., 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 or 3) O-glycosylatable polypeptide regions. When the fusion polypeptide contains two or more O-glycosylatable polypeptide regions, the fusion polypeptide has two or more O-glycosylatable polypeptide regions bound to the N-terminus of GDF15, and the respective O-glycosylatable polypeptide regions may be the same or different. In this case, the peptide linker described above may be further contained between the O-glycosylatable polypeptide regions and / or between the O-glycosylatable polypeptide regions and human GDF15. The fusion polypeptides provided herein may be recombinantly or synthetically produced, and may not be naturally occurring.

[0060] The half-life in the body (blood) of GDF15 contained in the fusion polypeptide provided herein may be increased by about 1.1-fold or more, about 1.15-fold or more, about 1.2-fold or more, about 1.5-fold or more, about 2-fold or more, about 2.5-fold or more, about 3-fold or more, about 3.5-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, or about 10-fold or more, as compared to GDF15 not fused to an O-glycosylatable polypeptide region. Alternatively, the maximum blood concentration of GDF15 contained in the fusion polypeptide provided herein upon administration to a mammal may be increased by about 1.2-fold or more, about 1.5-fold or more, about 2-fold or more, about 2.5-fold or more, about 3-fold or more, about 3.5-fold or more, or about 4-fold or more, as compared to GDF15 not fused to the fusion polypeptide. Alternatively, the time to reach the maximum blood concentration of GDF15 contained in the fusion polypeptide provided herein when administered to a mammalian body may be extended by about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, about 11-fold or more, about 12-fold or more, about 13-fold or more, about 14-fold or more, about 15-fold or more, about 18-fold or more, about 20-fold or more, or about 22-fold or more, as compared to unfused GDF15. Alternatively, the area under the blood concentration-time curve (AUC last ) and / or the area under the blood concentration-time curve (AUC inf ) may be increased by about 2-fold or more, about 2.5-fold or more, about 3-fold or more, about 3.5-fold or more, about 4-fold or more, about 4.5-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, about 11-fold or more, about 12-fold or more, about 13-fold or more, about 14-fold or more, or about 15-fold or more, as compared to GDF15 not fused to an O-glycosylatable polypeptide region.

[0061] Thus, due to the increased half-life of GDF15, the fusion polypeptide form of GDF15 to which an O-glycosylatable polypeptide domain is attached has the advantage that it can be administered at longer intervals compared to a form of GDF15 to which an O-glycosylatable polypeptide domain is not attached.

[0062] A fusion polypeptide comprising GDF15 and an O-glycosylatable polypeptide region can be produced by conventional chemical synthesis methods or by recombinant methods.

[0063] As used herein, the term "vector" refers to an expression means for expressing a gene of interest in a host cell, and is selected from the group consisting of, for example, a plasmid vector, a cosmid vector, and a virus vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. In one example, the vector usable for the recombinant vector may be prepared based on a plasmid (e.g., pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), a phage (e.g., λgt4λB, λ-Charon, λΔz1, M13, etc.) or a virus (e.g., SV40, etc.), but is not limited thereto.

[0064] In the recombinant vector, the nucleic acid molecule encoding the fusion polypeptide can be operatively linked to a promoter. The term "operatively linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter sequence) and another nucleic acid sequence. The control sequence can be "operatively linked" to control the transcription and / or translation of the other nucleic acid sequence.

[0065] The recombinant vector can typically be constructed as a vector for cloning or an expression vector for expression. The expression vector can be a vector commonly used in the art for expressing foreign proteins in plants, animals, or microorganisms. The recombinant vector can be constructed by various methods known in the art.

[0066] The recombinant vector can be expressed in a eukaryotic cell as a host. When expressing in a eukaryotic cell as a host, the recombinant vector includes the nucleic acid molecule to be expressed and the above-described promoter, ribosome binding site, secretion signal sequence (see Korean Patent Publication No. 2015-0125402), and / or transcription / translation termination sequence, and the replication origin operable in a eukaryotic cell can include, but is not limited to, an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, and / or a BBV replication origin. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus promoter, and an HSV tk promoter) may be used, and any secretory signal sequence that is normally usable as the secretory signal sequence may be used, for example, the secretory signal sequence described in Korean Patent Publication No. 2015-0125402 may be used, but is not limited thereto, and a polyadenylation sequence may be included as a transcription termination sequence.

