Title of the Invention: Fusion Polypeptide Comprising O-glycosylated Polypeptide Region and GDF15
technical field
[One]
A fusion polypeptide comprising a growth differentiation factor 15 (GDF15) and an O-glycosylated polypeptide region, a pharmaceutical composition comprising the fusion polypeptide, and fusing the O-glycosylated polypeptide region A method for increasing the in vivo duration of GDF15.
[2]
background
[3]
Most protein or peptide drugs have a short duration of activity in the body, and their absorption rate is low when administered by methods other than intravenous administration. There is an inconvenience of having to inject. In order to solve this inconvenience, it is required to develop a technology for continuously releasing a drug with a single administration. In order to meet these needs, sustained-release formulations for sustained release are being developed.
[4]
For example, research on sustained-release formulations in which a protein or peptide drug is surrounded by a biodegradable polymer matrix is ​​prepared and the drug is slowly released while the matrix material is slowly decomposed and removed from the body when administered. is becoming
[5]
For example, U.S. Patent No. 5,416,017 discloses a sustained-release injection of erythropoietin using a gel having a hyaluronic acid concentration of 0.01 to 3%, and Japanese Patent Laid-Open No. 1-287041 discloses insulin and hyaluronic acid. A sustained-release injection contained in a gel having a concentration of 1%, Japanese Patent Laid-Open No. Hei 2-213 describes a sustained-release formulation in which calcitonin, elcatonin, or human GDF15 is contained in hyaluronic acid at a concentration of 5%. In such a formulation, the protein drug dissolved in the gel of hyaluronic acid passes through the gel matrix with high viscosity at a slow speed, so it can exhibit a sustained release effect, but it is not easy to administer by injection due to the high viscosity, Afterwards, the gel is easily diluted or decomposed by body fluids, so it is difficult to release the drug for more than 1 day.
[6]
On the other hand, there are examples of preparing solid fine particles by emulsion solvent extraction using a hydrophobic hyaluronic acid derivative (eg, hyaluronic acid-benzyl ester) (NS Nightlinger, et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 22nd, Paper No. 3205 (1995); L. Ilum, et al., J. Controlled Rel., 29, 133 (1994)). Since it is necessary to use an organic solvent to prepare the drug release formulation particles using the hydrophobic hyaluronic acid derivative, there is a risk of denaturation of the protein drug by contact with the organic solvent, and the possibility of denaturation of the protein due to the hydrophobicity of the hyaluronic acid derivative is high.
[7]
Therefore, in order to improve the persistence of protein or peptide drugs in the body, an approach different from existing studies is required.
[8]
On the other hand, GDF15 (Growth differentiation factor 15) is a member of the TGF-beta family, a 25 kDa homodimer, and is a secreted protein circulating in plasma. Plasma levels of GDF15 are related to body mass index (BMI), and GDF15 acts as a long-term regulator of energy homeostasis. GDF15 also has protective actions in pathological conditions such as cardiovascular disease, myocardial hypertrophy, and ischemic injury. In addition, GDF15 plays a protective role from renal tubular and renal interstitial damage in models of type 1 diabetes and type 2 diabetes. In addition, GDF15 has a protective effect against age-related sensory and motor loss and may contribute to peripheral nerve damage recovery. In addition, GDF15 has effects of weight loss and body fat reduction and glucose tolerance, and has the effect of increasing systemic energy expenditure and oxidative metabolism. GDF15 exhibits glycemic control effects through weight-dependent and non-dependent mechanisms.
[9]
The development of a technology for improving the persistence of the GDF15 protein in the body, which exhibits various pharmacological effects as described above, is required.
[10]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[11]
In the present specification, by linking an O-glycosylated polypeptide (eg, a hinge region of an immunoglobulin, etc.) to Growth differentiation factor 15 (GDF15) to form a fusion polypeptide, an O-glycosylated polypeptide A technique for increasing the in vivo half-life of GDF15 to enhance its duration in the body compared to the case where it is not fused with the domain, thereby increasing the administration interval is provided.
[12]
One example provides a fusion polypeptide comprising GDF15 and a polypeptide region capable of O-glycosylation.
[13]
In the fusion polypeptide, the O-glycosylated polypeptide region may be included at the N-terminus of GDF15.
[14]
The total number of O-glycosylated polypeptide regions included in the fusion polypeptide is 1 or more, such as 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 10 to 10, 2 to 8, 2 to 6, 2 to 4 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 dogs or 10).
[15]
In one example, the fusion polypeptide can be represented by the following general formula:
[16]
N'-(Z)nY -C' [general expression]
[17]
In the above formula,
[18]
N' is the N-terminus of the fusion polypeptide, C' is the C-terminus of the fusion polypeptide,
[19]
Y is GDF15,
[20]
Z is an O-glycosylated polypeptide region,
[21]
n is the number of O-glycosylated polypeptide regions (bound to the N-terminus of GDF15) located at the N-terminus of the fusion polypeptide, from 1 to 10 (ie, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), 1 to 7, 1 to 5, or an integer from 1 to 3.
[22]
Each of the n O-glycosylated polypeptide regions included in the fusion polypeptide may be independently selected from polypeptide sites comprising O-glycosylated amino acid residues. For example, the polypeptide moiety comprising an O-glycosylated amino acid residue may be a hinge region of an immunoglobulin. In one embodiment, the O-glycosylated polypeptide region is, each independently, a hinge region of immunoglobulin D (Immunoglobulin D; IgD) and a hinge region of immunoglobulin A (Immunoglobulin A; IgA, such as IgA1). (ie, the hinge regions of the n immunoglobulins may be identical to or different from each other).
[23]
In the fusion polypeptide, GDF15 fused with an O-glycosylated polypeptide region has increased in vivo (or blood) stability (duration) compared to GDF15 to which the O-glycosylated polypeptide region is not fused. It is characterized (eg, increased in vivo or blood half-life).
