Fusion polypeptides comprising the Fc region of an immunoglobulin and GDF15

Abstract

To provide a fusion polypeptide including GDF15 (Growth / differentiation factor 15) and an Fc region of immunoglobulin, a pharmaceutical composition including the fusion polypeptide, and a method for increasing the in vivo duration of GDF15 including a step for fusing the Fc region of immunoglobulin.SOLUTION: There is provided a technique of connecting a GDF15 (Growth / differentiation factor 15) or a functional variant thereof with an Fc region of immunoglobulin to form a fusion polypeptide, by which an in vivo half life period of GDF15 increases to enhance the duration in a body comparing with a case where GDF15 or a functional variant thereof is not fused with the Fc region, and thus a pharmacological effect of GDF15 is enhanced and / or an administration interval is increased.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a fusion polypeptide comprising GDF15 (Growth / differentiation factor 15) and the Fc region of an immunoglobulin, a pharmaceutical composition comprising the fusion polypeptide, and a method for increasing the biological duration of GDF15, which comprises fusing the Fc region of an immunoglobulin to the fusion polypeptide. [Background technology]

[0002] Most protein or peptide drugs have a short period of activity in the body and poor absorption when administered by methods other than intravenous administration. Therefore, when long-term drug therapy is required, these drugs must be repeatedly injected at short intervals. To overcome this drawback, there is a need for a technology that can continuously release drugs after a single administration. To address this need, 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 protein or peptide drugs are prepared into fine particles surrounded by a biodegradable polymer matrix, and the drug is gradually released as the matrix material is gradually degraded and removed in the body upon administration.

[0004] Growth / differentiation factor 15 (GDF15), a member of the TGF-β family, is a 25 kDa homodimer and secreted protein that circulates in the blood. Circulating GDF15 levels are associated with body mass index (BMI), suggesting that GDF15 plays a role as a long-term regulator of energy homeostasis. GDF15 also protects against pathological conditions such as cardiovascular disease, myocardial hypertrophy, and ischemic injury. GDF15 also plays a protective role against tubular and interstitial injury in type 1 and type 2 diabetes models. GDF15 also protects against age-related sensory and motor neuron loss and contributes to recovery from peripheral nerve injury. GDF15 also promotes weight loss, body fat reduction, and glucose tolerance, increasing whole-body energy expenditure and oxidative metabolism. GDF15 regulates blood glucose levels through both weight-dependent and weight-independent mechanisms.

[0005] 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]

[0006] The present specification provides a technology that increases the half-life of GDF15 (Growth / differentiation factor 15) or a functional variant thereof to form a fusion polypeptide with the Fc region of an immunoglobulin, thereby enhancing the duration of GDF15 in the body compared to when it is not fused to the Fc region, thereby enhancing the pharmacological effect of GDF15 and / or increasing the administration interval.

[0007] One embodiment provides a fusion polypeptide comprising GDF15 or a functional variant thereof and an Fc region of an immunoglobulin.

[0008] In the fusion polypeptide, the Fc region of the immunoglobulin can be included (linked) at the N-terminus of the GDF15 or functional variant thereof. For example, the fusion polypeptide comprises, from the N-terminus to the C-terminus, (1) the Fc region of an immunoglobulin and (2) GDF15 or a functional variant thereof.

[0009] The immunoglobulin Fc region contained in the fusion polypeptide serves to increase the stability of GDF15 or its variant, for example, by extending its half-life (e.g., in vivo half-life). For example, the immunoglobulin Fc region is selected from the group consisting of an IgG1 Fc region and an IgG4 Fc region. The IgG1 Fc region comprises the IgG1 CH2 domain, CH3 domain, or CH2+CH3 domains, and may or may not comprise an IgG1 hinge region at the N-terminus. The IgG4 Fc region comprises the IgG4 CH2 domain, CH3 domain, or CH2+CH3 domains, and may or may not comprise an IgG4 hinge region at the N-terminus.

[0010] The fusion polypeptide may further comprise a peptide linker between the immunoglobulin Fc region and GDF15 or its functional variant. For example, the peptide linker is preferably a flexible linker so that the fused Fc region and GDF15 can freely perform their functions. For example, the peptide linker is not a rigid linker. For 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 an integer of 1 to 5 (1, 2, 3, 4, or 5)), but is not limited thereto.

[0011] In the fusion polypeptide, GDF15 or its functional variant fused to the Fc region of an immunoglobulin is characterized by increased stability (duration) in the body (or in the blood) (e.g., increased half-life in the body or in the blood) compared to GDF15 or its functional variant not fused to the Fc region of an immunoglobulin. Furthermore, GDF15 or its functional variant fused to the Fc region of an immunoglobulin is characterized by improved pharmacological effects (e.g., weight loss effects) compared to GDF15 or its functional variant not fused to the Fc region of an immunoglobulin.

[0012] The present invention provides a fusion polypeptide dimer comprising two of the above-described fusion polypeptides. The fusion polypeptide dimer may be a fusion polypeptide in which GDF15 or a functional variant thereof of the two fusion polypeptides is linked (bound) to each other.

[0013] The present invention provides a nucleic acid molecule encoding the fusion polypeptide.

[0014] The present invention provides a recombinant vector comprising the nucleic acid molecule.

[0015] The present invention provides a recombinant cell containing the recombinant vector.

[0016] The present invention provides a composition (pharmaceutical composition or health functional food composition) for weight loss, dietary regulation (food intake reduction), or prevention, amelioration, alleviation, and / or treatment of metabolic diseases, comprising one or more selected from the group consisting of the fusion polypeptide, fusion polypeptide dimer, fusion polypeptide-encoding nucleic acid molecule, recombinant vector containing the nucleic acid molecule, and recombinant cell containing the recombinant vector. The present invention also provides a method for weight loss, dietary regulation (food intake reduction), or prevention, amelioration, alleviation, and / or treatment of metabolic diseases, comprising administering a pharmaceutically effective amount of one or more selected from the group consisting of the fusion polypeptide, fusion polypeptide dimer, fusion polypeptide-encoding nucleic acid molecule, recombinant vector containing the nucleic acid molecule, and recombinant cell containing the recombinant vector to a subject in need thereof. The metabolic disease refers to any disease caused by metabolic disorders and is selected from the group consisting of obesity, diabetes (e.g., type 2 diabetes), nonalcoholic fatty liver disease (e.g., nonalcoholic steatohepatitis (NASH)), etc.

[0017] The present invention provides a method for producing GDF15 or a functional variant thereof with an increased in vivo (or blood) half-life, or a method for producing a fusion polypeptide containing GDF15 or a functional variant thereof with an increased in vivo (or blood) half-life, or a homodimer containing the fusion polypeptide, which comprises the step of expressing the recombinant vector in cells.