[0067] The recombinant cell may be obtained by introducing (transforming or transfecting) the recombinant vector into a suitable host cell. The host cell may be selected from among all eukaryotic cells in which the recombinant vector can be stably and continuously cloned or expressed. Eukaryotic cells that can be used as hosts include yeast (Saccharomyces cerevisiae), ... cerevisiae), insect cells, plant cells, and animal cells, including, but not limited to, mouse (e.g., COP, L, C127, Sp2 / 0, NS-0, NS-1, At20, or NIH3T3), rat (e.g., PC12, PC12h, GH3, or MtT), hamster (e.g., BHK, CHO, GS gene-deficient CHO, or DHFR gene-deficient CHO), monkey (e.g., COS (COS1, COS3, COS7, etc.), CV1 or Vero), human (e.g., HeLa, HEK-293, retina-derived PER-C6, cells derived from diploid fibroblasts, myeloma cells, or HepG2), other animal cells (e.g., MDCK, etc.), insect cells (e.g., Sf9 cells, Sf21 cells, Tn-368 cells, BTI-TN-5B1-4 cells, etc.), hybridomas, and the like.

[0068] By expressing the nucleic acid molecule encoding the fusion polypeptide provided herein in a suitable host cell as described above, it is possible to produce GDF15 or a fusion polypeptide containing the same, which has increased stability in vivo compared to the unfused form. The method for producing the fusion polypeptide can include a step of culturing a recombinant cell containing the nucleic acid molecule. The culturing step is carried out under normal culture conditions. The method can further include a step of isolating and / or purifying the fusion polypeptide from the culture after the culturing step.

[0069] The nucleic acid molecule or a recombinant vector containing the same can be delivered (introduced) into a host cell by a delivery method well known in the art, for example, when the host cell is a eukaryotic cell, the delivery method can be, but is not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, gene bombardment, etc.

[0070] The host cells transformed (introduced with a recombinant vector) can be easily selected by a method well known in the art using the phenotype expressed by the selection marker. For example, when the selection marker is a specific antibiotic resistance gene, recombinant cells into which a recombinant vector has been introduced can be easily selected by culturing the cells in a medium containing the antibiotic.

[0071] Said fusion polypeptides are used for the prevention and / or treatment of any disease which is associated with a deficiency and / or dysfunction of GDF15 or which can be treated, alleviated or ameliorated by the activity of GDF15.

[0072] Thus, in one example, there is provided 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. The pharmaceutical composition may be a pharmaceutical composition for preventing and / or treating a disease associated with a deficiency and / or dysfunction of GDF15 contained in the fusion protein or a disease in which the GDF15 has a therapeutic and / or preventive effect.

[0073] Another example provides a method for preventing and / or treating a disease associated with a deficiency and / or abnormality in GDF15 contained in the fusion protein or a disease for which the GDF15 has a therapeutic and / or preventive effect, 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 containing the nucleic acid molecule, and a recombinant cell containing the recombinant vector to a patient in need of the prevention and / or treatment of a disease associated with a deficiency and / or abnormality in GDF15 contained in the fusion protein or a disease for which the GDF15 has a therapeutic and / or preventive effect. The method may further comprise a step of identifying a patient in need of the prevention and / or treatment of a disease associated with a deficiency and / or abnormality in GDF15 contained in the fusion protein or a disease for which the GDF15 has a therapeutic and / or preventive effect prior to the administration step.

