RU2797520C2 - FUSION POLYPEPTIDE INCLUDING Fc REGION OF IMMUNOGLOBULIN AND GDF15 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to recombinant production of fusion proteins based on Growth/Differentiation Factor 15 (GDF15) and can be used in medicine to reduce body weight, control diet and prevent or treat a metabolic disease. A fusion polypeptide was constructed comprising GDF15 or a functional deleted variant thereof, to the N-terminus of which one strand of the human IgG4 Fc region was attached via a flexible peptide linker.

EFFECT: invention makes it possible to obtain a fusion polypeptide with an extended period of existence of GDF15 in vivo.

19 cl, 11 dwg, 13 tbl, 3 ex

Description

FIELD OF TECHNOLOGY

A fusion polypeptide is presented, comprising GDF15 (growth/differentiation factor 15) and an immunoglobulin Fc region, a pharmaceutical composition comprising a fusion polypeptide, and a method for increasing the duration of GDF15 in vivo, including a fusion with an immunoglobulin Fc region.

BACKGROUND OF THE INVENTION

Most protein or peptide drugs have a short period of activity in the body and have a low absorption rate when administered by a method other than intravenous, and therefore they present the inconvenience of administering these drugs repeatedly at short intervals when long-term treatment is required. To eliminate this inconvenience, it is required to develop a technique for the continuous release of drugs at a single dose. In response to such needs, sustained release formulations are being developed.

For example, sustained release formulations are being actively researched, where microparticles are prepared in the form of surrounding a protein or peptide drug in a biodegradable polymer matrix, and during their administration, the matrix material is slowly degraded and removed in the body, and the drug is slowly released.

On the other hand, GDF15 (growth/differentiation factor 15) is one of the TGF-beta family, and is a 25 kDa homodimer and is a secretory protein circulating in plasma. Plasma GDF15 levels are associated with BMI (body mass index) and GDF15 plays a long-term regulator of energy homeostasis. GDF15 also protects against pathological conditions such as cardiovascular disease, myocardial hypertrophy, ischemic injury, and the like. In addition, GDF15 protects against renal tubular injury and renal interstitial injury in models of type 1 and type 2 diabetes. Moreover, GDF15 has a protective effect against age-related loss of sensory and motor nerves and may promote repair of peripheral nerve damage. In addition, GDF15 has the effects of reducing body weight, reducing body fat and glucose tolerance, as well as increasing systemic energy intake and oxidative metabolism. GDF15 exerts glycemic control effects through weight-dependent and weight-independent mechanisms.

There is a need to develop a technique for increasing the persistence in the body of the GDF15 protein, which has various pharmacological effects.

DISCLOSURE

TECHNICAL PROBLEM

The present disclosure provides a technology for constructing a fusion polypeptide by linking GDF15 (growth/differentiation factor 15) or a functional variant thereof to the Fc region of an immunoglobulin; thereby increasing the in vivo duration by increasing the in vivo half-life of GDF15 compared to the case of no Fc region to enhance the pharmacological effect of GDF15 and/or increase the administration interval.

An embodiment provides a fusion polypeptide comprising GDF15 or a functional variant thereof and an immunoglobulin Fc region.

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

The immunoglobulin Fc region included in the fusion polypeptide has the role of enhancing the stability of GDF15 or a variant thereof and, for example, may have the effect of prolonging the half-life (eg, in vivo half-life). In one embodiment, the Fc region of an immunoglobulin may be selected from the group consisting of an Fc region of IgG1 and an Fc region of IgG4. An IgG1 Fc region may include a CH2 domain, a CH3 domain, or a CH2+CH3 domain of IgG1, and may or may not include an IgG1 hinge region at the N-terminus. The IgG4 Fc region may include the CH2 domain, the CH3 domain, or the CH2+CH3 domain of IgG4, and may or may not include the IgG4 hinge region at the N-terminus.

The fusion polypeptide may also include a peptide linker between the immunoglobulin Fc region and GDF15 or a functional variant thereof. In one embodiment, the peptide linker may be a flexible linker such that the Fc region and GDF15 that are fused to both ends of the linker can independently function. In a specific embodiment, the peptide linker may not be a rigid linker. For example, the peptide linker may be a GS linker that includes at least one Gly (G) and at least one Ser (S) in repeats, and, for example, may be represented as (GGGGS)n (n is the number of repeats of GGGGS (SEQ ID NO: 13) and is an integer from 1 to 10 or an integer from 1 to 5 (1, 2, 3, 4 or 5)), but is not limited to this.

In a fusion polypeptide, GDF15 or a functional variant thereof fused to the Fc region of an immunoglobulin has increased stability (lifetime) in vivo (or in blood) compared to GDF15 or a functional variant thereof not fused to an immunoglobulin Fc region (e.g., half-life in vivo or increases in the blood). In addition, GDF15 or a functional variant thereof fused to an immunoglobulin Fc region has an improved pharmacological effect (eg, weight loss effect) compared to GDF15 or a functional variant thereof not fused to an immunoglobulin Fc region.

Another embodiment provides a fusion polypeptide dimer comprising 2 of the fusion polypeptides. A fusion polypeptide dimer can be formed by linking (combining) GDF15 or a functional variant thereof from two fusion polypeptides.

Another embodiment provides a nucleic acid molecule encoding a fusion polypeptide.

Another embodiment provides a recombinant vector comprising a nucleic acid molecule.

Another embodiment provides a recombinant cell comprising a recombinant vector.

Another embodiment provides a composition (pharmaceutical composition or functional health product composition) for weight loss, diet control (reduction in food volume), or prevention, improvement, alleviation, and/or treatment of a metabolic disease, comprising at least one selected from the group , consisting of a fusion polypeptide, a dimer of a fusion polypeptide, a nucleic acid molecule encoding a fusion polypeptide, a recombinant vector comprising a nucleic acid molecule, and a recombinant cell comprising a recombinant vector. Another embodiment provides a method for reducing body weight, a method for controlling diet (reducing the amount of food), or a method for preventing, improving, alleviating, and/or treating a metabolic disease, comprising administering a pharmaceutically effective dose of at least one selected from the group consisting of a fused a polypeptide, a fusion polypeptide dimer, a nucleic acid molecule encoding a fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector, to a subject in need of prevention, improvement, alleviation, and/or treatment of a metabolic disease. Metabolic disease means all diseases caused by a metabolic disorder, and it can be 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.) , and so on.

Another embodiment provides a method for producing GDF15 or a functional variant thereof with an extended in vivo half-life (or in blood) comprising expressing the recombinant vector in a cell, or a method for producing a fusion polypeptide comprising GDF15 or a functional variant thereof with an extended in vivo half-life (or in blood), or a homodimer comprising a fusion polypeptide.

Another embodiment provides a method for increasing the in vivo lifespan of GDF15 or a functional variant thereof, comprising fusing (or linking or combining) GDF15 or a functional variant thereof to an immunoglobulin Fc region. In one particular embodiment, the fusion may include fusion (or linking or combining) 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 immunoglobulin Fc region, or a fusion polypeptide comprising them, comprising fusing (or linking or combining) an immunoglobulin Fc region with GDF15 or a functional variant thereof using a flexible linker (e.g., a GS linker). The fusion (or linking or combining) step can be carried out in vitro.

TECHNICAL SOLUTION

Hereinafter, the present invention will be described in more detail:

GDF15

GDF15 consists of amino acids 197 (A) to 308 (I), excluding signal peptide and propeptide at 308 amino acids in total (UniProt Q99988) (SEQ ID NO: 1; Fig. 2; mature form):

As used herein, GDF15 means a polypeptide essentially comprising amino acid sequence 197(A) to 308(I) of the full-length protein (UniProt Q99988) (SEQ ID NO: 1, see Fig. 2; ARNG DHCPLGPGRC CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA ANMHAQIKTS LHRLKPDTVP APCCVPASY N PMVLIQKTDT GVSLQTYDDL LAKDCHCI), or an amino acid sequence having sequence homology of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or above with an amino acid sequence in the range that maintains the unique activity and structure of GDF15 unless otherwise noted.

In the present invention, a functional variant of GDF15 may be a variant modified so that GDF15 maintains a unique activity and structure and is also useful for the formation of a dimeric structure. In one embodiment, a functional GDF15 variant may be an N-terminal deletion variant in which at least one (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ) of the 14 amino acid residues at the N-terminus of the GDF15 amino acid sequence of SEQ ID NO: 1 (namely, 14 amino acid residues in total from 1 to 14 in SEQ ID NO: 1) (e.g., at least one from the N-terminus in order), for example, all 14 amino acid residues are deleted (hereinafter, may be represented as 'ΔN14GDF15' or 'GDF(CRL)'). In one particular embodiment, a functional GDF15 variant 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 sequence homology of 80% or greater, 85% or higher , 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater with an amino acid sequence in the range that supports the unique activity and structure of GDF15.

Fc immunoglobulin

An immunoglobulin Fc to be fused with GDF15 or a functional variant thereof has the role of enhancing the stability of GDF15 or a functional variant thereof, and for example, may have the effect of prolonging the half-life (e.g., in vivo half-life) and/or reducing renal filtration, etc. In one embodiment, the Fc region of an immunoglobulin may be selected from an IgG1 Fc and an IgG4 Fc. An IgG1 Fc region may include a CH2 domain, a CH3 domain, or a CH2+CH3 domain of IgG1, and may or may not include an IgG1 hinge region at the N-terminus. The IgG4 Fc region may include the CH2 domain, the CH3 domain, or the CH2+CH3 domain of IgG4, and may or may not include the IgG4 hinge region at the N-terminus.