[0018] The present invention provides a method for increasing the biological duration of GDF15 or its functional variant, comprising fusing (or linking or conjugating) GDF15 or its functional variant with an Fc region of an immunoglobulin. In one embodiment, the fusing step can include fusing (or linking or conjugating) the Fc region of an immunoglobulin to the N-terminus of GDF15 or its functional variant with or without a linker. The present invention also provides a method for reducing the immunogenicity of GDF15, an Fc region of an immunoglobulin, or a fusion polypeptide comprising them, comprising fusing (or linking or conjugating) GDF15 or its functional variant with an Fc region of an immunoglobulin using a flexible linker (e.g., a GS linker). The fusing (or linking or conjugating) step is performed in vitro. [Means for solving the problem]

[0019] The present invention will now be described in more detail:

[0020] GDF15 GDF15 is composed of the 197th (A) to 308th (I) amino acids (SEQ ID NO: 1; see Figure 2; mature form) of the total 308 amino acids (UniProt Q99988), excluding the signal peptide and propeptide.

[0021] As used herein, unless otherwise specified, GDF15 refers to a polypeptide essentially comprising the amino acid sequence from amino acid 197 (A) to amino acid 308 (I) of the full-length protein (UniProt Q99988) (SEQ ID NO: 1, see Figure 2; ARNG DHCPLGPGRC 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 to said amino acid sequence, within the scope of maintaining the inherent activity and structure of GDF15.

[0022] As used herein, a functional variant of GDF15 may be a variant in which GDF15 has been mutated to further favor the formation of a dimer structure while maintaining its inherent activity and structure. For example, the functional variant of GDF15 may be an N-terminal deletion variant in which one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) of the N-terminal 14 amino acid residues (i.e., a total of 14 amino acid residues from positions 1 to 14 in SEQ ID NO: 1) (e.g., one or more from the N-terminus) of the amino acid sequence of GDF15 of SEQ ID NO: 1, for example, an N-terminal deletion variant in which all 14 amino acid residues are deleted (hereinafter referred to as "ΔN14GDF15" or "GDF(CRL)"). In one embodiment, the functional variant of GDF15 may be a polypeptide essentially comprising the amino acid sequence of SEQ ID NO: 2 (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 with said amino acid sequence, within the scope of maintaining the inherent activity and structure of GDF15.

[0023] Immunoglobulin Fc The Fc region of the immunoglobulin fused to GDF15 or its functional variants serves to increase the stability of the GDF15 or its functional variants, for example, by extending its half-life (e.g., in vivo half-life) and / or reducing renal filtration. For example, the Fc region of the immunoglobulin is selected from the Fc region of IgG1 and the Fc region of IgG4. The Fc region of IgG1 comprises the CH2 domain, CH3 domain, or CH2+CH3 domains of IgG1, and may or may not comprise the hinge region of IgG1 at the N-terminus. The Fc region of IgG4 comprises the CH2 domain, CH3 domain, or CH2+CH3 domains of IgG4, and may or may not comprise the hinge region of IgG4 at the N-terminus.

[0024] IgG1 is derived from primates such as humans or rodents such as mice or rats, for example, human IgG1 (UniprotKB P01857). IgG4 is derived from primates such as humans or rodents such as mice or rats, for example, human IgG4 (UniprotKB P01861).

[0025] The IgG1 Fc region comprises the CH2 domain, CH3 domain, or CH2+CH3 domains of IgG1, and may or may not include the hinge region of IgG1 at the N-terminus.The IgG4 Fc region comprises the CH2 domain, CH3 domain, or CH2+CH3 domains of IgG4, and may or may not include the hinge region of IgG4 at the N-terminus.

[0026] In one embodiment, the Fc region of IgG1 may be a polypeptide comprising the CH2 and CH3 domains of human IgG1 (SEQ ID NO: 3), or a polypeptide further comprising the hinge region of human IgG1 (SEQ ID NO: 4) at the N-terminus of the amino acid sequence of SEQ ID NO: 3. As used herein, the Fc region of IgG1 is understood to include 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 to the amino acid sequence of SEQ ID NO: 3 or the "N-terminus-(SEQ ID NO: 4)-(SEQ ID NO: 3)-C-terminus", within the scope of maintaining the inherent activity and structure of IgG1 Fc.

[0027] IgG4 Fc is a polypeptide comprising the CH2 and CH3 domains of human IgG4 (SEQ ID NO: 5) or a polypeptide further comprising a human IgG4 hinge region (SEQ ID NO: 10) at the N-terminus of the amino acid sequence of SEQ ID NO: 5. As used herein, the IgG4 Fc region is understood to include 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 to the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of "N-terminus-(SEQ ID NO: 10)-(SEQ ID NO: 5)-C-terminus" (or the amino acid sequence of an IgG4 Fc mutant described below), within the scope of maintaining the inherent activity and structure of IgG4 Fc.

[0028] When the Fc region of an immunoglobulin is used to extend the (in vivo) half-life of a protein linked to its N- or C-terminus, it is important to minimize any effector functions of the Fc in order to reduce side effects caused by the immunoglobulin Fc region. From this perspective, the human IgG4 Fc region is advantageous because it has lower binding affinity to FcγR and complement factors compared to other IgG subtypes. In addition, the effector functions of the human IgG4 Fc region are further reduced by including amino acid substitutions. The amino acid substitutions in the human IgG4 Fc region to reduce effector function can include one or more of the following: a substitution of the phenylalanine residue at position 234 of human IgG4 (UniprotKB P01861) with alanine, and a substitution of the leucine residue at position 235 with alanine (contained in the CH2-CH3 domain of IgG4 Fc; the amino acid residue numbers are according to the EU numbering system [Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, Bethesda, MD, NIH Publication no. 91-3242]). Furthermore, the IgG4 Fc region contains an amino acid substitution that stabilizes heavy chain dimer formation and prevents the formation of half-IgG4 Fc chains. Such an amino acid substitution includes a substitution of the serine amino acid residue at position 228 of the Fc region of human IgG4 (UniprotKB P01861) (contained in the hinge region according to the EU numbering system) with proline.

[0029] In one embodiment, the Fc region does not contain any mutations other than those described above to reduce immunogenicity, but is not limited to this. In one embodiment, the Fc region of the immunoglobulin comprises the CH2-CH3 domain of human IgG4 Fc (SEQ ID NO: 5 (generic formula); SEQ ID NO: 6 (wild type), or SEQ ID NO: 7, 8, or 9 (mutant)), or further comprises the hinge region of human IgG4 (SEQ ID NO: 10 (generic formula); SEQ ID NO: 11 (wild type), or SEQ ID NO: 12 (mutant)) N-terminal to the CH2-CH3 domain.

[0030] The Fc regions of immunoglobulins used herein are summarized in Table 1 below: [Table 1-1] [Table 1-2]

[0031] Fusion Polypeptides As used herein, a fusion polypeptide comprises an Fc region of an immunoglobulin and GDF15 or a functional variant thereof linked to the C-terminus of the Fc region of the immunoglobulin, wherein the Fc region of the immunoglobulin and GDF15 or a functional variant thereof are as described above.

[0032] In the fusion polypeptide, the Fc region of an immunoglobulin and GDF15 or its functional variant are linked covalently or non-covalently, or directly via a suitable linker (e.g., a peptide linker), or without a linker. For example, the peptide linker may be a polypeptide consisting of 1 to 20, 1 to 15, 1 to 10, 2 to 20, 2 to 15, or 2 to 10 amino acids, and there is no limitation on the type of amino acids contained therein. Herein, the peptide linker is preferably a flexible linker so that the fused Fc region (which provides stabilizing activities such as increased half-life and reduced renal filtration) and GDF15 can independently exert their functions. For example, the peptide linker is not a rigid linker. The peptide linker may contain, for example, one or more amino acid residues selected from the group consisting of Gly, Asn, Ser, Glu, and Lys, and may also 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.