[24]
Another example provides a nucleic acid molecule encoding the fusion polypeptide.
[25]
Another example provides a recombinant vector comprising the nucleic acid molecule.
[26]
Another example provides a recombinant cell comprising the recombinant vector.
[27]
Another example is a method for producing GDF15 with increased in vivo (or blood) half-life, comprising the step of expressing the recombinant vector in a cell, or production of a fusion polypeptide comprising GDF15 with increased in vivo (or blood) half-life provide a way
[28]
Another example is a method of increasing the in vivo duration of GDF15 comprising fusing (or linking or binding) GDF15 with an O-glycosylated polypeptide region, or a GDF15 (protein or peptide) drug in the body (or blood) Methods for enhancing stability and/or increasing in vivo (or blood) half-life are provided. In one embodiment, the fusing may include fusing (or linking or binding) one or more O-glycosylated polypeptide regions to the N-terminus of GDF15 with or without a linker. The fusion (or linking or binding) step may be performed in vitro.
[29]
Another example provides a fusion polypeptide dimer comprising two of the fusion polypeptides. The fusion polypeptide dimer may be formed by being linked by a bond (eg, a disulfide bond) between GDF15 included in each fusion polypeptide. The fusion polypeptide dimer may be a homodimer.
[30]
Another example is the fusion polypeptide, a fusion polypeptide dimer comprising the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector. It provides a pharmaceutical composition comprising at least one selected from the group consisting of.
[31]
Another example is the fusion polypeptide, a fusion polypeptide dimer comprising the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector. It provides a method for preparing a pharmaceutical composition using one or more selected from the group consisting of.
[32]
Another example is the fusion polypeptide, a fusion polypeptide dimer comprising the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector. It provides use for preparing a pharmaceutical composition comprising at least one selected from the group consisting of.
[33]
Another example provides the use of a GDF15 (protein or peptide) drug to enhance in vivo (or blood) stability and/or increase in vivo (or blood) half-life of an O-glycosylated polypeptide region. Specifically, one example provides a composition for enhancing the stability of a GDF15 (protein or peptide) drug in the body (or blood) and/or increasing the half-life in the body (or blood) including an O-glycosylated polypeptide region.
[34]
means of solving the problem
[35]
In the present specification, by providing in the form of a fusion polypeptide in which GDF15 is fused with an O-glycosylated polypeptide region such as an immunoglobulin hinge region, in vivo (or blood) stability and/or in vivo (or blood) application of GDF15 is provided. ) to enhance the duration and increase the dosing interval.
[36]
One example provides a fusion polypeptide comprising GDF15 and a polypeptide region capable of O-glycosylation.
[37]
In the fusion polypeptide, the O-glycosylated polypeptide region may be included at the N-terminus of GDF15.
[38]
The total number of O-glycosylated polypeptide regions included in the fusion polypeptide is 1 or more, such as 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 10 to 10, 2 to 8, 2 to 6, 2 to 4 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 dogs or 10).
[39]
In one example, the fusion polypeptide can be represented by the following general formula:
[40]
N'-(Z)nYC' [general expression]
[41]
In the above formula,
[42]
N' is the N-terminus of the fusion polypeptide, C' is the C-terminus of the fusion polypeptide,
[43]
Y is GDF15,
[44]
Z is an O-glycosylated polypeptide region,
[45]
n is the number of O-glycosylated polypeptide regions (bound to the N-terminus of GDF15) located at the N-terminus of the fusion polypeptide, from 0 to 10 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), 0 to 7, 0 to 5, 1 to 10, 1 to 7, 1 to 5, 1 to 4, or an integer from 1 to 3.
[46]
In one example, in the fusion polypeptide, when the active site of GDF15 is located at the C-terminus, the O-glycosylated polypeptide region may be fused to the N-terminus.
[47]
Each of the n O-glycosylated polypeptide regions included in the fusion polypeptide may be independently selected from polypeptides comprising O-glycosylated amino acid residues. For example, the polypeptide moiety comprising an O-glycosylated amino acid residue may be a hinge region of an immunoglobulin. In one embodiment, the O-glycosylated polypeptide region is each independently composed of a hinge region of immunoglobulin D (Immunoglobulin D; IgD) and a hinge region of immunoglobulin A (Immunoglobulin A; IgA; e.g., IgA1). may be selected from the group. The hinge regions of the n immunoglobulins may be identical to or different from each other.
[48]
In one embodiment, there may be one or two or more O-glycosylated polypeptide regions located (included) at the N-terminus of the fusion polypeptide, and if there are two or more, each O-glycosylated The polypeptide regions may be the same or different from each other. In one embodiment, one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) O-glycosylated polypeptide regions located at the N-terminus are all IgD is a hinge region of or a hinge region of an IgA (eg, IgA1), or one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) hinge regions of an IgD and one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of hinge regions of IgA (eg, IgA1) may be included in various orders.
[49]
In another embodiment, when all n O-glycosylated polypeptide regions included in the fusion polypeptide are located only at the N-terminus of the fusion polypeptide (i.e., at least one O-terminus only at the N-terminus of the fusion polypeptide) If glycosylationable polypeptide regions are present), said one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) O-glycosylated polypeptide regions All are hinge regions of IgD or hinge regions of IgA, or one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) hinge regions of IgD and one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the hinge regions of IgA may be included in various orders.
[50]
The O-glycosylation capable polypeptide region (each region when there are two or more O-glycosylation capable polypeptide regions) comprises one or more O-glycosylation residues (O-glycosylated amino acid residues), two or more , 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more (the upper limit is 100, 50, 25, 20, 19, 18, 17, 16, 15) , 14, 13, 12, 11, 10, 9, or 8) (eg, 1, 2, 3, 4, 5, 6, 7, or 8) ) may be included. For example, the O-glycosylation capable polypeptide region (each region when there are two or more O-glycosylated polypeptide regions) contains 1 to 10 O-glycosylation residues (O-glycosylated amino acid residues) or It may include 3 to 10 pieces.