The IgG1 may be from primates such as humans or rodents such as mice, rats and the like, and for example it may be human IgG1 (UniProtKB P01857). The IgG4 may be from primates such as humans or rodents such as mice, rats and the like, and for example it may be human IgG4 (UniProtKB P01861).

An IgG1 Fc region may include a CH2 domain, a CH3 domain, or a CH2+CH3 domain of IgG1, and may or may not include an IgG1 hinge region at the N-terminus. The IgG4 Fc region may include the CH2 domain, the CH3 domain, or the CH2+CH3 domain of IgG4, and may or may not include the IgG4 hinge region at the N-terminus.

In one specific embodiment, an IgG1 Fc may be a polypeptide comprising a CH2 domain and a CH3 domain of human IgG1 (SEQ ID NO: 3), or a polypeptide further comprising a human IgG1 hinge region (SEQ ID NO: 4) at the N-terminus of the amino acid sequence SEQ ID NO: 3. As described herein, the Fc region of IgG1 can be interpreted as including an amino acid sequence having a sequence homology of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or higher, 98% or higher, or 99% or higher with the amino acid sequence of SEQ ID NO: 3, or 'N-terminus-(SEQ ID NO: 4)-(SEQ ID NO: 3)-C-terminus' in range that maintains the unique activity and structure of IgG1.

An IgG4 Fc may be a polypeptide comprising a CH2 domain and a CH3 domain 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 described herein, the Fc region of IgG4 can be interpreted as comprising an amino acid sequence having sequence homology of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or higher or 99% or higher with the amino acid sequence of SEQ ID NO: 5, or 'N-terminus-(SEQ ID NO: 10)-(SEQ ID NO: 5)-C-terminus' (or the amino acid sequence of the Fc variant IgG4 described below) in a range that maintains the unique activity and structure of IgG4.

When the Fc region of an immunoglobulin is used to extend (in vivo) the half-life of the protein associated with its N-terminus or C-terminus, it is important to minimize any effector function of the Fc in order to reduce side effects from the Fc region of the immunoglobulin. In this aspect, the human IgG4 Fc region is advantageous due to its low binding capacity to FcγR and complement factors compared to other IgG subtypes. In addition, the Fc region of human IgG4 may have reduced effector function by including an amino acid substitution. Amino acid substitution of the Fc region of human IgG4 to reduce effector function may include at least one of substitution of phenylalanine, which is the 234th residue of human IgG4 (UniprotKB P01861), with alanine and substitution of the 235th residue of leucine with alanine (contained in IgG4 Fc domain CH2-CH3 site, amino acid residue number corresponds to EC numbering [Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, ^5th edition, US Dept. of Health and Human Services, Bethesda, MD, NIH Publication no. 91-3242]). In addition, the IgG4 Fc region may include an amino acid substitution that stabilizes heavy chain dimer formation and prevents formation of the IgG4 Fc half chain. Such amino acid substitution may include the replacement of the 228th amino acid residue of the human IgG4 Fc region (UniprotKB P01861) serine (according to EC numbering; located in the hinge region) with proline.

In one specific embodiment, to reduce immunogenicity, the Fc region may include, but is not limited to, mutations other than those described above.

In one specific embodiment, the immunoglobulin Fc region may include the CH2-CH3 Fc domain of human IgG4 (SEQ ID NO: 5 (general); SEQ ID NO: 6 (wild type) or SEQ ID NO: 7, 8, or 9 (variant)) or further include a human IgG4 hinge at the N-terminus of the CH2-CH3 domain (SEQ ID NO: 10 (general); SEQ ID NO: 11 (wild type) or SEQ ID NO: 12 (variant)).

Fc regions of immunoglobulins that can be used in the present invention are presented in the following Table 1:

Table 1 Region Amino acid sequence SEQID NO: Human IgG1-Fc (UniProtKB P01857) Core hinge area DKTHT CPPCP 4 CH2-CH3 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLY SKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGX (X is absent or is K) 3 Human IgG4-Fc (UniProtKB P01861) Hinge area (general) ESKYGPPCPX CP (X is S or P) 10 Hinge region (wild type) ESKYGPPCPS CP (X is S in SEQ ID NO: 10) eleven Hinged area (option) ESKYGPPCPP CP (X is P, in SEQ ID NO: 10) 12 CH2-CH3 (common) APEX1X2GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGX3 (X1 is F or A, X2 is L or A, X3 is absent or is K) 5 CH2-CH3 (wild type) APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFF LYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (X1 is F, X2 is L, X3 is K, in SEQ ID NO: 5) 6 CH2-CH3 (option) APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFF LYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG (X1 is A, X2 is A, X3 is absent, in SEQ ID NO: 5) 7 CH2-CH3 (option) APEALGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFF LYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG (X1 is A, X2 is L, X3 is absent, in SEQ ID NO: 5) 8 CH2-CH3 (option) APEFAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFF LYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG (X1 is F, X2 is A, X3 is absent, in SEQ ID NO: 5) 9

Fusion polypeptide

The fusion polypeptide provided herein comprises an immunoglobulin Fc region and GDF15 or a functional variant thereof linked to the C-terminus of an immunoglobulin Fc region. The Fc region of an immunoglobulin and GDF15 or a functional variant thereof are as described above.

In a Fc fusion polypeptide, the immunoglobulin region and GDF15, or a functional variant thereof, can be linked covalently or non-covalently, or linked using a suitable linker (eg, a peptide linker) or directly without a linker. In one embodiment, the peptide linker may be a polypeptide consisting of any number of amino acids 1 to 20, 1 to 15, 1 to 10, 2 to 20, 2 to 15, or 2 to 10, and the species contained in it amino acids are not limited. In the present disclosure, the peptide linker may be a flexible linker, so that the Fc region and GDF15, which are fused to both ends of the linker, can independently exert their function (for example, for the Fc region, stabilizing activity, such as increasing half-life, reducing renal filtration, etc.). In a specific embodiment, the peptide linker may not be a rigid linker. For example, the peptide linker may include at least one amino acid residue selected from the group consisting of Gly, Asn, Ser, Glu, and Lys, and may also include, but is not limited to, neutral amino acids such as Thr and/or Ala, and the amino acid sequence suitable for the peptide linker is known in the art.

In one embodiment, the peptide linker may be a GS linker repeatedly comprising one or more of Gly (G) and one or more of Ser (S), and for example, may be (GGGGS)n (n is the number of GGGGS repeats (SEQ ID NO: 13) and is an integer from 1 to 10 or an integer from 1 to 5 (1, 2, 3, 4 or 5)), but is not limited to this. In the GS linker, Gly (glycine), which is an amino acid having hydrogen (-H) as the R group, is non-polar and has a high degree of freedom (angle phi, psi) and excellent mobility; and Ser (Serine), which is an amino acid having -CH2-OH as the R group, has a small size and polarity to form a hydrogen bond with water, which is useful for maintaining stability, thereby helping to reduce non-specific interaction between the GS linker and GDF15 or Fc region. In addition, the GS linker has a flexible structure, which is advantageous for reducing immunogenicity. In addition, the GS linker may play a role in the spatial separation of the Fc region and GDF15 so that their functions do not interfere with each other. The GS linker may be 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). In a particular embodiment, the peptide linker that is included in the fusion polypeptide provided herein may include no amino acids other than the GS linker [(GGGGS)n (n is an integer from 1 to 10 or an integer from 1 to 5)], as described above.

In an embodiment, the fusion polypeptide may further include a peptide linker between the immunoglobulin Fc region and GDF15 or a functional variant thereof. In one embodiment, the peptide linker may be a GS linker comprising one or more Gly (G) and one or more Ser (S) repeats, and for example, may be (GGGGS)n (n is the number of GGGGS repeats (SEQ ID NO: 13) and is an integer from 1 to 10 or an integer from 1 to 5 (1, 2, 3, 4 or 5)), but is not limited to this.

The fusion polypeptide can be produced recombinantly or chemically synthesized. When produced recombinantly, the fusion polypeptide may be encoded by a single reading frame, the expression of which is controlled by a single regulatory transcription initiation factor (eg, promoter, etc.) (represented by "single strand").

In a GDF15 fusion polypeptide, or a functional variant thereof fused to an immunoglobulin Fc region, is characterized by increased in vivo (or blood) stability (lifetime) (e.g., increased in vivo or blood half-life) and/or reduced immunogenicity compared to case when GDF15 or a functional variant thereof is not fused to an immunoglobulin Fc region, and/or an immunoglobulin Fc region and GDF15 or a functional variant thereof are fused via a linker other than the GS linker as described above. In addition, GDF15 or a functional variant thereof fused to an immunoglobulin Fc region has an improved pharmacological effect (eg, weight loss effect) compared to the case where GDF15 or a functional variant thereof is not fused to an immunoglobulin Fc region.

Fusion polypeptide dimer

The functionally active form of GDF15 is a homodimeric form.