[0033] For 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 between 1 and 10 or between 1 and 5 (1, 2, 3, 4, or 5)), but is not limited thereto. In the GS linker, Gly (glycine) is an amino acid whose R group is -H. It is nonpolar, has a large degree of freedom (phi, psi angle), and exhibits excellent mobility. Ser (serine) is an amino acid whose R group is -CH2-OH. It is small but polar, forming hydrogen bonds with water, which is beneficial for maintaining linker stability and reducing nonspecific interactions between the GS linker and GDF15 or the Fc region. Furthermore, the GS linker has the advantage of low immunogenicity due to its flexible structure. The GS linker serves to spatially separate GDF15 and the Fc region so as not to interfere with their respective functions, and therefore the amino acid length of the GS linker is 15 to 25 amino acids (n = 3 to 5), 15 to 20 amino acids (n = 3 or 4), 15 to 25 amino acids (n = 4 or 5), or 20 amino acids (n = 4). For example, the peptide linker contained in the fusion polypeptide used herein does not contain any amino acid residues other than the GS linker [(GGGGS)n (n is an integer of 1 to 10 or an integer of 1 to 5)].

[0034] Therefore, the fusion polypeptide further comprises a peptide linker between the immunoglobulin Fc region and GDF15 or its functional variant, which is a GS linker containing one or more repeats of Gly (G) and one or more repeats of Ser (S), such as, but not limited to, (GGGGS)n (n is the number of repeats of 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)).

[0035] The fusion polypeptide may be recombinantly produced or chemically synthesized. If recombinantly produced, the fusion polypeptide is encoded by a single reading frame (referred to as "single strand") whose expression is regulated by a single transcription initiation regulatory element (e.g., a promoter).

[0036] In the fusion polypeptide, GDF15 or its functional variant fused to the Fc region of an immunoglobulin is characterized by increased stability (duration) in vivo (or in blood) (e.g., increased half-life in vivo or in blood) and / or reduced immunogenicity compared to GDF15 or its functional variant not fused to the Fc region of an immunoglobulin and / or GDF15 fused to the Fc region via a linker other than the GS linker. Furthermore, GDF15 or its functional variant fused to the Fc region of an immunoglobulin is characterized by improved pharmacological effects (e.g., weight loss effects) compared to GDF15 or its functional variant not fused to the Fc region of an immunoglobulin.

[0037] Fusion Polypeptide Dimer The functionally active form of GDF15 is the homodimeric form.

[0038] Thus, another embodiment of the present invention provides a fusion polypeptide dimer comprising two fusion polypeptides (a first fusion polypeptide and a second fusion polypeptide). The first fusion polypeptide comprises an Fc region of a first immunoglobulin and a first GDF15 or functional variant thereof, and the second fusion polypeptide comprises an Fc region of a second immunoglobulin and a second GDF15 or functional variant thereof. In the dimer, the first GDF15 or functional variant thereof and the second GDF15 or functional variant thereof are linked to each other via a covalent bond (e.g., a disulfide bond) in the first fusion polypeptide and a functional variant thereof in the second fusion polypeptide. Optionally, the Fc region of the first immunoglobulin and the Fc region of the second immunoglobulin are linked to each other via a covalent bond (e.g., a disulfide bond) or a non-covalent bond (e.g., a knob-and-hole bond, electrostatic interaction, hydrophobic interaction, etc.) in the dimer (see FIG. 1 ). The Fc regions of the immunoglobulins and GDF15 or its functional variants are as described above, and the Fc regions of the first immunoglobulin and the second immunoglobulin, and the first GDF15 or its functional variant and the second GDF15 or its functional variant may be identical to or different from each other.

[0039] For example, the Fc region of the first immunoglobulin and the Fc region of the second immunoglobulin contained in the fusion polypeptide dimer, the first GDF15 or its functional variant and the second GDF15 or its functional variant, and the first fusion polypeptide and the second fusion polypeptide formed by linking these are each in the form of a single chain (e.g., a polypeptide encoded by a single reading frame), and a dimeric structure of one or more of the Fc region of the first immunoglobulin and the Fc region of the second immunoglobulin, the first GDF15 or its functional variant and the second GDF15 or its functional variant, and the first fusion polypeptide and the second fusion polypeptide formed by linking these is excluded (e.g., a case in which the Fc region of the first or second immunoglobulin contained in the first or second fusion polypeptide is in the form of two chains (two molecules) linked by a disulfide bond, etc.).

[0040] In the dimeric structure of GDF15, the N-terminus of the monomer is exposed so that it can be fused to another protein, while the C-terminus is in a position that is not fused to another protein. Therefore, to form a fusion polypeptide while maintaining the dimeric form of GDF15, it is preferable that the fusion partner (i.e., the Fc region of an immunoglobulin) be linked to the N-terminus of GDF15.

[0041] GDF15 binds to its receptor, GFRAL (GDNF family receptor alpha-like; e.g., GenBank Accession no. NP_997293.2), to form a GDF15-GFRAL complex. In this complex, GDF15 and GFRAL monomers are bound to each other, with the N-terminus of GDF15 located in the middle of the GDF15 dimer. The four N-terminal amino acid residues fused to GDF15 are not observed in the X-ray structure and are therefore predicted to not form a specific structure. The N-terminal 14 amino acid residues of GDF15 are fixed by a Cys7-Cys14 disulfide bond and are not involved in the interaction between monomers within the GDF15 dimer. Furthermore, the N-terminal 14 amino acid residues of GDF15 are located at a distance that structurally prevents them from interacting (binding) with the GFRAL receptor, so it is predicted that deletion of the N-terminal 14 amino acid residues of GDF15 will not affect GDF15's binding to the GFRAL receptor.

[0042] The GDF15 monomer contains four intramolecular disulfide bonds and one interdimeric disulfide bond. The intramolecular disulfide bonds are thought to stabilize the structure, and the disulfide bond connecting Cys7 and Cys14 is located at the end of the monomeric structure and serves to anchor the N-terminal loop structure. Because the N-terminal 14 amino acid residues do not play a direct role in GFRAL receptor binding, deleting one or more of them does not affect the function or structure of GDF15. However, because Cys15 forms a disulfide bond with Cys88, deleting Cys15 does affect the three-dimensional structure of GDF15. Therefore, sites that can be removed while maintaining the function and structure of GDF15 are up to 14 amino acids from the N-terminus (i.e., one or more of the 14 amino acids from Ala1 to Cys14 in SEQ ID NO: 1, for example, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) from the N-terminus). For example, the GDF15 monomer is a wild-type (SEQ ID NO: 1) or a form in which the wild-type has the 14 N-terminal amino acids deleted (ΔN14GDF15; SEQ ID NO: 2).