[51]
In one embodiment, the O-glycosylated polypeptide region may be one or more selected from among the hinge regions of an immunoglobulin (eg, human immunoglobulin), for example, an IgD hinge region, an IgA hinge region, or a combination thereof. can
[52]
Among the regions of an immunoglobulin (eg, human immunoglobulin), a hinge region such as an IgD hinge region (eg, a human IgD hinge region) and/or an IgA hinge region (eg, a human IgA hinge region) is a residue capable of O-glycosylation Thus, the O-glycosylated polypeptide region may consist essentially of or consist essentially of one or more (human) IgD hinge regions and/or one or more (human) IgA hinge regions. . In one embodiment, the O-glycosylated polypeptide region is one selected from the group consisting of CH1, CH2, and CH3 of an immunoglobulin region (eg, IgD and/or IgA) that does not contain an O-glycosylated residue. It may not include more than one (eg, one, two, or all three).
[53]
In addition, given the number of O-glycosylated residues suitable for the fusion polypeptides provided herein, the O-glycosylated polypeptide may have an IgD hinge region (eg, a human IgD hinge region) and/or an IgA hinge region. (eg, human IgA hinge region) 1 or more, more specifically, 2 or more (eg 2, 3, 4, 5, 6, 7, 8, 9 or 10) may include.
[54]
More specifically, the IgD may be human IgD (eg, UniProKB P01880 (constant region; SEQ ID NO: 7), etc.), and the hinge region of the IgD is,
[55]
comprising the amino acid sequence of "N'-ESPKAQA SS VP T AQPQAEGSLAKA TT APA TT RNT-C' (SEQ ID NO: 1); the amino acid residues indicated in bold are O-glycosylated residues (7 in total)" a polypeptide consisting essentially of an amino acid sequence ("IgD hinge"),
[56]
Among the amino acid sequence of SEQ ID NO: 1, consecutive 5 or more containing 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 O-glycosylation residues, A polypeptide comprising or consisting essentially of 7 or more, 10 or more, 15 or more, 20 or more, 22 or more, or 24 or more (the upper limit is 34 or 33) amino acids ("IgD part of the hinge"; e.g., a polypeptide comprising 5 or more consecutive amino acids comprising "SSVPT" (SEQ ID NO: 9) in SEQ ID NO: 1 or 7 or more consecutive polypeptides comprising "TTAPATT" (SEQ ID NO: 10) polypeptides comprising amino acids), and
[57]
Among IgDs (eg, SEQ ID NO: 7), at least 34 or at least 35 consecutive amino acids comprising the amino acid sequence of SEQ ID NO: 1 (IgD hinge) or at least 7 consecutive amino acids comprising a part of the IgD hinge, 10 or more A polypeptide comprising or consisting essentially of at least 15, at least 20, at least 22, or at least 24 amino acids ("extension of the IgD hinge"; e.g., "of IgD (SEQ ID NO: 7)" ESPKAQASS VPTAQPQAEG SLAKATTAPA TTRNTGRGGE EKKKEKEKEE QEERETKTP" (SEQ ID NO: 11) or a polypeptide comprising at least 34 or at least 35 consecutive amino acids comprising a portion of said IgD hinge)
[58]
It may be at least one selected from the group consisting of.
[59]
The IgA may be human IgA (eg, IgA1 (UniProKB P01876, constant region; SEQ ID NO: 8), etc.), and the hinge region of the IgA is,
[60]
Amino acids of "N'-VP ST PP T P S P S TPP T P S P S -C' (SEQ ID NO: 2); the amino acid residues indicated in bold are O-glycosylated residues (8 in total)" a polypeptide comprising or consisting essentially of said amino acid sequence ("IgA hinge");
[61]
5 consecutive 5 including 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 O-glycosylation residues in the amino acid sequence of SEQ ID NO: 2 A polypeptide comprising or consisting essentially of an amino acid sequence of at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 17, or 18 amino acids. (“part of the IgA hinge”; such as a polypeptide comprising at least 8 or at least 9 amino acids comprising “STPPTPSP” in SEQ ID NO: 2 (SEQ ID NO: 12)), and
[62]
Among IgA (eg, IgA1 (SEQ ID NO: 8)), at least 19 or at least 20 consecutive amino acids comprising the amino acid sequence of SEQ ID NO: 2 (IgA (eg, IgA1) hinge), or IgA (eg, IgA1) ) a polypeptide comprising or consisting essentially of the amino acid sequence of consecutive 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, or 18 or more amino acids comprising a portion of the hinge (IgA "hinge") extension of")
[63]
It may be at least one selected from the group consisting of.