Accordingly, another embodiment of the present invention provides a fusion polypeptide dimer in which 2 fusion polypeptides (a first fusion polypeptide and a second fusion polypeptide) are combined. a second immunoglobulin and a second GDF15 or a functional variant thereof In a dimer, in the first fusion polypeptide and the second fusion polypeptide, the first GDF15 or a functional variant thereof and the second GDF15 or a functional variant thereof may be linked by a covalent bond (e.g., a disulfide bond, etc. Further, in a dimer, optionally, the Fc region of the first immunoglobulin and the Fc region of the second immunoglobulin may be linked by a covalent bond (e.g., disulfide bond, etc.) or a non-covalent bond (e.g., ridge & trough, electrostatic interaction, hydrophobic interaction etc.) (See Fig. 1) The immunoglobulin Fc region and GDF15 or a functional variant thereof are as described above, and the Fc region of the first immunoglobulin and the Fc region of the second immunoglobulin, and the first GDF15 or its functional variant and the second GDF15 or a functional variant thereof may be the same or different.

In one embodiment, the Fc region of a first immunoglobulin and the Fc region of a second immunoglobulin, and the first GDF15 or a functional variant thereof and the second GDF15 or a functional variant thereof, and the first fusion polypeptide and the second fusion polypeptide formed by linking them, included in the fusion polypeptide dimer, is single-stranded form (chain; for example, a polypeptide encoded by a single reading frame), respectively, and a structure in which at least one of the Fc region of the first immunoglobulin and the Fc region of the second immunoglobulin, and 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 them, is a dimeric form (e.g., the Fc region of the first or second immunoglobulin included in the first or second fusion polypeptide is linked by a disulfide bond of two strands (2 molecules), and etc., is excluded.

The N-terminus of the monomer in the dimeric structure of GDF15 is open for fusion with another protein, and the C-terminus is located where it cannot be fused with another protein. Therefore, in order to form a fusion polypeptide while maintaining the dimeric form of GDF15, the fusion partner (namely, the immunoglobulin Fc region) should preferably be linked to the N-terminus of GDF15.

GDF15 forms the GDF15-GFRAL complex structure by combining with its GFRAL receptor (GDNF family alpha-like receptor; eg GenBank accession number NP_997293.2). In the complex structure, the GDF15 monomer and the GFRAL monomer are combined in an appropriate manner, and the N-terminus of GDF15 is located in the middle part of the GDF15 dimer. The 4 amino acid residues at the N-terminus, which can be fused to GDF15, are not observed in the X-ray structure, and therefore it is assumed that it does not have a specific structure. The 14 amino acid residues at the N-terminus of GDF15 are fixed by a Cys7-Cys14 disulfide bond and are not involved in the interaction between monomers in the GDF15 dimer. In addition, the 14 amino acid residues at the N-terminus of GDF15 are structurally located at a distance that cannot interact (bind) with the GFRAL receptor, and thus it is expected that there is no effect on the binding of GDF15 to the GFRAL receptor, although the 14 amino acid residues at The N-terminus of GDF15 is deleted.

The GDF15 monomer has 4 disulfide bonds per molecule and one disulfide bond between dimer molecules. It is assumed that the disulfide bonds in the molecule stabilize the structure, and the disulfide bond linking Cys7-Cys14 is located at the edge of the monomeric structure and acts to fix the N-terminal loop structure. Since the 14 amino acid residues at the N-terminus do not play a direct role in GFRAL receptor binding, deletion of one or more of them will not affect the function and structure of GDF15, but Cys15 forms a disulfide bond with Cys88, and therefore, if Cys15 is deleted, this may affect 3D GDF15 structure. Therefore, the region to be deleted while maintaining the function and structure of GDF15 may be 14 amino acids at the N-terminus (namely, in SEQ ID NO: 1, at least one of the 14 amino acids from Ala1 to Cys14, for example, at least one from N- ends in order (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14)). In one embodiment, the GDF15 monomer may be wild-type (SEQ ID NO: 1) or a form in which 14 amino acids at the N-terminus are wild-type deleted (ΔN14GDF15; SEQ ID NO: 2).

In a complex structure in which wtGDF15 (SEQ ID NO: 1) or ΔN14GDF15 (the form in which 14 amino acids at the N-terminus are deleted in SEQ ID NO: 1) is combined with its GFRAL receptor, wt (wild-type) GDF15 is N-terminal (1-14 amino acid residues) portion is fixed by a Cys7-Cys14 disulfide bond, and the N-terminus is open in the bottom left and bottom right directions, respectively. When 14 amino acids are removed from the N-terminus in wtGDF15, the N-terminus is open in the upper left and upper right directions, respectively.

Due to this structure, when a fusion protein is formed by fusion of an immunoglobulin Fc protein with GDF15, ΔN14GDF15, in which 14 amino acid residues at the N-terminus are deleted, and the C-terminus of the Fc protein can be morphologically naturally linked (see Fig. 3a). On the other hand, in the case of wtGDF15, the N-terminus may be located in a direction that is slightly difficult to combine with the Fc protein (see Fig. 3b).

In a complex structure in which Fc-ΔN14GDF15 or Fc-wtGDF15 is combined with GFRAL, GDF15 and Fc are functionally dimeric, and Fc-ΔN14GDF15 has a structural arrangement to easily form an Fc dimer and a ΔN14GDF15 dimer, respectively. Therefore, Fc-ΔN14GDF15 may be advantageous over Fc-wtGDF15 in terms of dimer formation without interrupting GFRAL complex formation.

The mammalian in vivo (blood) half-life of GDF15 or a functional variant thereof incorporated into a fusion polypeptide or fusion polypeptide dimer provided herein can be increased by about 1.5 times or more, about 2 times or more, about 2.5 times or more About 3 times or more About 3.5 times or more About 4 times or more About 4.5 times or more About 5 times or more About 5 times 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, or about 10 times, compared to GDF15 or its non-fused functional variant with the Fc region of an immunoglobulin.

As described above, GDF15 or its functional variant in the form of a fusion polypeptide that is fused to the Fc region of an immunoglobulin has the advantage of longer intervals between administrations compared to GDF15 or its functional variant not fused to the Fc region of an immunoglobulin by increasing half-life of GDF15 or its functional variant.

Preparation of the fusion polypeptide

A fusion polypeptide comprising GDF15 or a functional variant thereof and an immunoglobulin Fc region may be prepared by conventional chemical synthesis or recombinant techniques and may not occur naturally.

In the present application, the term “vector” means an expression means for expressing a target gene in a host cell, and, for example, may be selected from the group consisting of a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage-based vector, a an adenovirus, a retrovirus vector, and an adeno-associated virus vector, and the like. In one embodiment, the vector used in the recombinant vector can be derived from 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.), phage (e.g. λgt4λB, λ-Charon, λΔz1, M13, etc.) or virus (e.g. SV40, etc.), but is not limited to this.

In a recombinant vector, a nucleic acid molecule encoding a fusion polypeptide may be operably linked to a promoter. The term “operably linked” means the functional linking of a nucleic acid expression control sequence (eg, a promoter sequence) and another nucleic acid sequence. A regulatory sequence may regulate transcription and/or translation of another nucleic acid sequence by being "operably linked".

The recombinant vector can generally be designed as a cloning vector or an expression vector. As an expression vector in the art, a conventional vector used to express a foreign protein in plants, animals, or microorganisms can be used. The recombinant vector can be constructed in various ways known in the art.

The recombinant vector can be used for expression using a eukaryotic host. When expressed using a eukaryotic host, the recombinant vector may include a nucleic acid molecule to be expressed and the above promoter, a ribosome binding site, a secretion signal sequence (see Korean Patent Publication No. 2015-0125402) and/or, in addition to the sequence a transcription/translation terminator, an origin of replication active in eukaryotes, such as an f1 origin, an SV40 origin, a pMB1 origin, an adeno origin, an AAV origin and/or a BBV origin, and the like. ., but is not limited to this. In addition, a promoter derived from the genome of a mammalian cell (for example, a metallothionein promoter), or a promoter derived from a mammalian virus (for example, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, fusion promoter (KR 10-1038126 or KR 10-1868139), tk promoter of HSV), and all secretion signal sequences commonly used as a secretion signal sequence can be used, and for example, the secretion signal sequence disclosed in Korean Patent Publication No. 2015-0125402 can be used, but without limitation, and it may include a polyadenylated sequence as a transcription terminator sequence.

A recombinant cell can be obtained by introducing (transforming or transfecting) a recombinant vector into a suitable host cell. The host cell may be selected from all eukaryotes that can stably and continuously clone or express the recombinant vector. The eukaryote used as the host includes yeast (Saccharomyces cerevisiae), insect cells, plant cells, animal cells, and the like. and e.g. includes mice (e.g. COP, L, C127, Sp2/0, NS-0, NS-1, At20 or NIH3T3), rats (e.g. PC12, PC12h, GH3 or MtT), hamsters (e.g. BHK , CHO, GS defective CHO or DHFR defective CHO), monkeys (e.g. COS (COS1, COS3, COS7, etc.), CV1 or Vero), humans (e.g. HeLa, HEK-293, obtained from retinal 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, etc.), but not limited to hybridomas and the like.