[0043] The complex structure shows wtGDF15 (SEQ ID NO: 1) or ΔN14GDF15 (SEQ ID NO: 1 with 14 amino acids deleted from the N-terminus) bound to its receptor, GFRAL. The N-terminus (amino acid residues 1-14) of wt (wild-type) GDF15 is fixed by a Cys7-Cys14 disulfide bond, with the N-terminus exposed on the lower left and lower right sides, respectively. When the N-terminal 14 amino acids are deleted from wtGDF15, the N-terminus is exposed on the upper left and upper right sides, respectively.

[0044] When the Fc protein of an immunoglobulin is fused with GDF15 to form a fusion protein, the C-terminus of the Fc protein naturally links to ΔN14GDF15, which has 14 amino acid residues removed from the N-terminus (see Figure 3a).However, in the case of wtGDF15, the N-terminus may be positioned in a somewhat difficult direction for linking to the Fc protein (see Figure 3b).

[0045] In the complex structure of Fc-ΔN14GDF15 or Fc-wtGDF15 bound to GFRAL, GDF15 and Fc are functionally dimeric, and in the case of Fc-ΔN14GDF15, the structural arrangement favors the formation of Fc dimers and ΔN14GDF15 dimers, respectively. Therefore, Fc-ΔN14GDF15 is more advantageous than Fc-wtGDF15 in terms of dimer formation, even though it does not interfere with complex formation with GFRAL.

[0046] The in vivo (blood) half-life in a mammal of GDF15 or a functional variant thereof contained in the fusion polypeptide or fusion polypeptide dimer provided herein may be increased by 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 4.5-fold or more, about 5-fold or more, about 5.5-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 compared to GDF15 or a functional variant thereof not fused to the Fc region of an immunoglobulin.

[0047] Thus, due to the increased half-life of GDF15 or its functional variant, GDF15 or its functional variant in the form of a fusion polypeptide to which the Fc region of an immunoglobulin is linked has the advantage of being able to extend the administration interval compared to GDF15 or its functional variant in the form to which the Fc region of an immunoglobulin is not linked.

[0048] Production of fusion polypeptides Fusion polypeptides comprising GDF15 or a functional variant thereof and an immunoglobulin Fc region are produced by conventional chemical synthesis methods or recombinant methods, and may not be naturally occurring.

[0049] 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 a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. For example, the vector used in the recombinant vector may be constructed 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.

[0050] In the recombinant vector, the nucleic acid molecule encoding the fusion polypeptide is operably 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 is "operably linked" so as to control the transcription and / or translation of the other nucleic acid sequence.

[0051] The recombinant vector can typically be constructed as a vector for cloning or an expression vector for expression. The expression vector can be any of those 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.

[0052] The recombinant vector is expressed in a eukaryotic host. When expressed in a eukaryotic host, the recombinant vector contains, in addition to the nucleic acid molecule to be expressed and the promoter, ribosome binding site, secretory signal sequence (see Korean Patent Publication No. 2015-0125402), and / or transcription / translation termination sequence described above, a replication origin operable in a eukaryotic cell, such as, but not limited to, an f1 origin, an SV40 origin, a pMB1 origin, an adenovirus origin, an AAV origin, and / or a BBV 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, a fusion promoter (KR10-1038126 or KR10-1868139), or an HSV tk promoter) may be used, and any secretory signal sequence that is commonly available may be used as the secretory signal sequence, for example, but is not limited to, the secretory signal sequence described in Korean Patent Publication No. 2015-0125402, and a polyadenylation sequence may be included as a transcription termination sequence.

[0053] The recombinant cell is obtained by introducing (transforming or transfecting) the recombinant vector into a suitable host cell. The host cell can be selected from all eukaryotic cells that allow stable and continuous cloning or expression of the recombinant vector. Eukaryotic cells that can be used as hosts include yeast (Saccharomyces 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, cornea-derived PER-C6, diploid fibroblast-derived cells, myeloma cells, or HepG2, other animal cells (e.g., MDCK), insect cells (e.g., Sf9 cells, Sf21 cells, Tn-368 cells, BTI-TN-5B1-4 cells, etc.), and hybridomas.

[0054] 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 its functional variants, or fusion polypeptides or fusion polypeptide dimers containing the same, with increased in vivo stability compared to unfused forms. The method for producing the fusion polypeptide or fusion polypeptide dimer may include culturing recombinant cells containing the nucleic acid molecule. The culturing step may be carried out under conventional culture conditions. Furthermore, the production method may further include a step of isolating and / or purifying the fusion polypeptide or fusion polypeptide dimer from the culture after the culturing step.

[0055] The nucleic acid molecule or a recombinant vector containing the same can be delivered (introduced) into a host cell using a delivery method well known in the art, such as, but not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and gene bombardment when the host cell is a eukaryotic cell.

[0056] The host cells transformed (transfected 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, if the selection marker is a specific antibiotic resistance gene, recombinant cells into which the recombinant vector has been introduced can be easily selected by culturing the cells in a medium containing the antibiotic.

[0057] Medical uses Provided are compositions (pharmaceutical compositions or health functional food compositions) for weight loss, dietary regulation (reduction of food intake), or prevention, amelioration, alleviation, and / or treatment of metabolic diseases, comprising one or more selected from the group consisting of a fusion polypeptide, a fusion polypeptide dimer, a fusion polypeptide-encoding nucleic acid molecule, a recombinant vector comprising said nucleic acid molecule, and a recombinant cell comprising said recombinant vector; and / or methods for weight loss, dietary regulation (reduction of food intake), or prevention, amelioration, alleviation, and / or treatment of metabolic diseases, comprising the step of administering a pharmaceutically effective amount of one or more selected from the group consisting of said fusion polypeptide, a fusion polypeptide dimer, a fusion polypeptide-encoding nucleic acid molecule, a recombinant vector comprising said nucleic acid molecule, and a recombinant cell comprising said recombinant vector to a subject in need thereof.

[0058] The metabolic disease means any disease caused by metabolic disorder, and is selected from the group consisting of obesity, diabetes (e.g., type 2 diabetes), non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), etc.

[0059] As used herein, the term "pharmaceutically effective amount" refers to the content or dosage of an active ingredient sufficient to achieve a desired effect. The content or dosage of the active ingredient (one or more selected from the group consisting of a fusion polypeptide comprising GDF15 or a functional variant thereof and an immunoglobulin Fc region, a fusion polypeptide dimer, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector) in the pharmaceutical composition may vary depending on factors such as the formulation method, 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 dose of the active ingredient may be in the range of, but is not limited to, 0.001 to 1000 mg / kg, 0.01 to 100 mg / kg, 0.01 to 50 mg / kg, 0.01 to 20 mg / kg, 0.01 to 10 mg / kg, 0.01 to 5 mg / kg, 0.1 to 100 mg / kg, 0.1 to 50 mg / kg, 0.1 to 20 mg / kg, 0.1 to 10 mg / kg, 0.1 to 5 mg / kg, 1 to 100 mg / kg, 1 to 50 mg / kg, 1 to 20 mg / kg, 1 to 10 mg / kg, or 1 to 5 mg / kg. In another example, the content of the active ingredient in the pharmaceutical composition is 0.01% by weight to 99.9% by weight, 0.01% by weight to 90% by weight, 0.01% by weight to 80% by weight, 0.01% by weight to 70% by weight, 0.01% by weight to 60% by weight, 0.01% by weight to 50% by weight, 0.01% by weight to 40% by weight, 0.01% by weight to 30% by weight, 1% by weight to 99.9% by weight, 1% by weight to 90% by weight, 1% by weight to 80% by weight, 1% by weight to 70% by weight, 1% by weight to 60% by weight, 1% by weight to 50% by weight, based on the total weight of the pharmaceutical composition. The amount % can be 1% to 40% by weight, 1% to 30% by weight, 5% to 99.9% by weight, 5% to 90% by weight, 5% to 80% by weight, 5% to 70% by weight, 5% to 60% by weight, 5% to 50% by weight, 5% to 40% by weight, 5% to 30% by weight, 10% to 99.9% by weight, 10% to 90% by weight, 10% to 80% by weight, 10% to 70% by weight, 10% to 60% by weight, 10% to 50% by weight, 10% to 40% by weight, or 10% to 30% by weight.