[0074] Examples of diseases associated with deficiency and / or abnormal function of GDF15 or diseases (or symptoms) for which GDF15 has a therapeutic and / or preventive effect include, but are not limited to, obesity, diabetes (type 1 diabetes, type 2 diabetes), cardiovascular disease, myocardial hypertrophy, liver disease (e.g., nonalcoholic steatohepatitis (NASH)), ischemic damage (ischemic brain damage, ischemic retinal damage), peripheral nerve damage, age-related sensory and / or motor nerve deficits, renal tubular and / or neo-epileptic damage, etc.

[0075] In another example, the pharmaceutical composition provided herein or a method including a step of administering the same may have one or more effects selected from the group consisting of weight loss, dietary regulation (reduction in food intake), body fat reduction, imparting and / or enhancing glucose tolerance, etc., in which case the pharmaceutical composition or method is applicable to uses for weight loss, body fat reduction, and / or imparting and / or enhancing glucose tolerance.

[0076] Thus, in one example, the composition includes one or more selected from the group consisting of the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector including the nucleic acid molecule, and a recombinant cell including the recombinant vector, and may be a pharmaceutical composition or a food composition (health functional food) for weight loss, dietary regulation (reducing food intake), body fat reduction, and imparting and / or improving glucose tolerance.

[0077] Another example provides a method for weight loss, dietary control (reduced food intake), body fat reduction, imparting and / or enhancing glucose tolerance, 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 to a patient in need of weight loss, dietary control (reduced food intake), body fat reduction, imparting and / or enhancing glucose tolerance. The method may further comprise identifying a patient in need of weight loss, dietary control (reduced food intake), body fat reduction, imparting and / or enhancing glucose tolerance prior to the administering step.

[0078] The pharmaceutical composition may contain at least one active ingredient selected from the group consisting of the fusion polypeptide, the fusion polypeptide dimer containing the fusion polypeptide, a nucleic acid molecule, a recombinant vector, and a recombinant cell in a pharmacologic effective amount. The pharmacologic effective amount means the content or dosage of the active ingredient that can achieve a desired effect. The content or dosage of the active ingredient in the pharmaceutical composition may be formulated in various ways depending on factors such as the formulation method, the administration method, the patient's age, weight, sex, pathological condition, diet, administration time, administration interval, administration route, excretion rate, and reaction sensitivity. For example, the single dosage of the active ingredient may be in the 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 is not limited thereto.

[0079] In addition, the pharmaceutical composition may further include a pharma- ceutically acceptable carrier in addition to the active ingredient. The carrier 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, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., which are commonly used in the formulation of drugs including proteins, nucleic acids, or cells, but are not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the preparation of pharmaceutical compositions.

[0080] The subject to which the pharmaceutical composition is administered may be a mammal, including a primate, such as a human, or a monkey, or a rodent, such as a mouse or a rat, or a cell, tissue, cell culture, or tissue culture derived therefrom.

[0081] The pharmaceutical composition may be administered orally or parenterally, or by contacting cells, tissues, or body fluids. Specifically, parenteral administration may be by intravenous injection, subcutaneous injection, intramuscular injection, peritoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration. When administered orally, proteins or peptides are digested, so oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach.

[0082] In addition, the pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be formulated into the form of an extract, powder, powder, granule, tablet or capsule, and may further contain a dispersant or stabilizer for formulation. Effect of the Invention

[0083] GDF15 fused to an O-glycosylatable polypeptide region provided herein has a long duration when administered to the body, allowing the administration interval to be increased, thereby enabling the administration dose to be reduced, thereby providing advantageous effects in terms of convenience of administration and / or economic aspects, and can be usefully applied in fields where GDF15 treatment is required. [Brief description of the drawings]