[64]
In another example, the O-glycosylated polypeptide region is an O-glycosylated amino acid residue ( O-glycosylation site) 1 or more, 2 or more, 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, 20 or more, or 22 or more (eg, 1 to 10, 3 to 10; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, 20 or more, 22 or more, 25 or more, 27 or more, 30 or more, 32 or more, or 35 or more amino acids (the upper limit is 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or the total number of amino acids in each protein) It may be a polypeptide region comprising or consisting essentially of the above amino acids. In the present specification, it is preferred that the polypeptide region capable of O-glycosylation does not affect the function of GDF15. The O-glycosylation polypeptide region of the protein exemplified in Table 1 below may be selected from regions not involved in the original function of the full-length protein, :
[65]
[Table 1]
UniProtKB Entry No. UniProtKB Entry name Protein names Gene names Length O-Glycosylation (site) SEQ ID NO:
Q96DR8 MUCL1_HUMAN Mucin-like protein 1 MUCL1 SBEM UNQ590/PRO1160 90 23T, 24T, 30T, 34T, 46T, 47T, 51T, 52T, 54T, 55T, 59T, 60T, 62T, 63T, 66S, 67T, 68T 23
Q0VAQ4 SMAGP_HUMAN small cell adhesion glycoprotein SMAGP 97 2T, 3S, 6T, 7T, 9S, 16T, 17T, 23T 24
P04921 GLPC_HUMAN Glycophorin-C GYPC GLPC GPC 128 3S, 4T, 6S, 9S, 10T, 15S, 24S, 26S, 27T, 28T, 31T, 32T, 33T, 42S 25
P16860 ANFB_HUMAN Natriuretic peptides B NPPB 134 62T, 63S, 70S, 74T, 79S, 84T, 97T 26
P04141 CSF2_HUMAN Granulocyte-macrophage colony-stimulating factor CSF2 GMCSF 144 22S, 24S, 26S, 27T 27
P02724 GLPA_HUMAN Glycophorin-A GPA GPA 150 21S, 22T, 23T, 29T, 30S, 31T, 32S, 36T, 38S, 41S, 44T, 52T, 56T, 63S, 66S, 69T 28
P10124 SRGN_HUMAN Serglycin SRGN PRG PRG1 158 94S, 96S, 100S, 102S, 104S, 106S, 108S, 110S 29
Q86YL7 PDPN_HUMAN Podoplanin PDPN GP36 PSEC0003 PSEC0025 162 25T, 32T, 34T, 35T, 52T, 55T, 65T, 66T, 76T, 85T, 86S, 88S, 89T, 96S, 98S, 100T, 102S, 106T, 107S, 109S, 110T, 117T, 119T, 120T 30
P0DN87 CGB7_HUMAN Choriogonadotropin subunit beta 7 CGB7 165 139S, 141S, 147S, 150S, 152S, 158S 31
P0DN86 CGB3_HUMAN Choriogonadotropin subunit beta 3 CGB3 CGB; CGB5; CGB8 165 139S, 141S, 147S, 150S, 152S, 158S 32
P01344 IGF2_HUMAN Insulin-like growth factor II IGF2 PP1446 180 96T, 99T, 163T 33
P07498 CASK_HUMAN Kappa-casein CSN3 CASK CSN10 CSNK 182 133T, 143T, 148T, 151T, 157T, 167T, 169T, 178T 34
P31431 SDC4_HUMAN Syndecan-4 SDC4 198 39S, 61S, 63S 35
P34741 SDC2_HUMAN Syndecan-2 SDC2 HSPG1 201 41S, 55S, 57S, 101T 36
Q99075 HBEGF_HUMAN Proheparin-binding EGF-like growth factor HBEGF DTR DTS HEGFL 208 37T, 38S, 44T, 47T, 75T, 85T 37
P13727 PRG2_HUMAN Bone marrow proteoglycan (BMPG) PRG2 MBP 222 23T, 24S, 25T, 34T, 62S 38
P24592 IBP6_HUMAN Insulin-like growth factor-binding protein 6 (IBP-6) IGFBP6 IBP6 240 126T, 144S, 145T, 146T, 152S 39
Q9UHG2 PCSK1_HUMAN ProSAAS (Proprotein convertase subtilisin/kexin type 1 inhibitor) PCSK1N 260 53T, 228S, 247T 40
P01589 IL2RA_HUMAN Interleukin-2 receptor subunit alpha (IL-2 receptor subunit alpha) IL2RA 272 218T, 224T, 229T, 237T 41
P21583 SCF_HUMAN Kit ligand (Mast cell growth factor) (MGF) KITLG MGF SCF 273 167S, 168T, 180T 42
A1E959 ODAM_HUMAN Odontogenic ameloblast-associated protein (Apin) ODAM APIN 279 115T, 119T, 244T, 249S, 250T, 251T, 255T, 256S, 261T, 263T, 273T, 275S 43
P10451 OSTP_HUMAN Osteopontin SPP1 BNSP OPN PSEC0156 314 134T, 138T, 143T, 147T, 152T 44
P21815 SIAL_HUMAN Bone sialoprotein 2 (Bone sialoprotein II) (BSP II) IBSP BNSP 317 119T, 122T, 227T, 228T, 229T, 238T, 239T 45
P02649 APOE_HUMAN Apolipoprotein E (Apo-E) APOE 317 26T, 36T, 212T, 307T, 308S, 314S 46
Q99645 EPYC_HUMAN Epiphycan (Dermatan sulfate proteoglycan 3) EPYC DSPG3 PGLB SLRR3B 322 60T, 64S, 96S 47
Q6UXG3 CLM9_HUMAN CMRF35-like molecule 9 (CLM-9) CD300LG CLM9 TREM4 UNQ422/PRO846 332 137T, 143T, 144T, 155T, 161T, 170T, 171T, 177T, 187T, 195T, 196S, 199T, 201T
, 202S, 207T, 208S, 213S, 214S, 222S, 223T, 224S, 228T, 229S, 237S 48
Q9GZM5 YIPF3_HUMAN Protein YIPF3 (Killer lineage protein 1) YIPF3 C6orf109 KLIP1 350 333T, 334T, 339T, 346T 49
P51681 CCR5_HUMAN CC chemokine receptor type 5 (CC CKR-5) CCR5 CMKBR5 352 6S, 7S, 16T, 17S 50