[0060] The administration interval (the time interval between two adjacent administrations) of the active ingredient or pharmaceutical composition containing the active ingredient provided herein can be adjusted depending on the concentration or condition of the active ingredient (e.g., the presence or absence of a mutation), or the patient's condition or symptoms, and can be, for example, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 6 weeks or more, 8 weeks or more, 10 weeks or more, or 12 weeks or more. The maximum administration interval can be, but is not limited to, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, or about 3 months, and can be increased or decreased depending on the concentration or condition of the active ingredient (e.g., the presence or absence of a mutation), or the patient's condition or symptoms, and in one embodiment, the administration interval is appropriately determined within about 1 week to about 3 months.

[0061] In addition to the active ingredient, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The carrier may be one or more selected from the group consisting of, but not limited to, those commonly used in formulating drugs containing proteins, nucleic acids, or cells, such as lactose, textrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition may also further 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, which are commonly used in the manufacture of pharmaceutical compositions.

[0062] The subject to which the pharmaceutical composition is administered may be a mammal, including a primate, such as a human, a monkey, or a rodent, such as a mouse or a rat, or a cell, tissue, cell culture, or tissue culture derived therefrom.

[0063] The pharmaceutical composition may be administered orally or parenterally, or by contacting with cells, tissues, or body fluids. Specifically, parenteral administration may be by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration. Since proteins or peptides are digested during oral administration, oral compositions must be formulated to coat or protect the active agent from gastrointestinal degradation.

[0064] 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, granules, tablet or capsule, and may further contain a dispersant or stabilizer for formulation.

[0065] Another embodiment provides a method for increasing the biological duration of GDF15 or a functional variant thereof, comprising fusing (or linking or conjugating) GDF15 or a functional variant thereof to an Fc region of an immunoglobulin. In one embodiment, the fusing step may comprise fusing (or linking or conjugating) the Fc region of an immunoglobulin to the N-terminus of GDF15 or a functional variant thereof with or without a linker. Another embodiment provides a method for reducing the immunogenicity of GDF15, an Fc region of an immunoglobulin, or a fusion polypeptide comprising them, comprising fusing (or linking or conjugating) GDF15 or a functional variant thereof to the Fc region of an immunoglobulin via a flexible linker. The flexible linker is as described above. The fusing (or linking or conjugating) step is performed in vitro. [Effects of the Invention]

[0066] The GDF15 or its functional variants fused to the Fc region of immunoglobulin provided by the present invention have a longer duration when administered to the body than when not fused to the Fc region of immunoglobulin, allowing for longer administration intervals and thereby reducing the administration volume, thereby providing advantageous effects in terms of ease of administration and / or economic aspects, and also has excellent pharmacological effects, making it useful for application in fields requiring treatment with GDF15 or its functional variants. [Brief explanation of the drawings]

[0067]

Figure 1

Figure 2

Figure 3a

Figure 3b

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

[0068] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention and the scope of the present invention is not limited to these examples.

[0069] Example 1: Production of fusion polypeptides 1.1. Cloning and Cultivation of the Gene Encoding the Fusion Polypeptide Fusion polypeptides IgG1-GDF(CRL), HIgG1-GDF(CRL), IgG4-GDF, HIgG4-GDF, IgG4-GDF(CRL), and HIgG4-GDF(CRL) (see FIG. 1) were prepared by fusing immunoglobulin Fc with or without a hinge (IgG1-Fc (without hinge: SEQ ID NO: 3; HIgG1-Fc (with hinge: [SEQ ID NO: 4]-[SEQ ID NO: 3]), Mutated IgG4-Fc (without hinge: SEQ ID NO: 7), or Mutated HIgG4-Fc (with hinge: [SEQ ID NO: 12]-[SEQ ID NO: 7]) with the target polypeptide, mature GDF15 (referred to as GDF or GDF15; SEQ ID NO: 1) or a GDF15 mutant (referred to as GDF(CRL); 14 amino acids deleted at the N-terminus: SEQ ID NO: 2). The amino acid sequences of each portion contained in the fusion polypeptides are summarized in Tables 2, 3, and 4 below.

[0070] [Table 2]

[0071] [Table 3]

[0072] [Table 4]

[0073] 1.1.1 Preparation 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: 14) was synthesized by Bioneer, Inc., with reference to the amino acid sequence of UniprotKB Q99968. SEQ ID NO: 14 (339 bp) 1 GCCCGGAACG GCGACCACTG CCCCCTGGGG CCCGGACGGT GC TGCCGGCT 51 GCACACCGTG CGGGCCTCCC TGGAGGACCT GGGCTGGGCC GACTGGGTGC 101 TGTCCCCAAG GGAGGTGCAA GTGACCATGT GCATCGGCGC CTGCCCATCT 151 CAGTTCCGGG CCGCCAACAT GCACGCTCAG ATCAAGACCA GCCTGCACCG 201 GCTGAAGCCC GACACCGTGC CCGCCCCCTG CTGCGTGCCC GCCTCCTACA 251 ACCCCATGGT GCTGATTCAG AAGACCGACA CCGGCGTGAG CCTGCAGACC 301 TACGACGACC TGCTGGCCAA GGACTGCCAC TGCATCTAA (*The underlined part is the GDF(CRL))

[0074] IgG1-Fc The gene encoding human IgG1 Fc with or without hinge was obtained by PCR using a plasmid containing the genes encoding human IgG1 core hinge and IgG1 Fc. SEQ ID NO: 15 (678 bp) 1 GACAAAACTC ACACATGCCC ACCGTGCCCA GCACCTGAAC TCCTGGGGGG 51 ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT 101 CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC 151 CCTGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC 201 CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC CGTGTGGTCA 251 GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGAGTACAAG 301 TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC 351 CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTATACC CTGCCCCCAT 401 CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA 451 GGCTTCTATC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC 501 GGAGAACAAC TACAAGACCA CGCCTCCCGT GCTGGACTCC GACGGCTCCT 551 TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG 601 AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC 651 GCAGAAGAGC CTCTCCCTGT CTCCGGGT (*The underlined part is the gene encoding the IgG1 Core Hinge)