[0084] [Figure 1] FIG. 1 is a schematic diagram showing the amino acid sequence of GDF15. [Figure 2a] FIG. 1 is a schematic diagram of a fusion polypeptide comprising His Taq according to one embodiment. [Figure 2b] FIG. 1 shows a schematic diagram of the structure of a fusion polypeptide according to one embodiment. [Diagram 3] FIG. 1 is a schematic diagram showing the structures of various forms of fusion polypeptides. [Figure 4] FIG. 1 shows the results of SDS-PAGE analysis of the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 synthesized in one embodiment. [Diagram 5] FIG. 1 shows the results of Q-TOF Mass Spectrometry analysis of the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 synthesized in one embodiment. [Figure 6] 1 is a graph showing changes in body weight following a single administration of the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 to mice. [Figure 7] 7 is a graph showing the results on the fourth day extracted from the results in FIG. 6. [Figure 8] 1 is a graph showing the change in food intake in mice administered the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15. [Figure 9]1 is a graph showing changes in body weight when fusion polypeptides HT-ID2-GDF15 and HT-ID3-GDF15 were repeatedly administered to mice. [Figure 10a] 10 is a graph showing the results of days 7 and 14 extracted from the results of FIG. 9. [Figure 10b] 10 is a graph showing the results of days 21 and 28 extracted from the results of FIG. 9. [Figure 11] 1 is a graph showing cumulative food intake on days 7, 14, 21, and 28 following repeated administration of the fusion polypeptides HT-ID2-GDF15 and HT-ID3-GDF15 to mice, respectively. [Figure 12] 1 is a graph showing the change in blood fusion polypeptide concentration over time when the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 were administered to SD rats. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] The present invention will now be described in more detail with reference to the following examples. However, these are merely for the purpose of illustrating the present invention, and the scope of the present invention is not limited to these examples.

[0086] Example 1: Production of fusion polypeptides 1.1. Production of Fusion Polypeptides Containing GDF15 IgD hinge (ESPKAQA SS VP T AQPQAEGSLAKA TT APA TT We prepared fusion polypeptides IgD-GDF15 (ID1-GDF15), IgD-IgD-GDF15 (ID2-GDF15), and IgD-IgD-IgD-GDF15 (ID3-GDF15) (see Figures 2a and 2b) in which a combination of IgD hinges (RNT; SEQ ID NO: 1; the underlined parts are potential O-glycosylation sites) or multiple (1, 2, or 3) IgD hinges was fused to GDF15 (SEQ ID NO: 3, Figure 1).

[0087] For the convenience of purification, a fusion polypeptide containing a His-tag (SEQ ID NO: 15) and a TEV cleavage site (SEQ ID NO: 16) was also prepared. The amino acid sequences of each part contained in the fusion polypeptide are summarized in Table 2 below.

[0088] [Table 2]

[0089] 1.1.1. Construction of Recombinant Expression Vectors 1.1.1.1.Mature GDF15 To obtain a gene encoding mature GDF15, a gene encoding mature GDF15 (SEQ ID NO: 5) was synthesized (Bioneer Co., Ltd.) with reference to the amino acid sequence information of UniprotKB Q99968. SEQ ID NO:5 (339bp) [Table A]

[0090] 1.1.1.2.IgD Hinge (ID) To obtain a gene encoding a human IgD hinge, a gene (SEQ ID NO: 6) encoding three human IgD hinges (hereinafter referred to as "ID3") was synthesized by Bioneer with reference to the amino acid sequence information of UniprotKB P01880. SEQ ID NO:6 (306bp) [Table B] (Polynucleotides 1-102 (underlined), 103-204 (bold), and 205-306 (bold + underlined) each encode an IgD hinge.)

[0091] 1.1.1.3. Construction of expression vectors pDHDD-D1G1 (containing the KR10-1868139B1 promoter), a variant of pcDNA3.1(+) (Invitrogen, Cat. No. V790-20), was cleaved with BamHI and NotI, and a gene designed to encode a fusion protein having the following structure (see FIG. 3) was inserted by combining the above genes (the gene encoding mature GDF15 and the gene encoding ID3) to prepare each recombinant vector.