By expressing the nucleic acid molecule encoding the fusion polypeptide of the present application in the above appropriate host cell, as compared to the non-fused form, GDF15 or a functional variant thereof, or a fusion polypeptide, or a fusion polypeptide dimer comprising them, can be obtained with improved stability. in vivo. A method for producing a fusion polypeptide or fusion polypeptide dimer may include culturing a recombinant cell comprising the nucleic acid molecule. The cultivation can be carried out under normal cultivation conditions. In addition, the production method may further include isolating and/or purifying the fusion polypeptide or fusion polypeptide dimer from the culture after culturing.

To deliver (introduce) a nucleic acid molecule or a recombinant vector comprising it into a host cell, delivery methods widely known in the art can be used. The delivery method can use, for example, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection and gene bombardment, and the like when the host cell is a eukaryote, but not limited to.

The method for selecting a transformed (introducing a recombinant vector) host cell can be easily carried out according to methods well known in the art using the phenotype expressed by the selectable marker. For example, when the selectable marker is a specific antibiotic resistance gene, a recombinant cell into which the recombinant vector has been introduced can be easily selected by culturing in a medium containing that antibiotic.

Medical Applications

Presented is a composition (pharmaceutical composition or composition of functional health products) for reducing body weight, controlling diet (reducing food volume) or preventing, improving, alleviating and/or treating a metabolic disease, comprising at least one selected from the group consisting of a fused a polypeptide, a dimer of a fusion polypeptide, a nucleic acid molecule encoding a fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector; and / or a method for reducing body weight, a method for controlling a diet (reducing the volume of food) or a method for preventing, improving, alleviating and / or treating a metabolic disease, comprising administering a pharmaceutically effective dose of at least one selected from the group consisting of a fusion polypeptide, a fusion polypeptide dimer, a nucleic acid molecule encoding a fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector, to a subject in need of prevention, improvement, alleviation and/or treatment of a metabolic disease.

Metabolic disease means all diseases caused by metabolic disorders and may be selected from the group consisting of obesity, diabetes (eg, type II diabetes), non-alcoholic fatty liver disease (eg, non-alcoholic steatohepatitis (NASH), etc.), etc. .P.

In the context of the present application, a pharmaceutically effective dose means the content or dose of the active ingredient capable of providing the desired effect. Content or dose of an active ingredient (at least one 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 a fusion polypeptide, a recombinant vector comprising a nucleic acid molecule , and a recombinant cell comprising a recombinant vector) in a pharmaceutical composition may be administered differently depending on factors such as formulation method, route of administration, patient age, body weight, sex, incidence, nutrition, time of administration, interval between administrations, route administration, excretion rate and response sensitivity. For example, a single dose 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, 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, from 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, but not limited to. In another embodiment, the content of the active ingredient in the pharmaceutical composition may be 0.01% of the mass. - 99.9% wt., 0.01% wt. - 90% wt., 0.01% wt. - 80% wt., 0.01% wt. - 70% wt., 0.01% wt. - 60% wt., 0.01% wt. - 50% wt., 0.01% wt. - 40% wt., 0.01% wt. - 30% wt., 1% wt. - 99.9% wt., 1% wt. - 90% wt., 1% wt. - 80% wt., 1% wt. - 70% wt., 1% wt. - 60% wt., 1% wt. - 50% wt., 1% wt. - 40% wt., 1% wt. - 30% wt., 5% wt. - 99.9% wt., 5% wt. - 90% wt., 5% wt. - 80% wt., 5% wt. - 70% wt., 5% wt. - 60% wt., 5% wt. - 50% wt., 5% wt. - 40% wt., 5% wt. - 30% wt., 10% wt. - 99.9% wt., 10% wt. - 90% wt., 10% wt. - 80% wt., 10% wt. - 70% wt., 10% wt. - 60% wt., 10% wt. - 50% wt., 10% wt. - 40% of the mass. or 10% of the mass. - 30% of the mass. based on the total weight of the pharmaceutical composition.

The dosing interval (time interval between two successive doses) of the active ingredient or pharmaceutical composition comprising the same provided herein may be adjusted depending on the concentration or status (variation, etc.) of the active ingredient or the condition or symptoms of the patients, and, for example, it can be 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 interval between administrations can be about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months or about 3 months, but is not limited to this, and it can be increased or decreased depending on the concentration or status (variations etc.) the active ingredient or the condition or symptoms of patients, and the like. In one particular embodiment, the interval between administrations may suitably be set to range from about 1 week to about 3 months.

In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier in addition to the active ingredient. The carrier is generally used to formulate drugs comprising protein, nucleic acids, or cells, and may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, but not limited to alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition may further include at least one selected from the group consisting of diluent, excipient, lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative, and the like commonly used to prepare the pharmaceutical composition.

The subject of administration of the pharmaceutical composition may be mammals, including primates such as humans, monkeys, etc., rodents such as mice, rats, etc. and the like, or cells, tissue, cell culture or tissue culture isolated from them.

The pharmaceutical composition can be administered orally or parenterally, or administered by contact with a cell, tissue, or body fluid. In particular, in the case of parenteral administration, it can be administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration, and the like. In the case of oral administration, the oral composition should be formulated to provide coverage of the active substance or protect it from degradation in the stomach during digestion of proteins or peptides.

In addition, the pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule or the like, and may further include a dispersing agent or stabilizer for composition formulation.

Yet another embodiment provides a method for increasing the in vivo lifetime of GDF15 or a functional variant thereof, comprising fusing (or linking or combining) GDF15 or a functional variant thereof to an immunoglobulin Fc region. In one specific embodiment, the fusion may include the fusion (or linking or joining) of the immunoglobulin Fc region 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 immunoglobulin Fc region, or a fusion polypeptide comprising them, comprising fusing (or linking or combining) GDF15 or a functional variant thereof to an immunoglobulin Fc region using a flexible linker. The fusion (or linking or combining) step can be carried out in vitro.

BENEFICIAL EFFECTS OF THE INVENTION

GDF15 fused to an immunoglobulin Fc region or a functional variant thereof provided in the present invention has a longer life span when administered to an organism compared to a case where it is not fused to an immunoglobulin Fc region, and thus the interval between administrations can be increased, and thus the dose can be reduced, and therefore it has a favorable effect in terms of ease of administration and/or economic aspect, and also has an excellent pharmacological effect, and thus it can be successfully used in areas requiring treatment with GDF15 or a functional variant.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a mimetic diagram of a fusion polypeptide according to one example.

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

Fig. 3a and Fig. 3b shows the structure of the complex in which Fc-ΔN14GDF15 (3a) or Fc-wtGDF15 (3b) binds to GFRAL.

Fig. 4 is a mimetic diagram schematically showing the fusion polypeptide gene according to the example.

Fig. 5 is a graph showing the change in body weight in the IgG1-GDF (CRL) administration group and the HIgG1-GDF (CRL) administration group.

Fig. 6 is a graph showing the percent change in body weight 7 days after administration in the IgG1-GDF (CRL) administration group and the HIgG1-GDF (CRL) administration group.

Fig. 7 is a graph showing the change in body weight in the IgG4-GDF15 administration group and the IgG4-GDF(CRL) administration group.

Fig. 8 is a graph showing the percent change in body weight at 7 days (blue bar) and 14 days (red bar) after administration in the IgG4-GDF15 administration group and the IgG4-GDF(CRL) administration group.

Fig. 9 is a graph showing the cumulative food intake 7 days after administration in the IgG1-GDF (CRL) administration group and the HIgG1-GDF (CRL) administration group.

Fig. 10 is a graph showing the cumulative food intake at 7 days, 14 days and 19 days after administration in the IgG4-GDF15 administration group and the IgG4-GDF(CRL) administration group.

Fig. 11 is a graph showing the change in blood (serum) concentration of a fusion polypeptide as a function of time elapsed after administration of the fusion polypeptide.

DETAILED DESCRIPTION OF EMBODIMENTS

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

Example 1 Preparation of a Fusion Polypeptide

1.1. Cloning and cultivation of the gene encoding the fusion polypeptide

Received IgG1-GDF(CRL), HIgG1-GDF(CRL), IgG4-GDF, HIgG4-GDF, IgG4-GDF(CRL) and HIgG4-GDF(CRL) (see Fig. 1), which were fused polypeptides, fused to mature GDF15 (represented by GDF or GDF15; SEQ ID NO: 1), or a GDF15 variant (represented by GDF(CRL); 14 amino acids at the N-terminus were deleted: SEQ ID NO: 2) that had the target immunoglobulin Fc polypeptide ( IgG1-Fc (excluding hinge: SEQ ID NO: 3; HIgG1-Fc (including hinge; [SEQ ID NO: 4]-[SEQ ID NO: 3]), mutated IgG4-Fc (excluding hinge; SEQ ID NO: 7) or mutated HIgG4-Fc (including hinge region: [SEQ ID NO: 12]-[SEQ ID NO: 7]), including or not including the hinge region.The amino acid sequences of each part included in the fusion polypeptide, are presented in Table 2, Table 3 and Table 4 below.