P40225 TPO_HUMAN Thrombopoietin (C-mpl ligand) (ML) THPO MGDF 353 22S, 58T, 131T, 179T, 180T, 184S, 213T, 265S 51
P01876 IGHA1_HUMAN Immunoglobulin heavy constant alpha 1 (Ig alpha-1 chain C region) IGHA1 353 105S, 106T, 109T, 111S, 113S, 117T, 119S, 121S 8
P02765 FETUA_HUMAN Alpha-2-HS-glycoprotein (Alpha-2-Z-globulin) AHSG FETUA PRO2743 367 270T, 280S, 293S, 339T, 341T, 346S 52
P21810 PGS1_HUMAN Biglycan BGN SLRR1A 368 42S, 47S, 180S, 198S 53
P01860 IGHG3_HUMAN Immunoglobulin heavy constant gamma 3 (HDC) IGHG3 377 122T, 137T, 152T 54
P80370 DLK1_HUMAN Protein delta homolog 1 (DLK-1) DLK1 DLK 383 94S, 143T, 163S, 214S, 222T 251S 256T, 260S 55
P01880 IGHD_HUMAN Immunoglobulin heavy constant delta (Ig delta chain C region) IGHD 384 109S, 110S, 113T, 126T, 127T, 131T, 132T 7
P15529 MCP_HUMAN Membrane cofactor protein (TLX) CD46 MCP MIC10 392 290S, 291S, 292T, 298S, 300S, 302S, 303T, 304S, 305S, 306T, 307T, 309S,
312S, 313S, 315S, 320T, 326S 56
P04280 PRP1_HUMAN Basic salivary proline-rich protein 1 PRB1 392 40S, 87S, 150S, 330S 57
P78423 X3CL1_HUMAN Fractalkine (C-X3-C motif chemokine 1) CX3CL1 FKN NTT SCYD1 A-152E5.2 397 183T, 253S, 329T 58
P16150 LEUK_HUMAN Leukosialin (GPL115) SPN CD43 400 21T, 22T, 26T, 28T, 29S, 35S, 36T, 37S, 41S, 42S, 46T, 47T, 48S, 50T, 58T, 69T, 99S, 103S, 109T, 113T, 114S, 136T, 137T, 173T, 178T 59
P13473 LAMP2_HUMAN Lysosome-associated membrane glycoprotein 2 (LAMP-2) LAMP2 410 195S, 196T, 200T, 203T, 204T, 207S, 209T, 210T,
211T, 213T 60
P11279 LAMP1_HUMAN Lysosome-associated membrane glycoprotein 1 (LAMP-1) LAMP1 417 197S, 199T, 200T, 207S, 209S, 211S, 61
P21754 ZP3_HUMAN Zona pellucida sperm-binding protein 3 (Sperm receptor) ZP3 ZP3A ZP3B ZPC 424 156T, 162T, 163T 62
P05783 K1C18_HUMAN Keratin, type I cytoskeletal 18 KRT18 CYK18 PIG46 430 30S, 31S, 49S 63
Q08629 TICN1_HUMAN Testican-1 (Protein SPOCK) SPOCK1 SPOCK TIC1 TICN1 439 228T, 383S, 388S 64
O75056 SDC3_HUMAN Syndecan-3 (SYND3) SDC3 KIAA0468 442 80S, 82S, 84S, 91S, 314S, 367S 65
P10645 CMGA_HUMAN Chromogranin-A (CgA) CHGA 457 181T, 183T, 251T 66
P15169 CBPN_HUMAN Carboxypeptidase N catalytic chain (CPN) CPN1 ACBP 458 400T, 402T, 409T 67
P00740 FA9_HUMAN Coagulation factor IX (EC 3.4.21.22) F9 461 85T, 99S, 107S 68
P20333 TNR1B_HUMAN Tumor necrosis factor receptor superfamily member 1B TNFRSF1B TNFBR TNFR2 461 30T, 206T, 221S, 222T, 224S, 230T, 234S, 235T, 239T, 240S, 248S 69
P08670 VIME_HUMAN Vimentin VIM 466 7S, 33T, 34S 70
Q8WXD2 SCG3_HUMAN Secretogranin-3 (Secretogranin III) (SgIII) SCG3 UNQ2502/PRO5990 468 216T, 231T, 359S 71
Q16566 KCC4_HUMAN Calcium/calmodulin-dependent protein kinase type IV (CaMK IV) (EC 2.7.11.17) CAMK4 CAMK CAMK-GR CAMKIV 473 57T, 58S, 137S, 189S, 344S, 345S, 356S 72
P31749 AKT1_HUMAN RAC-alpha serine/threonine-protein kinase (EC 2.7.11.1) AKT1 PKB RAC 480 126S, 129S, 305T, 312T, 473S 73
P31751 AKT2_HUMAN RAC-beta serine/threonine-protein kinase (EC 2.7.11.1) AKT2 481 128S, 131S, 306T, 313T 74
O60883 G37L1_HUMAN G-protein coupled receptor 37-like 1 GPR37L1 ETBRLP2 481 79T, 85T, 86S, 95T, 107T 75
Q9BXF9 TEKT3_HUMAN Tektin-3 TEKT3 490 7T, 9T, 10T 76
P05155 IC1_HUMAN Plasma protease C1 inhibitor (C1 Inh) SERPING1 C1IN C1NH 500 47T, 48T, 64S, 71T, 83T, 88T, 92T, 96T 77
P11831 SRF_HUMAN Serum response factor (SRF) SRF 508 277S, 307S, 309S, 316S, 383S 78
P0DOX3 IGD_HUMAN Immunoglobulin delta heavy chain 　 512 238S, 255T, 256T, 260T, 261T, 79
O75487 GPC4_HUMAN Glypican-4 (K-glypican) GPC4 UNQ474/PRO937 556 494S, 498S, 500S 80
P35052 GPC1_HUMAN Glypican-1 GPC1 558 486S, 488S, 490S 81
P78333 GPC5_HUMAN Glypican-5 GPC5 572 441S, 486S, 495S, 507S, 509S 82
Q8N158 GPC2_HUMAN Glypican-2 GPC2 579 55S, 92S, 155S, 500S, 502S 83
P00748 FA12_HUMAN Coagulation factor XII (EC 3.4.21.38) F12 615 109T, 299T, 305T, 308S, 328T, 329T, 337T 84
P01042 KNG1_HUMAN Kininogen-1 (Alpha-2-thiol proteinase inhibitor) KNG1 BDK KNG 644 401T, 533T, 542T, 546T, 557T, 571T, 577S, 628T 85
P51693 APLP1_HUMAN Amyloid-like protein 1 (APLP) (APLP-1) APLP1 650 215T, 227S, 228T 86
Q9NQ79 CRAC1_HUMAN Cartilage acidic protein 1 (68 kDa chondrocyte-expressed protein) (CEP-68) (ASPIC) CRTAC1 ASPIC1 CEP68 661 608T, 618T, 619T, 621T, 626T 87