[0092] 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 site]-(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)"

[0093] 1.1.2. Expression of Fusion Polypeptides 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 used in ExpiCHO-S TMThe fusion polypeptides GDF15, HT-GDF15, ID1-GDF15, HT-ID1-GDF15, ID2-GDF15, HT-ID2-GDF15, ID3-GDF15, and HT-ID3-GDF15 were expressed by introducing the fusion polypeptides into cells (Thermo Fisher Scientific) and culturing them for 12 days (Fed-Batch Culture; Day 1 & Day 5 Feeding) in ExpiCHO Expression Medium (Thermo Fisher Scientific; 400 mL).

[0094] 1.1.3. Purification of fusion polypeptides The fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 produced by expression of the recombinant vector were purified and analyzed for sialic acid content and O-glycan site occupancy using Q-TOF mass spectrometry.

[0095] Specifically, the fusion polypeptide was purified by successive ultrafiltration / diafiltration, immobilized metal affinity chromatography (IMAC), and anion exchange chromatography (AEX). First, the cell-free culture medium of the fusion protein was filtered through a 0.22 μm filter. The filtrate was concentrated using a TFF System and then buffer-exchanged with a tromethamine buffer solution. HiTrap TM A column packed with Chelating HP (GE Healthcare Life Sciences) resin was used. TMThe column was then equilibrated by passing equilibration buffer (20 mM Tris pH 8.0, 0.5 M NaCl, 5 mM imidazole) through the column. The above-mentioned ultrafiltration / diafiltration process solution was then poured into the column, and the column was washed again by passing equilibration buffer through it. After the column washing was completed, elution buffer (20 mM Tris pH 8.0, 0.5 M NaCl, 0.5 M imidazole) was passed through the column to elute the target protein.

[0096] The resulting eluate was concentrated using an Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge, and then buffer-exchanged with tromethamine buffer. The process solution thus prepared was poured into an equilibrated anion exchange column, and the column was washed with equilibration buffer (20 mM Tris pH 8.0). After washing the column, elution buffer (20 mM Tris pH 8.0, 0.5 M NaCl) was poured through the column under concentration gradient conditions to elute the target protein. The eluted fractions with high fusion polypeptide concentration and high purity were collected and frozen for storage.

[0097] For animal experiments, samples were concentrated and buffer exchanged with Phosphate Buffered Saline (PBS, 10 mM sodium phosphate, 150 mM NaCl pH 7.4) using an Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge.

[0098] Quantitative analysis of the fusion polypeptide was performed by measuring the absorbance at 280 nm and 340 nm using a UV Spectrophotometer (G113A, Agilent Technologies) and calculating according to the following formula: The absorption coefficient of each substance was calculated theoretically using the amino acid sequence.

number

[0099] [Table 3]

[0100] The purified fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 were analyzed for sialic acid content and O-glycan site occupancy after reduction using Q-TOF mass spectrometry.

[0101] The results of SDS-PAGE analysis of the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 are shown in Figure 4, and the results of Q-TOF Mass Spectrometry analysis are shown in Figure 5 and Table 4. The sialic acid content is also shown in Table 4.

[0102] [Table 4]

[0103] Example 2. Pharmacological effects of fusion polypeptides (in vivo) 2.1. Single dose 2.1.1 Testing process The pharmacological effect of the fusion polypeptide produced and purified in Example 1 was tested in mice (C57BL / 6J, 6-week-old, male, 100 mice; Raon Bio Co., Ltd.).

[0104] In this example, a DIO mouse model (Mouse, C57BL / 6J-DIO, male, 100 mice, 14 weeks old (fed with obesity diet for 8 weeks)) was used, in which obesity was induced in the C57BL / 6J mice by feeding them a high-fat diet for 8 weeks. The DIO mouse model shows clinical characteristics of type 2 diabetes, such as hyperlipidemia, insulin resistance, and hyperglycemia, and is an animal model that is widely used to evaluate the efficacy of diabetes and insulin resistance improvement. A large amount of comparative basic data has been accumulated for the study of metabolic diseases such as obesity, diabetes, and hyperlipidemia, and therefore this model was selected as being appropriate for the pharmacological effect test in this example.