table 2
IgG1-GDF(CRL) (excluding hinge) and HIgG1-GDF(CRL) (including hinge) (N-terminal to C-terminal direction) Amino acid sequence SEQID NO: Signal peptide MHRPEAMLLL LTLALLGGPT WA 17 IgG1 Fc Hinge area DKTHTCPPCP 4 CH2-CH3 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
ALHNHYTQKS LSLSPG 3 Linker GGGGSGGGGS GGGGSGGGGS 18 GDF(CRL) CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA ANMHAQIKTS
LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL LAKDCHCI 2

Table 3
IgG4-GDF15 (excluding hinge) and HIgG4-GDF15 (including hinge) (N-terminal to C-terminal direction) Amino acid sequence SEQID NO: Signal peptide MHRPEAMLLL LTLALLGGPT WA 17 Mutant IgG4 Fc Hinge area ESKYGPPCPP CP 12 CH2-CH3 APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD
GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS
SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE
ALHNHYTQKS LSLSLG 7 Linker GGGGSGGGGS GGGGSGGGGS 18 GDF15 ARNGDHCPLG PGRCCRLHTV RASLEDLGWA DWVLSPREVQ VTMCIGACPS
QFRAANMHAQ IKTSLHRLKP DTVPAPCCVP ASYNPMVLIQ KTDTGVSLQT
YDDLLAKDCH CI 1

Table 4
IgG4-GDF(CRL) (excluding hinge) and HIgG4-GDF(CRL) (including hinge) (N-terminal to C-terminal direction) Amino acid sequence SEQID NO: Signal peptide MHRPEAMLLL LTLALLGGPT WA 17 Mutant IgG4 Fc Hinge area ESKYGPPCPP CP 12 CH2-CH3 APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK
CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG
NVFSCSVMHE ALHNHYTQKS LSLSLG 7 Linker GGGGSGGGGS GGGGSGGGGS 18 GDF15(CRL) CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA ANMHAQIKTS
LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL LAKDCHCI 2

1.1.1 Obtaining a recombinant expression vector

1.1.1.1. Mature GDF15

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

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 GDF(CRL))

1.1.1.2. IgG1-Fc

A gene encoding a human IgG1 Fc including a hinge or a gene encoding a human IgG1 Fc not including a hinge was obtained using a plasmid including a gene encoding a human IgG1 core hinge and an IgG1 Fc by PCR.

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 GGCTTTCTATC 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 region)

1.1.1.3. IgG4-Fc

A gene encoding a human IgG4 Fc including a hinge or a gene encoding a human IgG4 Fc not including a hinge was obtained using a plasmid including a gene encoding a human IgG4 hinge and an IgG4 Fc by PCR.

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 CCCTGTCCCCT GGGC

(The underlined part is the gene encoding the IgG4 hinge region)

1.1.1.4. Obtaining an expression vector

pDHDD-D1G1 (including the KR10-1868139B1 promoter), which was a pcDNA3.1(+) variant (Invitrogen, Cat. No. V790-20), was cut with BamHI and NotI, and genes (mature GDF15, IgG1-Fc, IgG4-Fc) were combined with them to insert a gene having the following structure (see Fig. 4), thereby obtaining each recombinant vector.

pHIgG1-GDF(CRL)

'(N-terminal)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG1 Core hinge region (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)'

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)'

pHIgG4-GDF15

'(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG4 Hinge region (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)'

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)'

pHIgG4-GDF(CRL)

'(N-terminus)-[BamHI restriction site-signal peptide (SEQ ID NO: 17)-IgG4 Hinge region (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)'

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)'

1.1.2. Cultivation of the gene encoding the fusion polypeptide

The resulting recombinant expression vectors pHIgG1-GDF(CRL), pIgG1-GDF(CRL), pHIgG4-GDF(CRL), pIgG4-GDF(CRL), pHIgG4-GDF and pIgG4-GDF were introduced into an ExpiCHO-S™ cell (Thermo Fisher Scientific ) and cultured in ExpiCHO expression medium (Thermo Fisher Scientific; 400 mL) for 12 days (fed culture; fed on day 1 and day 5) to express HIgG1-GDF(CRL), IgG1-GDF(CRL) fusion polypeptides , HIgG4-GDF(CRL), IgG4-GDF(CRL), HIgG4-GDF and IgG4-GDF.

1.2. Purification of the fusion polypeptide

The fusion polypeptide was purified from the cell culture prepared in Example 1.1 using protein A affinity chromatography.

First, the cell-derived fusion polypeptide culture solution was filtered through a 0.22 μm filter. A MabSelect SuRe™ pcc resin column (GE Healthcare Life Sciences) was stocked with AKTA™ Pure (GE Healthcare Life Sciences) and phosphate buffered saline (PBS, 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) was passed through for column balancing. After introducing the culture solution filtered through a 0.22 μm filter, PBS was again passed into the balanced column to wash the column. After completion of the column wash, elution buffer (0.1 M sodium citrate, pH 3.5) was injected into the column to elute the target fusion polypeptide. To the eluting solution was immediately added 1M Tris pH 8.5 to obtain a neutral pH. From the eluted fractions, those with high fusion polypeptide concentration and high purity were collected and stored frozen.

For animal experiments using the Amicon Ultra Filter Device (MWCO 10K, Merck) and centrifuge, a sample of the eluted fraction containing the fusion polypeptide was concentrated with PBS or 20 mM Tris pH 8.0, 150 mM NaCl and buffer exchanged.

The fusion polypeptide was quantified by measuring the absorbance at 280 nm and 340 nm in a UV spectrophotometer (G113A, Agilent Technologies) and calculating the protein concentration using the following equation. The value theoretically calculated using the amino acid sequence was used as the extinction coefficient of each substance (Table 5).

* Extinction coefficient (0.1%): theoretical absorbance at 280 nm, assuming a protein concentration of 0.1% (1 g/l) and all cysteines in the primary sequence are oxidized to form a disulfide bond, which is calculated using the tool ProtParam tool (https://web.expasy.org/protparam/).

Table 5
Fusion Polypeptide Extinction Coefficient Sample name Extinction coefficient (0.1%, 1 mg/ml) IgG1-GDF (CRL) 1.317 HIgG1-GDF (CRL) 1.22 IgG4-GDF (CRL) 1.334 IgG4-GDF 1.287

Example 2 Pharmacological Effect of Fusion Polypeptide (in vivo)

2.1. Test method

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

In this example, a DI0 mouse model (Mouse, C57BL/6J-DIO, male, 100 mice, 14 weeks (8 weeks of obesity-producing diet) was used in which C57BL/6J mice were fed a high fat diet for 8 weeks to The DIO mouse model exhibits the clinical characteristics of type 2 diabetes such as hyperlipidemia, insulin resistance, hyperglycemia, etc., and therefore it is an animal model widely used to assess improvement in diabetes and insulin resistance, and baseline data comparable to metabolic studies. diseases such as obesity, diabetes, hyperlipidemia, and the like are accumulated in large numbers, and therefore it is suitable for testing the pharmacological effect of this example, and thus this model was chosen.

A mouse model that was fed an obesity-promoting diet for 8 weeks was subjected to a 2-week inspection and cleaning period, and during this period, general symptoms were observed once a day to check on health and whether it was suitable for the experiment to select healthy individuals. animals. During the cleaning period, the individual was tagged by making a mark with a red oil pen on the animal's tail at the time of registration (tail mark), and a temporary identification card of the individual (test name, individual number and time of admission) was attached to the breeding box at the time of examination and cleaning. When separating the group, the tail of the animal was marked with a black oil pen, and an individual identification card (trial name, group information, individual number, sex, entry time, administration period) was attached to each cage.

To minimize test animal stress upon subcutaneous administration of test substance (fusion polypeptide), sterile distilled saline was administered subcutaneously at 200 µl/animal to all animals using a 1 ml syringe 3 days prior to administration of test substance for pre-adaptation preparation for subcutaneous administration.

With regard to healthy animals in which no abnormalities were found during the inspection and cleaning period, body weight and food intake were measured for all individuals after the completion of the cleaning period.

Body weight and food intake were measured, and the groups were separated so that the average of the two measurements between groups was the same depending on body weight. The administration of the test substance was started the day after the separation of the groups. The remaining animals, which were not selected, were excluded from the test system after the end of the separation of the groups.

The information on the high-fat diet (obesity-promoting diet; High Fat Diet (HFD)) given to C57BL/6J-DIO was as follows:

5.24 kcal/g, fat 60% wt., proteins 20% wt., and calories derived from carbohydrates 20% wt.; Research Diet Inc., U.S.A.; Product No. High fat diet (Fat 60 kcal%, D12492).

The food was given in the mode of free food (food during the period of cleaning and testing).

As for drinking water, tap water was filtered using a flow-through filter sterilizer and then irradiated with ultraviolet light, and it was freely consumed using a polycarbonate drinking water bottle (250 ml).

2.1.1. HIgG1-GDF(CRL) and IgG1-GDF(CRL)

Administration of test substances (HIgG1-GDF(CRL) and IgG1-GDF(CRL)) and control substance GDF15 (R&D Systems) was carried out the day after group separation, with administration times at 9 am each day. Both the control substance and the test substances were administered subcutaneously. The route of administration of the control substance and test substances was chosen as the subcutaneous route, depending on the intended clinical route of administration.