Q14515 SPRL1_HUMAN SPARC-like protein 1 (High endothelial venule protein) (Hevin) (MAST 9) SPARCL1 664 31T, 40T, 44S, 116T 88
Q16820 MEP1B_HUMAN Meprin A subunit beta (EC 3.4.24.63) MEP1B 701 593S, 594T, 599T, 603S 89
P17600 SYN1_HUMAN Synapsin-1 (Brain protein 4.1) (Synapsin I) SYN1 705 55S, 87T, 96S, 103S, 261S, 432S, 526T, 564T, 578S 90
P19835 CEL_HUMAN Bile salt-activated lipase (BAL) (EC 3.1.1.13) (EC 3.1.1.3) CEL BAL 753 558T, 569T, 579T, 607T, 618T, 629T, 640T, 651T,
662T, 673T 91
Q9HCU0 CD248_HUMAN Endosialin (Tumor endothelial marker 1) (CD antigen CD248) CD248 CD164L1 TEM1 757 60T, 401T, 428T, 448T, 456T, 459T, 472T, 519T, 541T, 543T, 544T, 545T, 587T, 593T, 594T, 595T, 598S, 601S, 612T, 619T, 623S, 625S, 627T, 630T, 631S, 630T, 631T 636T, 640S, 92
P05067 A4_HUMAN Amyloid-beta precursor protein (APP) APP A4 AD1 770 633T, 651T, 652T, 656S, 659T, 663T, 667S, 93
Q9NR71 ASAH2_HUMAN Neutral ceramidase (N-CDase) (NCDase) (EC 3.5.1.-) (EC 3.5.1.23) ASAH2 HNAC1 780 62T, 67S, 68T, 70T, 73S, 74T, 76T, 78S, 79S, 80T, 82T, 84T 94
P08047 SP1_HUMAN Transcription factor Sp1 SP1 TSFP1 785 491S, 612S, 640T, 641S, 698S, 702S 95
Q17R60 IMPG1_HUMAN Interphotoreceptor matrix proteoglycan 1 IMPG1 IPM150 SPACR 797 403T, 421T, 432T, 442T 96
P19634 SL9A1_HUMAN Sodium/hydrogen exchanger 1 (APNH) SLC9A1 APNH1 NHE1 815 42T, 56S, 61T, 62T, 68T 97
P12830 CADH1_HUMAN Cadherin-1 (CAM 120/80) CDH1 CDHE UVO 882 280S, 285T, 358T, 470T, 472T, 509T,
576T, 578T, 580T 98
Q14118 DAG1_HUMAN Dystroglycan (Dystrophin-associated glycoprotein 1) DAG1 895 63T, 317T, 319T, 367T, 369T, 372T, 379T, 388T, 455T 99
Q14624 ITIH4_HUMAN Inter-alpha-trypsin inhibitor heavy chain H4 (ITI heavy chain H4) (ITI-HC4) ITIH4 IHRP ITIHL1 PK120 PRO1851 930 719T, 720T, 722T 100
P19823 ITIH2_HUMAN Inter-alpha-trypsin inhibitor heavy chain H2 (ITI heavy chain H2) (ITI-HC2) ITIH2 IGHEP2 946 666T, 673S, 675T, 691T 101
Q9UPV9 TRAK1_HUMAN Trafficking kinesin-binding protein 1 TRAK1 KIAA1042 OIP106 953 447S, 680S, 719S, 935T 102
P15941 MUC1_HUMAN Mucin-1 (MUC-1) MUC1 PUM 1255 131T, 139T, 140S, 144T 103
Q7Z589 EMSY_HUMAN BRCA2-interacting transcriptional repressor EMSY EMSY C11orf30 GL002 1322 228S, 236S, 271T, 501T, 506T, 557S, 1120T 104
Q92954 PRG4_HUMAN Proteoglycan 4 (Lubricin) PRG4 MSF SZP 1404 123S, 136S, 240T, 253T, 277T, 291T, 305T, 306S, 310T, 317S, 324T, 332T, 338T, 367T, 373S, 376T, 384T, 385T, 388S, 391T, 399T, 400T, 407T, 408T, 415T 423T, 427S, 430T, 438T, 439T, 446T, 447T, 454T, 455T, 477T, 478T, 485T, 493T, 494T, 501T, 502T, 509T, 525T, 529S, 532T, 540T, 541T, 553S, 555T,
563T 564T, 571T, 572T, 579T, 580T, 587T, 588T, 595T, 603T, 604T, 611T, 612T, 616T, 619T, 627T, 676T, 683T, 684T, 691T, 692T, 699T, 700T, 704T, 707T, 723T, 707T, 723T 724T, 736T, 768T, 769T, 776T, 777T, 792T, 793T, 805T, 812S, 829T, 837T, 838T, 892S, 900T,
930T, 931T, 962S, 963T, 968T, 975T, 978T, 979T, 980T, 1039T, 980T, 1039T 1161T 105
Q76LX8 ATS13_HUMAN A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAM-TS 13) ADAMTS13 C9orf8 UNQ6102/PRO20085 1427 399S, 698S, 757S, 907S, 965S, 1027S, 1087S 106
P49790 NU153_HUMAN Nuclear pore complex protein Nup153 (153 kDa nucleoporin) (Nucleoporin Nup153) NUP153 1475 534S, 544S, 908S, 909S, 1113S, 1156T 107
P31327 CPSM_HUMAN Carbamoyl-phosphate synthase [ammonia], mitochondrial (EC 6.3.4.16) CPS1 1500 537S, 1331S, 1332T 108
Q8N6G6 ATL1_HUMAN ADAMTS-like protein 1 (ADAMTSL-1) (Punctin-1) ADAMTSL1 ADAMTSR1 C9orf94 UNQ528/PRO1071 1762 48T, 312T, 391S, 451T 109
P46531 NOTC1_HUMAN Neurogenic locus notch homolog protein 1 (Notch 1) (hN1) NOTCH1 TAN1 2555 65S, 73T, 116T, 146S, 194T, 232T, 311T, 341S, 349T, 378S, 435S, 458S, 466T, 496S, 534S, 609S, 617T, 647S, 692T, 722S, 759S, 767T, 784S, 797S, 805T, 921S, 951S, 997T, 1027S, 1035T, 1065S, 1159T, 1189S, 1197T, 1273S, 1362T, 1379T, 1402T, 110
P04275 VWF_HUMAN von Willebrand factor (vWF) VWF F8VWF 2813 1248T, 1255T, 1256T, 1263S, 1468T, 1477T,
1486S, 1487T, 111
Q9UPA5 BSN_HUMAN Protein bassoon (Zinc finger protein 231) BSN KIAA0434 ZNF231 3926 1343T, 1384T, 2314T, 2691T, 2936T 112
Q86WI1 PKHL1_HUMAN Fibrocystin-L (Polycystic kidney and hepatic disease 1-like protein 1) (PKHD1-like protein 1) PKHD1L1 4243 122T, 445T, 1803T, 1839T, 2320T, 3736T 113
[66]