[0105] The mouse model fed the obese diet for 8 weeks underwent a 2-week quarantine and acclimation period, during which general symptoms were observed once a day to confirm whether the animals were healthy and suitable for the experiment, and healthy animals were selected. During the acclimation period, the animals were individually marked on their tails with a red oil-based pen upon acquisition, and temporary individual identification cards (study name, individual number, date of entry) were attached to the breeding cage during the quarantine and acclimation period. When the animals were separated into groups, they were individually marked on their tails with a black oil-based pen, and individual identification cards (study name, group information, individual number, sex, date of entry, administration period) were attached to each cage.

[0106] In order to minimize the stress that the experimental animals would experience during subcutaneous administration of the test substance (fusion polypeptide), all animals were pre-trained to adapt to subcutaneous administration by subcutaneously administering 200 μL / head of sterile distilled saline using a 1 mL syringe from 3 days before administration of the test substance.

[0107] For healthy animals for which no abnormalities were found during the quarantine and acclimation periods, the body weight and food intake of all individuals were measured after the acclimation period ended.

[0108] Body weight and food intake were measured, and the groups were separated based on body weight so that the averages of the two measurements were similar between groups. Test substance administration was started the day after group separation. The remaining unselected animals were removed from the test system after group separation.

[0109] The information on the high fat diet (HFD) fed to the C57BL / 6J-DIO mice is as follows: 5.24 kcal / g, 60% fat by weight, 20% protein by weight, and 20% carbohydrate-derived calories by weight; Research Diet Inc., USA; Product No. High fat diet (Fat 60 kcal%, D12492).

[0110] The diet was fed ad libitum (fed during the habituation and testing periods).

[0111] Drinking water was provided by filtering tap water through a filter and sterilizing device, irradiating it with ultraviolet light, and allowing the animals to drink the water ad libitum from 250 mL polycarbonate water bottles.

[0112] The test substances HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 and the control substance Semaglutide (Bachem) were administered from the day after group separation at 9:00 a.m. every day. The control and test substances were all administered subcutaneously. The administration route for the control and test substances was selected as subcutaneous route based on the planned clinical administration route.

[0113] The volume of the control and test substances was 5mL / kg, and the volume of each substance was calculated based on the most recent body weight. The test substances were administered subcutaneously once on the first day of the test using a disposable syringe (1mL). The test substances were administered only once on the first day of the test. For comparison, a control group administered with the control substance Semaglutide was prepared, and the Semaglutide-administered control group was administered with Semaglutide once a day, and all administrations began at 9:00 a.m.

[0114] The composition of the test groups and the dosages are summarized in Table 5 below:

[0115] [Table 5]

[0116] The observation, measurement and examination schedule for the test group was set to Day 0 on the day administration started, and 7 days from the day administration started was set to 1 week of administration.

[0117] The inspection schedule is summarized in Table 6:

[0118] [Table 6]

[0119] All animals were observed once a day for general clinical symptoms, and twice a day for the presence or absence of moribund or dead animals. Such observations were carried out from the first day of administration until the end of administration. Any abnormal symptoms observed were recorded on a record sheet.

[0120] The body weight of each mouse was measured on the first day of administration of the test substance (before administration), and thereafter, the body weight was measured every day (up to 9 days). The volume of the test substance to be administered was determined based on the most recently measured body weight.

[0121] After administering the test substance to the mice, the amount of food intake was measured every day. The amount of food given to each cage was measured using an electronic balance, and the remaining amount was measured to calculate the daily food intake. Individuals that chewed the food excessively were excluded from the measurement.

[0122] All experimental results obtained in this example are expressed as mean ± standard error and were analyzed using Prism5 (version 5.01). One-way analysis of variance (ANOVA) was performed on all data, and when significance was observed, Dunnett's test was performed to examine the test group with significant difference from the control group (significance level: 5%, 1%, 0.1% on both sides).

[0123] 2.1.2. Weight loss test results The changes in body weight measured in Example 2.1.1 are shown in Figures 6 and 7, and Table 7 (Body Weight (Group, % of initial)).