The dose of both the control substance and the test substances was 5 ml/kg, and the individual dose was calculated based on the recently measured body weight, and they were injected subcutaneously once on the day of the start of the test using a disposable syringe (1 ml). The test substances were administered only once on the day of the start of the test. For comparison, the control group receiving the GDF15 control substance and the comparison group receiving GDF15 were administered once a day for 5 days, 5 times in total, and all administrations were made from 9 am.

Composition of the test group, dose administered, etc. are presented in the following Table 6:

Table 6
Composition of the IgG1-GDF fusion polypeptide (CRL) administration group High fat diet Test substance Route of administration Administered dose (nmol/kg) Injection volume (ml/kg) Number of animals X Carrier Subcutaneous - 5 5 O Carrier, qd Subcutaneous - 5 5 O Solvent qw Subcutaneous - 5 5 O GDF15 Subcutaneous 2.86 5 3 O IgG1-GDF (CRL) Subcutaneous 1 5 5 O IgG1-GDF (CRL) Subcutaneous 10 5 5 O HIgG1-GDF (CRL) Subcutaneous 1 5 5 O HIgG1-GDF (CRL) Subcutaneous 10 5 5

A schedule of observation, measurements and examination for the study group was established: Day 0 to start administration, and 7 days from the start of administration were set as 1 week of administration.

The study schedule is presented in Table 7:

Table 7
Study Schedule Subject of study Cleaning period (week) Period (day) 1 2 0 1 2 3 4 5 6 7 Feeding a high-fat diet • • • • • • • • • • Adaptation to oral and subcutaneous administration • Introduction • Body weight measurement • • • • • • • • • Measuring food intake • • • • • • • •

For all animals, general clinical symptoms were observed once a day, and the presence of dying and dead animals was confirmed twice a day, and this examination was carried out from day 1 of administration to the end of administration. During the examination, only the case when an abnormal symptom occurred was recorded.

The body weight of each mouse was measured on the day the test substances were administered (before administration), and then the body weight was measured every day (measured for a maximum of 9 days). The dose of test substances was determined based on the most recent measured body weight.

In addition, after administration of the test substances to mice, the food intake was measured every day, and the amount of food was measured using an electronic scale in each breeding box, and then the residual amount was measured to calculate the daily food intake. Individuals who consumed too much food were excluded from the measurement.

All experimental results obtained in this example were presented as mean ± standard deviation and were verified 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 applied to find a study group with a significant difference from the control group (significance level: both sides 5% and 1%, 0.1%).

2.1.2. IgG4-GDF and IgG4-GDF (CRL)

Test substances (IgG4-GDF and IgG4-GDF(CRL)) and control substance Semaglutide (Bachem) were administered subcutaneously. The dose of all control substance and test substances was 5 ml/kg, and the individual dose was calculated based on the recently measured body weight, and they were injected subcutaneously once on the day of the start of the test using a disposable syringe. The test substances were administered only once on the day of the start of the test, and a control group was prepared for comparison, which was administered the control substance Semaglutide. As for the comparison group administered with Semaglutide, Semaglutide was administered every day once a day. All injections were carried out from 9 am.

Composition of the test group, dose administered, etc. are presented in the following Table 8:

Table 8
Composition of the IgG4-GDF15 fusion polypeptide administration group High fat diet Test substance Route of administration Administered dose (nmol/kg) Injection volume (ml/kg) Number of animals O Solvent qw Subcutaneous - 5 5 O Semaglutide, qd Subcutaneous 3 5 5 O IgG4-GDF Subcutaneous 1 5 5 O IgG4-GDF Subcutaneous 10 5 5 O IgG4-GDF (CRL) Subcutaneous 1 5 5 O IgG4-GDF (CRL) Subcutaneous 10 5 5

The examination, measurement and study schedule for the test groups were set to Day 0 for the start of administration and were performed in the same manner as in 2.1.1.

The body weight of each mouse was measured on the day the administration of the test substances was started (before administration), and then the body weight was measured every day (measured for a maximum of 19 days). The dose of test substances was determined based on the most recent measured body weight.

In addition, after administration of the test substances to mice, the food intake was measured every day, and the amount of food was measured using an electronic scale in each breeding box, and then the residual amount was measured to calculate the daily food intake. Individuals who consumed too much food were excluded from the measurement.

2.2. Result of weight loss test

2.2.1. HIgG1-GDF(CRL) and IgG1-GDF(CRL)

The body weight change measured in Example 2.1.1 is shown in FIG. 5 and FIG. 6, and in Table 9 (Body weight (Group, % of baseline).

Table 9 Group Day 0 1 2 3 4 5 6 7 8 9 Low fat diet Vehicle control group Average value 100 102 101 101 101 101 101 101 101 102 (Daily input.) S.E. 0 1 1 1 1 1 1 1 1 1 DIO Vehicle treated control group Average value 100 100 100 100 100 100 101 101 101 101 (Daily input.) S.E. 0 0 0 0 1 1 1 1 0 0 DIO Vehicle treated control group Average value 100 100 99 100 100 100 101 101 102 102 (Single entry) S.E. 0 0 0 0 1 1 1 1 1 1 GDF15, 2.86 nmol/kg Average value 100 99 98 97 96 95 95 95 96 97 (Daily input.) S.E. 0 0 0 1 1 1 1 2 2 2 IgG1-GDF(CRL), 1 nmol/kg Average value 100 99 98 96 96 96 95 95 95 95 (Single entry) S.E. 0 1 1 0 1 1 1 1 1 1 IgG1-GDF(CRL), 10 nmol/kg Average value 100 98 97 95 94 93 91 90 90 90 (Single entry) S.E. 0 0 1 1 1 1 1 1 1 1 HIgG1-GDF(CRL), 1 nmol/kg Average value 100 100 98 98 97 96 97 96 96 97 (Single entry) S.E. 0 0 0 0 0 0 0 0 0 1 HIgG1-GDF(CRL), 10 nmol/kg Average value 100 99 98 97 95 95 94 93 93 93 (Single entry) S.E. 0 0 0 0 1 1 1 1 1 1 (In Table 9, Day 0: first administration of GDF15 control substance, single administration of IgG1-GDF (CRL) fusion polypeptide; Fat-free diet: no high-fat diet group; S.E.=standard error)

Fig. 5 and Table 9 show the change in body weight after a single administration of the IgG1-GDF fusion protein compared to the negative control group (vehicle administration group) and the positive control group (GDF15 daily administration group). In addition, FIG. 6 is a graph extracting and showing the result after 7 days from the results in FIG. 5.

As shown in the results, it can be confirmed that there was little change in body weight in the negative control group (vehicle administration group), while the effect of weight loss disappeared from Day 6, the first day after administration was stopped, in the case of the GDF15 daily administration group ( cessation of administration from Day 5). On the other hand, it can be confirmed that the effect of reducing body weight was exhibited immediately after a single administration on Day 0 in the case of a fusion polypeptide in which GDF15(CRL) was fused to IgG1 or Fc including the hinge region, and the effect of reducing body weight was not reduced. and was constantly present during the whole test period (9 days), and over time, the concentration-related effect of body weight loss increased, and the effect of body weight loss depended on the concentration. It can be said that this weight loss effect exhibited by the fusion polypeptide is comparable to GDF15 when administered once a day throughout the entire test period.

2.2.2. IgG4-GDF and IgG4-GDF(CRL)

The body weight change measured in Example 2.1.2 is shown in FIG. 7 and in Table 10 (Body weight (Group, % of baseline).

Table 10 Group Day 0 1 2 3 4 5 6 7 8 9 DIO Vehicle treated control group
(Single introduction) Average value 100 100 100 99 100 100 100 100 101 101 S.E. 0 0 1 0 0 0 0 1 0 1 Semaglutide, 3 nmol/kg
(Daily Introduction) Average value 100 94 91 90 87 87 85 86 83 85 S.E. 0 0 1 2 2 3 3 3 3 3 IgG4-GDF15, 1 nmol/kg
(Single introduction) Average value 100 99 97 96 95 93 93 92 92 91 S.E. 0 0 1 1 1 1 1 1 1 1 IgG4-GDF15, 10 nmol/kg
(Single introduction) Average value 100 98 97 96 94 93 93 92 91 90 S.E. 0 0 1 1 1 1 1 1 2 2 IgG4-GDF(CRL), 1 nmol/kg
(Single introduction) Average value 100 99 98 97 96 95 94 93 92 92 S.E. 0 0 0 0 1 1 1 1 1 1 IgG4-GDF(CRL), 10 nmol/kg
(Single introduction) Average value 100 98 97 95 94 92 91 90 88 87 S.E. 0 0 0 1 1 1 1 1 1 1 Group Day 10 eleven 12 13 14 15 16 17 18 19 DIO Vehicle treated control group
(Single introduction) Average value 102 102 103 104 104 104 105 105 105 105 S.E. 1 1 0 1 1 1 1 1 1 1 Semaglutide, 3 nmol/kg
(Daily Introduction) Average value 84 82 81 82 81 79 79 78 77 77 S.E. 3 3 3 3 3 2 2 2 2 1 IgG4-GDF15, 1 nmol/kg
(Single introduction) Average value 91 91 93 94 94 94 95 96 96 96 S.E. 1 1 1 1 1 1 1 2 1 2 IgG4-GDF15, 10 nmol/kg
(Single introduction) Average value 90 90 91 92 92 92 92 93 94 94 S.E. 2 1 2 2 2 2 2 2 2 2 IgG4-GDF(CRL), 1 nmol/kg
(Single introduction) Average value 91 91 91 91 91 91 91 92 92 92 S.E. 1 1 1 1 1 1 1 2 2 2 IgG4-GDF(CRL), 10 nmol/kg
(Single introduction) Average value 85 85 84 83 83 82 83 84 84 85 S.E. 1 1 1 1 2 2 2 2 3 2 (In Table 10, Day 0: first administration of control substance (Semaglutide) and test substances; control substance was administered every day; Test substances were administered once; S.E. = standard error)

Fig. 7 and FIG. 8 and Table 10 show the change in body weight after a single administration of IgG4-GDF15 IgG4-GDF fusion protein (CRL) compared to a negative control group (vehicle administration group) and a positive control group (Semaglutide daily administration group). In addition, FIG. 8 is a graph extracting and showing the result after 7 days and 14 days from the results in FIG. 7.