[0075] 1.1.1.3.IgG4-Fc The gene encoding human IgG4 Fc with or without hinge was obtained by PCR using a plasmid containing the genes encoding human IgG4 hinge and IgG4 Fc. SEQ ID NO: 16 (684 bp) 1 GAGTCCAAAT ATGGTCCCCC ATGCCCACCC TGCCCA GCAC CTGAGGCCGC 51 CGGGGGACCG TCAGTCTTCC TCTTCCCCCC AAAACCCAAG GACACCCTCA 101 TGATCTCCCG GACCCCTGAG GTCACGTGCG TGGTGGTGGA CGTGTCCCAG 151 GAGGACCCCG AGGTGCAGTT CAACTGGTAC GTGGACGGCG TGGAGGTGCA 201 CAACGCCAAG ACCAAGCCCC GGGAGGAGCA GTTCAACTCC ACCTACCGGG 251 TGGTGTCCGT GCTGACCGTG CTGCACCAGG ACTGGCTGAA CGGCAAGGAG 301 TACAAGTGCA AGGTGTCCAA CAAGGGCCTG CCCTCCTCCA TCGAGAAGAC 351 CATCTCCAAG GCCAAGGGCC AGCCCCGGGA GCCCCAGGTG TACACCCTGC 401 CCCCCTCCCA GGAGGAGATG ACCAAGAACC AGGTGTCCCT GACCTGCCTG 451 GTGAAGGGCT TCTACCCCTC CGACATCGCC GTGGAGTGGG AGTCCAACGG 501 CCAGCCCGAG AACAACTACA AGACCACCCC CCCCGTGCTG GACTCCGACG 551 GCTCCTTCTT CCTGTACTCC CGGCTGACCG TGGACAAGTC CCGGTGGCAG 601 GAGGGCAACG TGTTCTCCTG CTCCGTGATG CACGAGGCCC TGCACAACCA 651 CTACACCCAG AAGTCCCTGT CCCTGTCCCT GGGC (*The underlined part is the gene encoding the IgG4 hinge)

[0076] 1.1.1.4. Production 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 the above-mentioned genes (mature GDF15, IgG1-Fc, IgG4-Fc) were combined and genes having the following structures (see Figure 4) were inserted to prepare the respective recombinant vectors.

[0077] pHIgG1 - GDF(CRL) '(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG1 Core Hinge (SEQ ID NO: 4)-IgG1 CH2-CH3 (SEQ ID NO: 3)-GS Linker (SEQ ID NO: 18)-GDF(CRL) (SEQ ID NO: 2)-NotI restriction site]-(C-terminus)'

[0078] pIgG1 - GDF(CRL) '(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG1 CH2-CH3 (SEQ ID NO: 3)-GS Linker (SEQ ID NO: 18)-GDF(CRL) (SEQ ID NO: 2)-NotI restriction site]-(C-terminus)'

[0079] pHIgG4 - GDF15 '(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG4 Hinge (SEQ ID NO: 12)-IgG4 CH2-CH3 (SEQ ID NO: 7)-GS Linker (SEQ ID NO: 18)-GDF15 (SEQ ID NO: 1)-NotI restriction site]-(C-terminus)'

[0080] pIgG4 - GDF15 '(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG4 CH2-CH3 (SEQ ID NO: 7)-GS Linker (SEQ ID NO: 18)-GDF15 (SEQ ID NO: 1)-NotI restriction site]-(C-terminus)'

[0081] pHIgG4 - GDF(CRL) '(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG4 Hinge (SEQ ID NO: 12)-IgG4 CH2-CH3 (SEQ ID NO: 7)-GS Linker (SEQ ID NO: 18)-GDF(CRL) (SEQ ID NO: 2)-NotI restriction site]-(C-terminus)'

[0082] pIgG4 - GDF(CRL) '(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG4 CH2-CH3 (SEQ ID NO: 7)-GS Linker (SEQ ID NO: 18)-GDF(CRL) (SEQ ID NO: 2)-NotI restriction site]-(C-terminus)'

[0083] 1.1.2. Cultivation of the gene encoding the fusion polypeptide The prepared recombinant expression vectors pHIgG1-GDF(CRL), pIgG1-GDF(CRL), pHIgG4-GDF(CRL), pIgG4-GDF(CRL), pHIgG4-GDF, and pIgG4-GDF were transformed into ExpiCHO-S TM The cells were transfected with the vector and cultured in ExpiCHO Expression Medium (Thermo Fisher Scientific; 400 mL) for 12 days (Fed-Batch Culture; Day 1 & Day 5 Feeding) to express the fusion polypeptides HIgG1-GDF(CRL), IgG1-GDF(CRL), HIgG4-GDF(CRL), IgG4-GDF(CRL), HIgG4-GDF, and IgG4-GDF.

[0084] 1.2. Purification of fusion polypeptides The fusion polypeptide was purified from the cell culture prepared in Example 1.1 above using Protein A affinity chromatography.

[0085] First, the cell-free culture medium containing the fusion polypeptide was filtered through a 0.22 μm filter. TM A column packed with pcc (GE Healthcare Life Sciences) resin was used. TM The column was attached to a PBS (GE Healthcare Life Sciences) and equilibrated with Phosphate Buffered Saline (PBS, 10 mM sodium phosphate, 150 mM NaCl, pH 7.4). Culture medium filtered through a 0.22 μm filter was injected onto the equilibrated column, and the column was washed again with PBS. After washing, the desired fusion polypeptide was eluted by pouring elution buffer (0.1 M sodium citrate, pH 3.5) through the column. The eluate was immediately adjusted to a neutral pH by adding 1 M Tris, pH 8.5. The eluted fractions with a high concentration of fusion polypeptide and high purity were collected and stored frozen.

[0086] For animal experiments, the eluted fraction samples containing the fusion polypeptide were concentrated and buffer-exchanged with PBS or 20 mM Tris pH 8.0, 150 mM NaCl using an Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge.

[0087] 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 the protein concentration using the following formula: The extinction coefficient of each substance was theoretically calculated using the amino acid sequence (Table 5).

number

[0088] [Table 5]

[0089] Example 2. Pharmacological effects of fusion polypeptides (in vivo) 2.1. 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.).

[0090] In this example, we used a DIO mouse model (C57BL / 6J-DIO, male, 100 mice, 14 weeks old (8 weeks of high-fat diet feeding)) in which obesity was induced in C57BL / 6J mice. The DIO mouse model exhibits clinical characteristics of type 2 diabetes, such as hyperlipidemia, insulin resistance, and hyperglycemia, and is therefore widely used to evaluate the efficacy of improving diabetes and insulin resistance. Furthermore, a large amount of basic data has been accumulated for comparison in research on metabolic diseases such as obesity, diabetes, and hyperlipidemia. Therefore, this model was selected because it is suitable for the pharmacological efficacy test of this embodiment.