[0124] [Table 7]

[0125] Figure 6 and Table 7 show the changes in body weight following a single administration of the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15, in comparison with the negative control group (vehicle administration group) and the positive control group (daily administration of Semaglutide group). Figure 7 is a graph showing the results on day 4 from the results shown in Figure 6.

[0126] As can be seen from the above results, the negative control group (vehicle administration group) showed almost no change in body weight, whereas the positive control group (daily administration of Semaglutide) showed a sustained weight loss effect from day 1 after administration. In addition, in the case of the fusion polypeptide in which GDF15 is fused with IgD Hinge, a weight loss effect was observed immediately after a single administration on day 0, and the weight loss effect continued without decreasing until days 3 to 4.

[0127] 2.1.3. Dietary intake test results The changes in food intake measured in Example 2.1.1 above are shown in Table 8 and Figure 8 (cumulative intake up to day 6), respectively.

[0128] [Table 8]

[0129] As can be seen from the above results, the group administered the fusion polypeptide in which GDF15 is fused with IgD Hinge showed a food intake reduction effect up to the sixth day in response to the fusion polypeptide compared to the negative control group (vehicle administration group), and this effect of reducing food intake by the fusion polypeptide was comparable to that of the positive control group, Semaglutide, administered once a day throughout the test period.

[0130] 2.2 Repeated administration 2.2.1 Testing process Most of the test procedures were the same as those in Example 2.1.1 above, except for the administration volume, number of animals and administration period.

[0131] The test substance was administered twice a week (days 0, 4, 7, 11, 14, 18, 21, and 25) for a total of eight doses. For comparison, a control group was administered the control substance Semaglutide every day, and the control group was administered Semaglutide once a day, with all doses starting at 9:00 a.m.

[0132] The composition of the test groups and the dosage are summarized in Table 9 below:

[0133] [Table 9]

[0134] 2.2.2. Weight loss test results The changes in body weight measured in Example 2.2.1 are shown in Figures 9, 10a, 10b, and Table 10 (Body Weight (Group, % of initial)).

[0135] [Table 10-1] [Table 10-2]

[0136] Figure 9 and Table 10 show the changes in body weight after repeated administration of the fusion proteins HT-ID2-GDF15 and HT-ID3-GDF15 (Table 9), in comparison with the negative control group (vehicle administration group) and the positive control group (daily administration of Semaglutide group). Figure 10a shows the results of days 7 and 14 from the results of Figure 9, and Figure 10b shows the results of days 21 and 28 from the results of Figure 9.

[0137] As can be seen from the above results, the negative control group (vehicle administration group) showed almost no change in body weight, whereas the positive control group (daily administration of Semaglutide) showed a sustained weight loss effect from day 1 after administration. In addition, in the case of the fusion polypeptide in which GDF15 is fused with IgD Hinge, a weight loss effect was observed immediately after a single administration on Day 0, and the weight loss effect continued throughout the test period without any decrease, demonstrating that the weight loss effect is concentration-dependent.

[0138] 2.2.3. Dietary intake study results The changes in food intake measured in Example 2.2.1 above are shown in Table 11 and Figure 11 (cumulative intake up to days 7, 14, 21 and 28), respectively.

[0139] [Table 11-1] [Table 11-2]

[0140] As can be seen from the above results, the group administered the fusion polypeptide in which GDF15 was fused with IgD Hinge showed a reduction in food intake throughout the test period in response to the fusion polypeptide compared to the negative control group (vehicle administration group), and showed a concentration-dependent tendency.

[0141] Example 3. Pharmacokinetic testing of fusion polypeptides 3.1. Preparation of serum from test and control groups To evaluate the pharmacokinetic properties of each fusion polypeptide when subcutaneously administered to rats, the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15, and HT-ID3-GDF15 were subcutaneously administered at 2 mg / kg to SD rats (Coretech Co., Ltd., male, 7 weeks old, approximately 250 g; n=3 for each; test group), and approximately 200 ul of blood was collected from the tail vein at the designated time. The blood was collected at 1, 2, 4, 8, 24, 48, 72, 96, 168, 240, and 336 hours after administration of the fusion polypeptide. As a control group for comparing the pharmacokinetic properties, GDF15 (R&D Systems) was subcutaneously administered at 2 mg / kg in the same manner as above to prepare a GDF15 administration group.