As shown in the results, it can be confirmed that the body weight increased slightly in the case of the negative control group (vehicle administration group), while the body weight loss was constant in the case of the positive control group (semaglutide daily administration group). With a single injection of IgG4-GDF15, regardless of the dose, the effect of weight loss lasted up to 9 days. On the other hand, it can be confirmed that the effect of reducing body weight was constantly manifested up to 10 days with a single administration of 1 nmol/kg IgG4-GDF(CRL) and up to 15 days with a single administration of 10 nmol/kg, and over time associated with concentration the effect of weight loss was increased, and the effect of weight loss was concentration dependent. In addition, it was confirmed that a GDF (CRL) fusion polypeptide with an Fc (mutant) IgG4 without a hinge region had an excellent body weight loss effect at the same dose compared to a GDF (CRL) fusion polypeptide with an Fc (mutant) IgG4 including a hinge region. region. It can be said that the weight-reducing effect of this IgG4 Fc fusion polypeptide was comparable to when Semaglutide was administered once a day throughout the entire test period.

2.3. Result of the food intake test

2.3.1 . HIgG1-GDF(CRL) and IgG1-GDF(CRL)

The change in food intake measured in Example 2.1.1 is shown in Table 11 and in FIG. 9 (cumulative food intake over 6 days), respectively.

Table 11 Group Day 0 1 2 3 4 5 6 7 8 9 Low fat diet Vehicle control group Average value 4.5 4.6 4.6 4.3 4.4 4.6 4.2 4.5 3.9 4.7 (Daily input.) S.E. 0.2 0.2 0.1 0.2 0.1 0.3 0.2 0.2 0.1 0.3 DIO Vehicle treated control group Average value 2.8 2.8 3.1 3.0 3.1 2.9 3.0 2.9 2.9 3.3 (Daily input.) S.E. 0.3 0.2 0.2 0.3 0.3 0.2 0.3 0.2 0.1 0.2 DIO Vehicle treated control group Average value 3.1 2.9 3.1 3.2 3.3 3.2 3.3 3.2 3.0 3.5 (Single entry) S.E. 0.1 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.2 GDF15, 2.86 nmol/kg Average value 2.8 2.4 2.7 2.4 2.5 2.6 2.7 3.0 3.3 3.5 (Daily input.) S.E. 0.4 0.3 0.2 0.4 0.2 0.3 0.2 0.3 0.2 0.3 IgG1-GDF(CRL), 1 nmol/kg Average value 3.0 2.1 2.2 2.2 2.6 2.6 2.6 2.8 2.7 3.0 (Single entry) S.E. 0.2 0.2 0.1 0.1 0.2 0.1 0.0 0.1 0.1 0.3 IgG1-GDF(CRL), 10 nmol/kg Average value 2.7 2.0 2.5 2.0 1.9 1.9 2.1 2.1 2.3 2.8 (Single entry) S.E. 0.3 0.1 0.1 0.3 0.2 0.2 0.2 0.1 0.1 0.3 HIgG1-GDF(CRL), 1 nmol/kg Average value 3.0 2.4 2.4 2.5 2.7 2.6 2.9 2.7 2.9 3.0 (Single entry) S.E. 0.2 0.1 0.1 0.0 0.1 0.0 0.1 0.1 0.1 0.1 HIgG1-GDF(CRL), 10 nmol/kg Average value 3.8 2.2 2.2 2.3 2.4 2.4 2.3 2.4 2.9 3.3 (Single entry) S.E. 0.5 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.3 0.3

As shown as a result, it can be confirmed that the administration group of a fusion polypeptide in which GDF (CRL) is fused to an IgG1 Fc including a hinge or an IgG1 Fc not including a hinge has an effect of reducing food intake during the test (9 days ), compared with the negative control administration group (vehicle administration group), and the food intake reduction effect was concentration dependent. This food intake reducing effect of the fusion polypeptide can be considered comparable to when GDF15 is administered once a day for the duration of the test.

2.3.2. IgG4-GDF and IgG4-GDF(CRL)

The change in food intake measured in Example 2.1.2 is shown in Table 12 and in FIG. 10 (cumulative consumption over 7 days, 14 days and 19 days).

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

As shown as a result, it can be confirmed that the fusion polypeptide administration group in which GDF15 or GDF (CRL) is fused to IgG4 (mutant) not including the hinge region showed the effect of reducing food intake during the test (19 days), compared with a negative control administration group (vehicle administration group). This effect of reducing food intake of the fusion polypeptide can be considered comparable to when Semaglutide is administered once a day throughout the entire test period. When comparing administration groups of a 10 nmol/kg fusion polypeptide in which GDF15 or GDF (CRL) is fused to an IgG4 Fc (mutant) that does not include the hinge region, in the case of the administration group of the full-length fusion polypeptide, GDF15-fusion, the ability to suppress food intake was maintained for about 10 days, and in the case of the fusion polypeptide administration group, GDF(CRL)-fusion, the ability to suppress food intake was maintained for about 2 weeks. In other words, it was confirmed that the GDF (CRL) fusion polypeptide had a somewhat superior body weight loss effect at a dose of 10 nmol/kg compared to the full length GDF15 fusion polypeptide.

Example 3 Fusion Polypeptide Pharmacodynamic Test (IgG4-GDF or IgG4-GDF(CRL))

3.1. Obtaining serum samples of the study group and the control group

To evaluate pharmacodynamic performance, when each polypeptide was administered subcutaneously to rats, IgG4-GDF or IgG4-GDF (CRL) polypeptide was administered subcutaneously at 2 mg/kg, respectively, to SD rats (Koatech, male, 7 weeks old, about 250 g; n= 3 each; study group), and approximately 200 ml of blood was collected via the tail vein at the set time. Blood collection was performed prior to fusion polypeptide administration and 1, 2, 4, 8, 24, 48, 72, 96, 168, 240 and 336 hours after administration. As a control group for comparison of pharmacodynamic characteristics, GDF15 (R&D Systems) was administered subcutaneously at 2 mg/kg in the same manner as above to prepare the GDF15 administration group.

After administration to SD rats as above, blood collected at a specific time point was centrifuged to obtain serum and ELISA was performed using a human GDF15 immunoassay (SGD150, R&D Systems) to measure serum concentration over time for each polypeptide. Using these data, parameter values including AUC (area under the curve) were obtained using PK analysis software (WinNonlin (Certara L.P.) et al.).

3.2 Result of the pharmacodynamic test

The resulting pharmacodynamic parameters of the fusion polypeptide are shown in Table 13, and the concentration of the fusion polypeptide over time is shown in FIG. eleven:

Table 13 PK parameter Group 1 Group 2 Group 3 GDF15 IgG4-GDF IgG4-GDF(CRL) _Cmax (µg/ml) 0.443 10.6 9.73 T _max (hour) 1 96 48 AUC _last (µg⋅h/mL) 4.86 2144 2004 AUC _inf (µg⋅h/ml) 4.88 2854 2593 t _1/2 (hour) 19.0 101 114 AUC _extp (%) 0.463 14.6 18.2 (C _max : maximum blood concentration, T _max : time to reach maximum blood concentration, AUC _inf : area under the blood concentration versus time curve calculated by extrapolating from the last measured time of blood collection to infinite time, AUC _last : area under blood concentration-time curve to last measured time of blood collection, T _1/2 : half-life, AUC _Extp (%): [(AUC _inf -AUC _last )/AUC _inf ]*100)

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

From the foregoing description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be embodied in other specific forms without changing its technical nature or essential characteristics. In this regard, the embodiments described above are to be understood as illustrative and not restrictive in all respects. The scope of the present invention is to be considered as including all altered or modified forms arising from the spirit and scope of the claims to be described below and their equivalent concepts, and not the above detailed description.

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SEQUENCE LIST

<110> LG CHEM, LTD.