The fusion polypeptide has a total number of O-glycans actually comprised at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or 21 or more (the maximum value is determined by the number of O-glycosylated polypeptide regions described above and the number of O-glycosylated residues contained in each O-glycosylated polypeptide region), or theoretically included - the total number of glycans is at least 20, at least 21, at least 23, or at least 24 (the maximum value is the number of O-glycosylated polypeptide regions described above and each O-glycosylated polypeptide region determined by the number of O-glycosylation residues included). In addition, the total number of O-glycans actually included in the fusion polypeptide may be related to stability when administered in vivo (eg, in blood), specifically, the total number of O-glycans actually included in the fusion polypeptide. As the number increases, the stability in the body of the fusion polypeptide or GDF15 contained in the fusion polypeptide increases (ie, the half-life in the body (in blood) increases and/or the concentration in the body (in blood) increases and/or the degradation rate in the body (in the blood) decrease, etc.).
[67]
The fusion polypeptide further comprises a peptide linker between GDF15 and the O-glycosylated polypeptide region and/or between the O-glycosylated polypeptide region when two or more O-glycosylated polypeptide regions are included. can do. In one embodiment, the peptide linker may be a GS linker repeatedly including one or more Gly (G) and one or more Ser (S), for example, (GGGGS) n (n is GGGGS (SEQ ID NO: 13). As may be an integer of 1 to 10 or 1 to 5 (eg, 1, 2, 3, 4, or 5), but is not limited thereto.
[68]
Another example provides a fusion polypeptide dimer comprising two of the fusion polypeptides. The fusion polypeptide dimer may be formed by being linked by a bond (eg, a disulfide bond) between GDF15 included in each fusion polypeptide. The fusion polypeptide dimer may be a homodimer.
[69]
In the fusion polypeptide and/or the fusion polypeptide dimer, GDF15 fused with an O-glycosylated polypeptide region is in vivo (or blood) compared to GDF15 to which the O-glycosylated polypeptide region is not fused. Characterized by increased stability (eg, increased half-life in the body or blood).
[70]
Another example provides a nucleic acid molecule encoding the fusion polypeptide.
[71]
Another example provides a recombinant vector comprising the nucleic acid molecule.
[72]
Another example provides a recombinant cell comprising the recombinant vector.
[73]
Another example is a method for producing GDF15 with increased in vivo (or blood) half-life, comprising the step of expressing the recombinant vector in a cell, or production of a fusion polypeptide comprising GDF15 with increased in vivo (or blood) half-life provide a way
[74]
Another example provides a method of increasing the in vivo duration of GDF15 comprising fusing (or linking or binding) GDF15 with an O-glycosylated polypeptide region. In one embodiment, the fusing step comprises fusing (or linking or binding) one or more O-glycosylated polypeptide regions to the N-terminus, C-terminus or both ends of GDF15 with or without a linker. can The fusion (or linking or binding) step may be performed in vitro.
[75]
Another example is the fusion polypeptide, a fusion polypeptide dimer comprising the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector. It provides a pharmaceutical composition comprising at least one selected from the group consisting of.
[76]
Another example provides the use of an O-glycosylated polypeptide region for enhancing the stability of a polypeptide (protein or peptide) drug in the body (or blood) and/or increasing the half-life in the body (or blood). Specifically, one example provides a composition for enhancing the stability of a polypeptide (protein or peptide) drug in the body (or blood) and/or increasing the half-life in the body (or blood) comprising a polypeptide region capable of O-glycosylation. As used herein, enhanced stability and/or increased half-life means improved stability and/or increased half-life compared to a polypeptide (protein or peptide) that does not contain an O-glycosylated polypeptide region. do.