[0142] After administration to SD rats as described above, blood samples were collected at each time point and centrifuged to obtain serum. ELISA was performed using Human GDF15 Immunoassay (SGD150, R&D Systems) to measure serum concentrations of each polypeptide over time. Using the data, parameter values ​​including AUC (area under the curve) were calculated using PK analysis software (WinNonlin (Certara LP), etc.).

[0143] 3.2. Pharmacokinetic study results The pharmacokinetic parameters of the fusion polypeptide obtained above are shown in Table 12, and the concentration changes of the fusion polypeptide as a function of time are shown in FIG.

[0144] [Table 12]

[0145] As can be seen from the above results, the half-life of the fusion polypeptide fused with IgD Hinge was increased compared to GDF15 (half-life: 19 hours). In particular, the AUC last In the case of GDF15, the increase was up to 16.7-fold compared to GDF15.

[0146] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. A fusion polypeptide dimer comprising two fusion polypeptides, The fusion polypeptide comprises: Human Growth / Differentiation Factor 15 (GDF15), and A total of 1 to 3 O-glycosylatable polypeptide regions linked to the N-terminus of the GDF15. Each of each of the one to three O-glycosylatable polypeptide regions is a polypeptide containing from three to ten O-glycosylatable amino acid residues; Each of the 1 to 3 O-glycosylatable polypeptide regions comprises an immunoglobulin D (IgD) hinge region having the amino acid sequence of SEQ ID NO:1; The fusion polypeptide dimer is a homodimer.

2. The fusion polypeptide dimer of claim 1, wherein the fusion polypeptide is represented by the formula: N'-(Z)n-Y-C' In the above formula, N' is the N-terminus of the fusion polypeptide and C' is the C-terminus of the fusion polypeptide; Y is human GDF15, Z is an IgD hinge region consisting of the amino acid sequence of SEQ ID NO:1; n is the number of polypeptide regions capable of O-glycosylation attached to the N-terminus of GDF15 and is an integer from 1 to 3.

3. The area under the blood concentration-time curve (AUC) up to the last measurable blood sampling time point when human GDF15 bound to the O-glycosylatable polypeptide region in the fusion polypeptide was administered in vivo ｌａｓｔ 3. The fusion polypeptide dimer of claim 1 or 2, wherein the amount of GDF15 bound to the O-glycosylated polypeptide region is increased by more than 2-fold compared to human GDF15 that is not bound to the O-glycosylated polypeptide region.

4. A nucleic acid molecule encoding the fusion polypeptide contained in the fusion polypeptide dimer described in claim 1 or 2.

5. A recombinant vector comprising the nucleic acid molecule of claim 4.

6. A recombinant cell comprising the recombinant vector of claim 5.

7. A method for producing a fusion polypeptide dimer according to claim 1 or 2, comprising human GDF15 and an O-glycosylatable polypeptide region, comprising culturing the recombinant cell according to claim 6.

8. linking in vitro a total of 1 to 3 O-glycosylatable polypeptide regions to the N-terminus of human GDF15; each of the one to three O-glycosylatable polypeptide regions is a polypeptide containing from three to ten O-glycosylatable amino acid residues; Each of the 1 to 3 O-glycosylatable polypeptide regions consists of a hinge region of immunoglobulin D (IgD) consisting of the amino acid sequence of SEQ ID NO: 1; A composition comprising a fusion polypeptide dimer according to claim 1 or 2 for use in a method for increasing the in vivo stability of GDF15.

9. A pharmaceutical composition for preventing or treating a disease associated with a deficiency or dysfunction of GDF15, comprising the fusion polypeptide dimer according to claim 1 or 2.

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