[0091] The mouse model fed a high-fat diet for 8 weeks underwent a 2-week quarantine and acclimation period, during which time it was observed daily for general symptoms and healthy animals were selected to confirm their fitness for the experiment. During the acclimation period, animals were individually marked with a red permanent marker on their tails upon acquisition, and temporary identification cards (study name, individual number, and date of entry) were attached to their cages during the quarantine and acclimation period. Upon separation into groups, animals were individually marked with a black permanent marker on their tails, and individual identification cards (study name, group information, individual number, sex, date of entry, and administration period) were attached to each cage.

[0092] To minimize the stress that the experimental animals experienced during subcutaneous administration of the test substance (fusion polypeptide), all animals were trained to adapt to subcutaneous administration by subcutaneously administering 200 μL / head of sterile distilled saline using a 1 mL syringe starting 3 days before the administration of the test substance.

[0093] For healthy animals in which no abnormalities were found during the quarantine and acclimatization periods, the body weight and food intake of all individuals were measured after the acclimatization period.

[0094] Body weight and food intake were measured, and the animals were separated into groups based on body weight so that the average of the two measurements between groups was the same. Test substance administration began the day after group separation. The remaining unselected animals were removed from the test system after group separation.

[0095] 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).

[0096] The diet was fed ad libitum (fed during the purification and testing periods).

[0097] The drinking water was obtained by filtering tap water through a filter sterilizer, irradiating it with ultraviolet light, and then providing the animals with free access to drinking water in polycarbonate drinking bottles (250 mL).

[0098] 2.1.1.HIgG1-GDF(CRL)and IgG1-GDF(CRL) The test substances (HIgG1-GDF(CRL) and IgG1-GDF(CRL)) and the control substance GDF15 (R&D Systems) were administered starting the day after group separation, at 9:00 AM every day. Both the control and test substances were administered subcutaneously. The subcutaneous route of administration for the control and test substances was selected based on the planned clinical administration route.

[0099] Both the control and test substances were administered at a volume of 5 mL / kg. The individual doses were calculated based on the most recent body weight and administered subcutaneously using a disposable syringe (1 mL) once on the first day of the study. The test substance was administered only once on the first day of the study. For comparison, a control group administered the control substance GDF15 was prepared. The comparison group administered GDF15 was administered once a day for five days, a total of five times, all starting at 9:00 AM.

[0100] The composition of the test groups and the dosages are summarized in Table 6 below: [Table 6]

[0101] The observation, measurement and examination schedule for the test group was set as Day 0, with the first day of administration being the first week of administration, which lasted for 7 days.

[0102] The inspection schedule is summarized in Table 7: [Table 7]

[0103] All animals were observed once a day for general clinical symptoms and twice a day for moribundity and death. These observations were carried out from the first day of administration until the end of administration. Any abnormal symptoms observed were recorded on a chart.

[0104] The body weight of each mouse was measured on the first day of administration of the test substance (before administration), and then every day thereafter (up to 9 days). The volume of the solution to be administered with the test substance was determined based on the most recently measured body weight.

[0105] After administering the test substance to the mice, the amount of food intake was measured daily. The amount of food given was measured using an electronic scale for each cage, and the remaining amount was measured to calculate the daily food intake. Mice that chewed the food excessively were excluded from the measurement.

[0106] All experimental results obtained in this embodiment are expressed as mean ± standard error and were analyzed using Prism 5 (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 determine whether the test group was significantly different from the control group (significance levels: 5%, 1%, and 0.1%, two-sided).

[0107] 2.1.2.IgG4-GDF and IgG4-GDF(CRL) All test substances (IgG4-GDF and IgG4-GDF(CRL)) and the control substance Semaglutide (Bachem) were administered subcutaneously. The administration volume for both the control and test substances was 5 mL / kg, with the individual administration volume calculated based on the most recent body weight. The test substances were administered subcutaneously once on the first day of the study using a disposable syringe (1 mL). For comparison, a control group administered the control substance Semaglutide was prepared. The control group administered Semaglutide once daily, with all administrations starting at 9:00 AM.

[0108] The composition of the test groups and the dosages are summarized in Table 8 below: [Table 8]

[0109] The observation, measurement and testing schedule for the test group was set to Day 0, the day administration began, and was carried out in the same manner as in Section 2.1.1.

[0110] The body weight of each mouse was measured on the first day of administration of the test substance (before administration), and then every day thereafter (up to 19 days). The volume of the solution to be administered with the test substance was determined based on the most recently measured body weight.

[0111] After administering the test substance to the mice, the amount of food intake was measured daily. The amount of food given was measured using an electronic scale for each cage, and the remaining amount was measured to calculate the daily food intake. Mice that chewed the food excessively were excluded from the measurement.

[0112] 2.2. Weight loss test results 2.2.1.HIgG1-GDF(CRL)and IgG1-GDF(CRL) The changes in body weight measured in Example 2.1.1 above are shown in Figures 5 and 6, and Table 9 (Body Weight (Group, % of initial)). [Table 9]

[0113] Figure 5 and Table 9 show the changes in body weight following a single administration of the fusion protein IgG1-GDF(CRL) compared with the negative control group (vehicle administration group) and the positive control group (daily administration of GDF15 group). Figure 6 is a graph showing the results on day 7 extracted from the results in Figure 5.

[0114] As can be seen from the results, the negative control group (vehicle-treated group) showed little change in body weight, while the GDF15 daily treatment group (treatment discontinued on Day 5) showed no weight loss from Day 6, the first day after treatment was discontinued. In contrast, the fusion polypeptide in which GDF15(CRL) was fused to IgG1 or Fc containing Hinge showed an immediate weight loss effect after a single administration on Day 0, and the weight loss effect continued unabated throughout the test period (9 days). The weight loss effect increased with time, confirming that the weight loss effect was concentration-dependent. The weight loss effect of these fusion polypeptides can be said to be comparable to that of once-daily administration of GDF15 during the test period.

[0115] 2.2.2.IgG4-GDF and IgG4-GDF(CRL) The changes in body weight measured in Example 2.1.2 are shown in Figure 7 and Table 10 (Body Weight (Group, % of initial)). [Table 10]

[0116] Figures 7, 8, and Table 10 show the changes in body weight following a single administration of the fusion proteins IgG4-GDF and IgG4-GDF(CRL), compared with the negative control group (vehicle-administered group) and the positive control group (daily Semaglutide-administered group). Figure 8 is a graph showing the results on days 7 and 14 extracted from the results in Figure 7.

[0117] As can be seen from the results, the negative control group (vehicle-administered group) experienced a slight weight gain, while the positive control group (daily Semaglutide-administered group) experienced sustained weight loss. A single dose of IgG4-GDF15 maintained weight loss for up to 9 days, regardless of dose. In contrast, a single dose of 1 nmol / kg of IgG4-GDF(CRL) maintained weight loss for up to 10 days, and a single dose of 10 nmol / kg maintained weight loss for up to 15 days, demonstrating that the weight loss effect increased with time, demonstrating a concentration-dependent effect. Furthermore, the Hinge-free IgG4 Fc(Mutated)-GDF(CRL) fusion polypeptide demonstrated superior weight loss at the same dose compared to the Hinge-containing IgG4 Fc(Mutated)-GDF(CRL) fusion polypeptide. The weight loss effect of the IgG4 Fc fusion polypeptide was comparable to that of once-daily Semaglutide administered during the study period.