<120> FUSION POLYPEPTIDE INCLUDING FC REGION OF IMMUNOGLOBULIN AND

GDF15

<130> OPP20200894KR

<150> KR 10-2019-0047558

<151> 2019-04-23

<160> 18

<170> KopatentIn 3.0

<210> 1

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> mature GDF15 (wild type)

<400> 1

Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg

1 5 10 15

Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp

20 25 30

Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys

35 40 45

Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser

50 55 60

Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro

65 70 75 80

Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val

85 90 95

Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile

100 105 110

<210> 2

<211> 98

<212> Protein

<213> Artificial sequence

<220>

<223> DeltaN14GDF15

<400> 2

Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala

1 5 10 15

Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly

20 25 30

Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys

35 40 45

Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys

50 55 60

Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr

65 70 75 80

Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His

85 90 95

Cys Ile

<210> 3

<211> 217

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG1-Fc (UniProtKB P01857) CH2-CH3

<400> 3

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 4

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG1-Fc (UniProtKB P01857) core hinge region

<400> 4

Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10

<210> 5

<211> 217

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG4-Fc (UniProtKB P01861) CH2-CH3 (total)

<220>

<221> MOD_RES

<222> (4)

<223> Xaa is Phe or Ala

<220>

<221> MOD_RES

<222> (5)

<223> Xaa represents Leu or Ala

<220>

<221> MOD_RES

<222> (217)

<223> Xaa absent or Lys

<400> 5

Ala Pro Glu Xaa Xaa Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly Xaa

210 215

<210> 6

<211> 217

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG4-Fc (UniProtKB P01861) CH2-CH3 (wild type)

<400> 6

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215

<210> 7

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG4-Fc (UniProtKB P01861) CH2-CH3 (mutant)

<400> 7

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly

210 215

<210> 8

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG4-Fc (UniProtKB P01861) CH2-CH3 (mutant)

<400> 8

Ala Pro Glu Ala Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly

210 215

<210> 9

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG4-Fc (UniProtKB P01861) CH2-CH3 (mutant)

<400> 9

Ala Pro Glu Phe Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly

210 215

<210> 10

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG4-Fc (UniProtKB P01861) hinge region (general)

<220>

<221> MOD_RES

<222> (10)

<223> Xaa is Ser or Pro

<400> 10

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Xaa Cys Pro

1 5 10

<210> 11

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG4-Fc (UniProtKB P01861) hinge region (wild type)

<400> 11

Glu Ser Lys Tyr Gly Pro Cys Pro Ser Cys Pro

1 5 10

<210> 12

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> human IgG4-Fc (UniProtKB P01861) hinge region (mutated)

<400> 12

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 13

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> peptide linker

<400> 13

Gly Gly Gly Gly Ser

15

<210> 14

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> GDF15 encoding gene

<400> 14

gcccggaacg gcgaccactg ccccctgggg cccggacggt gctgccggct gcacaccgtg 60

cgggcctccc tggaggacct gggctgggcc gactgggtgc tgtccccaag ggaggtgcaa 120

gtgaccatgt gcatcggcgc ctgcccatct cagttccggg ccgccaacat gcacgctcag 180

atcaagacca gcctgcaccg gctgaagccc gacaccgtgc ccgccccctg ctgcgtgccc 240

gcctcctaca accccatggt gctgattcag aagaccgaca ccggcgtgag cctgcagacc 300

tacgacgacc tgctggccaa ggactgccac tgcatctaa 339

<210> 15

<211> 678

<212> DNA

<213> Artificial sequence

<220>

<223> IgG1-Fc encoding gene

<400> 15

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtatacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggt 678

<210> 16

<211> 684

<212> DNA

<213> Artificial sequence

<220>

<223> IgG4-Fc encoding gene

<400> 16

gagtccaaat atggtccccc atgcccaccc tgcccagcac ctgaggccgc cgggggaccg 60

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 120

gtcacgtgcg tggtggtgga cgtgtcccag gaggaccccg aggtgcagtt caactggtac 180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaactcc 240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 300

tacaagtgca aggtgtccaa caagggcctg ccctcctcca tcgagaagac catctccaag 360

gccaagggcc agccccggga gccccaggtg tacaccctgc ccccctccca ggaggagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctacccctc cgacatcgcc 480

540

gactccgacg gctccttctt cctgtactcc cggctgaccg tggacaagtc ccggtggcag 600

gagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagtccctgt ccctgtccct gggc 684

<210> 17

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 17

Met His Arg Pro Glu Ala Met Leu Leu Leu Leu Thr Leu Ala Leu Leu

1 5 10 15

Gly Gly Pro Thr Trp Ala

20

<210> 18

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> peptide linker

<400> 18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<---

Claims (41)

1. A fusion polypeptide having an increased lifetime of GDF15 in vivo, comprising GDF15 (Growth/Differentiation Factor 15) or a functional variant thereof and an immunoglobulin Fc region, wherein The Fc region of an immunoglobulin is a single strand of the Fc region of human IgG4 and is linked to the N-terminus of GDF15 or a functional variant via a flexible peptide linker, a functional variant of GDF15 is a deletion variant in which at least one of the 14 amino acids at positions 1 to 14 of the amino acid sequence of SEQ ID NO: 1 is deleted, the human IgG4 Fc region contains the amino acid sequence of SEQ ID NO: 7, and the flexible peptide linker is represented by (GGGGS)n, where n is 1, 2, 3, 4, or 5. 2. The fusion polypeptide of claim 1, wherein the human IgG4 Fc region further comprises the amino acid sequence of SEQ ID NO: 10 at the N-terminus of the amino acid sequence of SEQ ID NO: 7. 3. The fusion polypeptide of claim 2, wherein the human IgG4 Fc region further comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 at the N-terminus of the amino acid sequence of SEQ ID NO: 7. 4. The fusion polypeptide of claim 1, wherein the GDF15 functional variant comprises the amino acid sequence of SEQ ID NO: 2. 5. Fusion polypeptide according to any one of paragraphs. 1-4, wherein GDF15 or a functional variant thereof associated with an immunoglobulin Fc region in the fusion polypeptide has at least a 1.5-fold increased in vivo half-life compared to GDF15 or a functional variant thereof that is not associated with an immunoglobulin Fc region . 6. A fusion polypeptide dimer having an increased lifetime of GDF15 in vivo, comprising two fusion polypeptides according to any one of paragraphs. 1-4. 7. A nucleic acid molecule encoding a fusion polypeptide according to any one of paragraphs. 1-4. 8. Recombinant expression vector comprising the nucleic acid molecule according to claim 7. 9. A recombinant cell for producing a fusion polypeptide according to claim 1, including a recombinant vector according to claim 8, where the cell is not a human embryonic cell. 10. A method for producing a fusion polypeptide according to claim 1, wherein the fusion polypeptide comprises GDF15 or a functional variant thereof and an IgG4 immunoglobulin Fc region, wherein the method comprises culturing a recombinant cell according to claim 9. 11. A method for producing a fusion polypeptide according to claim 1, comprising linking one chain of the human IgG4 Fc region to the N-terminus of GDF15 or its functional variant through a flexible peptide linker, where the functional variant of GDF15 is a deletion variant in which at least one of the 14 amino acids positions 1 to 14 of the amino acid sequence of SEQ ID NO: 1 is deleted, the Fc region of human IgG4 contains the amino acid sequence of SEQ ID NO: 7, and a flexible peptide linker represented by (GGGGS)n, where n is 1, 2, 3, 4, or 5. 12. The method of claim 11, wherein the human IgG4 Fc region further comprises the amino acid sequence of SEQ ID NO: 10 at the N-terminus of the amino acid sequence of SEQ ID NO: 7. 13. The method of claim 12, wherein the human IgG4 Fc region further comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 at the N-terminus of the amino acid sequence of SEQ ID NO: 7. 14. The method of claim 11, wherein the GDF15 functional variant comprises the amino acid sequence of SEQ ID NO: 2. 15. The method according to any one of paragraphs. 11-14, wherein GDF15 or a functional variant thereof associated with an immunoglobulin Fc region in the fusion polypeptide has at least a 1.5-fold increased in vivo half-life compared to GDF15 or a functional variant thereof that is not associated with an immunoglobulin Fc region . 16. Composition for weight loss, comprising an effective amount of at least one selected from the group consisting of: fused polypeptide according to any one of paragraphs. 1-4, a fusion polypeptide dimer according to claim 6, comprising two of the fusion polypeptides, a nucleic acid molecule according to claim 7 encoding a fusion polypeptide, a recombinant vector according to claim 8, including a nucleic acid molecule, and a recombinant cell according to claim 9, including a recombinant vector; And a pharmaceutically acceptable carrier. 17. A diet control composition comprising an effective amount of at least one selected from the group consisting of: fused polypeptide according to any one of paragraphs. 1-4, a dimer of a fusion polypeptide according to claim 6, comprising two of the fusion polypeptides, a nucleic acid molecule according to claim 7 encoding a fusion polypeptide, a recombinant vector according to claim 8, including a nucleic acid molecule, and a recombinant cell according to claim 9, including a recombinant vector; And a pharmaceutically acceptable carrier. 18. A pharmaceutical composition for the prevention or treatment of a metabolic disease, comprising an effective amount of at least one selected from the group consisting of: fused polypeptide according to any one of paragraphs. 1-4, a dimer of a fusion polypeptide according to claim 6, comprising two of the fusion polypeptides, a nucleic acid molecule according to claim 7 encoding a fusion polypeptide, a recombinant vector according to claim 8, including a nucleic acid molecule, and a recombinant cell according to claim 9, including a recombinant vector; And a pharmaceutically acceptable carrier. 19. The pharmaceutical composition of claim 18, wherein the metabolic disease is obesity, diabetes, or non-alcoholic fatty liver disease.

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