[0118] 2.3. Dietary intake test results 2.3.1. IgG1-GDF(CRL) and IgG1-GDF(CRL) The changes in food intake measured in Example 2.1.1 above are shown in Table 11 and Figure 9 (cumulative intake up to day 6), respectively. [Table 11]

[0119] As can be seen from the results, the groups treated with the fusion polypeptide fused to IgG1 Fc with or without the GDF(CRL) Hinge showed a reduced food intake effect over the 9-day test period compared to the negative control group (vehicle-treated group), and the reduced food intake effect was concentration-dependent. This reduced food intake effect of the fusion polypeptide is comparable to that of GDF15 administered once daily during the test period.

[0120] 2.3.2.IgG4-GDF and IgG4-GDF(CRL) The changes in food intake measured in Example 2.1.2 above are shown in Table 12 and Figure 10 (cumulative intake up to day 7, days 14 and 19). [Table 12]

[0121] As can be seen from the results, the groups administered with fusion polypeptides containing GDF15 or GDF(CRL) fused to Hinge-less IgG4 Fc(mutated) demonstrated a significant reduction in food intake over the 19-day study period compared to the negative control group (vehicle-treated group). The food intake reduction effect of these fusion polypeptides was comparable to that of semaglutide administered once daily during the study period. When comparing the groups administered with 10 nmol / kg of fusion polypeptides containing GDF15 or GDF(CRL) fused to Hinge-less IgG4 Fc(mutated), the food intake suppression effect of the full-length GDF15 fusion polypeptide group lasted for approximately 10 days, while the food intake suppression effect of the GDF(CRL) fusion polypeptide group lasted for approximately 2 weeks. This indicates that the GDF(CRL) fusion polypeptide at a dose of 10 nmol / kg was slightly more effective at reducing body weight than the full-length GDF15 fusion polypeptide.

[0122] Example 3. Pharmacokinetic study of fusion polypeptides (IgG4-GDF or IgG4-GDF(CRL)) 3.1. Preparation of serum from test and control groups To evaluate the pharmacokinetic properties of each polypeptide when administered subcutaneously to rats, the polypeptides IgG4-GDF and IgG4-GDF(CRL) were administered subcutaneously at 2 mg / kg to SD rats (CoreTech, male, 7 weeks old, approximately 250 g; n = 3 per group; test group). Approximately 200 μl of blood was collected via the tail vein at designated times. Blood was collected before and after administration of the fusion polypeptide, and at 1, 2, 4, 8, 24, 48, 72, 96, 168, 240, and 336 hours. A control group for comparison of pharmacokinetic properties was prepared by subcutaneously administering 2 mg / kg of GDF15 (R&D Systems) in the same manner as above.

[0123] 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 a Human GDF15 Immunoassay (SGD150, R&D Systems) to measure serum concentrations of each polypeptide over time. Parameter values, including AUC (area under the curve), were calculated using PK analysis software (e.g., WinNonlin (Certara LP)).

[0124] 3.2 Pharmacokinetic study results The pharmacokinetic parameters of the fusion polypeptide obtained above are shown in Table 13, and the concentration change of the fusion polypeptide over time is shown in Figure 11: [Table 13]

[0125] As can be seen from the above results, the safety in blood (serum) of the IgG4-GDF15 (half-life: 101 hours) and IgG4-GDF(CRL) (half-life: 114 hours) fusion proteins was confirmed to be significantly increased (more than five times) compared to GDF15 (half-life: 19 hours).

[0126] From the above description, it will be understood by those skilled in the art that the present invention can be implemented in other embodiments without changing the technical spirit or essential features of the present invention. 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, rather than the detailed description, and their equivalent concepts.

Claims

1. A fusion polypeptide dimer comprising two fusion polypeptides, each of the fusion polypeptides GDF15 (Growth / differentiation factor 15) or the GDF15 variant of SEQ ID NO: 2, and comprising the Fc region of an immunoglobulin, the immunoglobulin Fc region is a single-chain human IgG4 Fc region and is linked to the N-terminus of the GDF15 or GDF15 variant via a flexible peptide linker; the human IgG4 Fc region comprises the amino acid sequence of SEQ ID NO:7; The flexible peptide linker is represented by (GGGGS)n (n is 1, 2, 3, 4 or 5); The two fusion polypeptides form a dimer between GDF15 or a GDF15 variant, The GDF15 comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1; The GDF15 variant comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 2; A fusion polypeptide dimer, wherein the GDF15 or GDF15 variant linked to the human IgG4 Fc region has an increased weight loss effect or an increased duration of the weight loss effect compared to the GDF15 or GDF15 variant linked to a human IgG1 Fc region having the amino acid sequence of SEQ ID NO:

3.

2. The fusion polypeptide dimer of claim 1, which is a homodimer.

3. A fusion polypeptide dimer as described in claim 1 or 2, wherein GDF15 or GDF15 variant linked to the Fc region of an immunoglobulin in the fusion polypeptide has an in vivo half-life that is increased by 1.5 times or more compared to GDF15 or GDF15 variant that does not bind to the Fc region of an immunoglobulin.

4. A nucleic acid molecule encoding the fusion polypeptide dimer of 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 the fusion polypeptide dimer of claim 1, comprising culturing the recombinant cell of claim 6.

8. 1. A method for enhancing the in vivo stability of GDF15 or a GDF15 variant, comprising linking a single-chain human IgG4 Fc region to the N-terminus of GDF15 or a GDF15 variant via a flexible peptide linker, the human IgG4 Fc region comprises the amino acid sequence of SEQ ID NO:7; The flexible peptide linker is represented by (GGGGS)n [(SEQ ID NO: 13)n; n is 1, 2, 3, 4, or 5]; the GDF15 or GDF15 variant forms a dimer; The GDF15 comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1; The GDF15 variant comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 2; A method for increasing the in vivo stability of GDF15 or a GDF15 variant, wherein the GDF15 or GDF15 variant linked to the human IgG4 Fc region has an enhanced weight loss effect or an enhanced duration of the weight loss effect compared to the GDF15 or GDF15 variant linked to the human IgG1 Fc region having the amino acid sequence of SEQ ID NO:

3.

9. The method for enhancing the in vivo stability of GDF15 or a GDF15 variant according to claim 8, wherein the GDF15 or GDF15 variant forms a homodimer.

10. A method for increasing the in vivo stability of GDF15 or a GDF15 mutant described in claim 8 or 9, wherein the GDF15 or GDF15 mutant linked to the human IgG4 Fc region has an in vivo half-life that is increased by 1.5 times or more compared to GDF15 or a GDF15 mutant that does not bind to the human IgG4 Fc region.

11. A composition for weight loss comprising the fusion polypeptide dimer of claim 1 or 2.

12. A dietary regulating composition comprising the fusion polypeptide dimer of claim 1 or 2.

13. A pharmaceutical composition for preventing or treating a metabolic disease, comprising the fusion polypeptide dimer of claim 1 or 2.

14. The pharmaceutical composition of claim 13, wherein the metabolic disease is obesity, diabetes, or non-alcoholic fatty liver disease.

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