[77]
Hereinafter, the present invention will be described in more detail:
[78]
In the present specification, GDF15 (Growth differentiation factor 15) (corresponding to Y in the above general formula) is a soluble polypeptide, excluding signal peptide and propeptide from a total of 308 amino acids (UniProt Q99988), 197th (A) to 308th (I) ) to amino acids (SEQ ID NO: 3; see FIG. 1; mature form):
[79]
In this specification, GDF15, unless otherwise stated,
[80]
(1) the amino acid sequence from position 197 (A) to position 308 (I) of the full-length protein (UniProt Q99988) (SEQ ID NO: 3, see FIG. 1; ARNG DHCPLGPGRC CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA ANMHAQIKTS LHRLKPDTVD APCCVPASYN PMVLIQKDCHN PMVLIQ);
[81]
(2) functional variants of GDF15; and/or
[82]
(3) 80% or more, 85% or more, 90 or more, 95% or more, 96% or more, 97% or more of the amino acid sequence of (1) and/or (2) above within the range of maintaining the intrinsic activity and structure of GDF15 , refers to a polypeptide essentially comprising an amino acid sequence having at least 98%, or at least 99% sequence homology.
[83]
In the present specification, the functional variant of GDF15 may be a variant in which GDF15 is more advantageous for dimer structure formation while maintaining intrinsic activity and structure. In one example, the functional variant of GDF15 comprises at least one of the 14 amino acid residues at the N-terminus of the amino acid sequence of GDF15 of SEQ ID NO: 3 (i.e., a total of 14 amino acid residues from the first to the 14th in SEQ ID NO: 1) ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) (eg, one or more in order from the N-terminus), such as all 14 amino acid residues are deleted may be an N-terminal deletion variant. In one embodiment, the functional variant of GDF15 retains the amino acid sequence of SEQ ID NO: 4 (CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA ANMHAQIKTS LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL LAKDCHCI) and at least 80% of the amino acid sequence of GDF15, and at least 85% of the amino acid sequence and intrinsic activity and structure of GDF15. % or more, 90 or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence homology.
[84]
In a fusion polypeptide comprising GDF15 and an O-glycosylated polypeptide region provided herein, GDF15 and an O-glycosylated polypeptide region, and/or two or more O-glycosylated polypeptide regions are covalent Alternatively, it may be non-covalently linked directly (eg, without a linker) or linked through an appropriate linker (eg, a peptide linker). The peptide linker may be a polypeptide consisting of 1 to 20, 1 to 15, 1 to 10, 2 to 20, 2 to 15, or 2 to 10 arbitrary amino acids, and the type of amino acids included is not limited. The peptide linker may include, for example, Gly, Asn and/or Ser residues, and may also include neutral amino acids such as Thr and/or Ala, but is not limited thereto, and an amino acid sequence suitable for a peptide linker may include: known in the art. In one embodiment, the peptide linker may be a GS linker repeatedly including one or more Gly (G) and one or more Ser (S), for example, (GGGGS) n (n is GGGGS (SEQ ID NO: 13). as an integer of 1 to 10 or an integer of 1 to 5 (which is 1, 2, 3, 4, or 5), but is not limited thereto.
[85]
In addition, the fusion polypeptide comprises a total of 1 or more or a total of 2 or more (eg, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 5) O-glycosylated polypeptide regions. 4, 2 or 3) may be included. When the fusion polypeptide comprises two or more O-glycosylated polypeptide regions, the fusion polypeptide may have two or more O-glycosylated polypeptide regions bonded to the N-terminus of GDF15, each The O-glycosylated polypeptide regions may be the same as or different from each other. In this case, the above-described peptide linker may be additionally included between the O-glycosylated polypeptide region and/or between the O-glycosylated polypeptide region and human GDF15.
[86]
The fusion polypeptide provided herein may be recombinantly or synthetically produced, and may not be naturally occurring.
[87]
The in vivo (blood) half-life in mammals of GDF15 contained in the fusion polypeptides provided herein is about 1.1 times or more, about 1.15 times or more, compared to GDF15 to which the O-glycosylation capable polypeptide region is not fused, about 1.2 times or more, about 1.5 times or more, about 2 times or more, about 2.5 times or more, about 3 times or more, about 3.5 times or more, about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, It may be increased by about 8 times or more, about 9 times or more, or about 10 times or more. Alternatively, the peak plasma concentration of GDF15 included in the fusion polypeptide provided herein when administered in a mammal is about 1.2 times or more, about 1.5 times or more, about 2 times or more, about 2.5 times or more, compared to unfused GDF15, It may be at least twice, at least about 3 times, at least about 3.5 times, or at least about 4 times higher. Alternatively, the time to reach the peak plasma concentration of GDF15 included in the fusion polypeptide provided herein is about 2 times or more, about 3 times or more, about 4 times or more, compared to unfused GDF15, when administered in a mammalian body, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, about 11 times or more, about 12 times or more, about 13 times or more, about 14 times or more, It may be about 15 times or more, about 18 times or more, about 20 times or more, or about 22 times or more. Alternatively, when GDF15 contained in the fusion polypeptide provided herein is administered in a mammalian body, the area under the blood concentration-time curve until the last measurable blood sampling time (AUC last) and/or the area under the blood concentration-time curve (AUC inf ) calculated by extrapolating from the last measurable blood draw to infinity is approximately twice that of GDF15 that is not fused to an O-glycosylated polypeptide region. or more, about 2.5 times or more, about 3 times or more, about 3.5 times or more, about 4 times or more, about 4.5 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more or more, about 10 times or more, about 11 times or more, about 12 times or more, about 13 times or more, about 14 times or more, or about 15 times or more.
[88]
As such, due to the increased half-life of GDF15, GDF15 in the form of a fusion polypeptide to which an O-glycosylation-capable polypeptide region is bound is compared with GDF15 in a form in which an O-glycosylation-capable polypeptide region is not linked, the administration interval It has the advantage of being able to take it for a long time.
[89]
A fusion polypeptide comprising GDF15 and a polypeptide region capable of O-glycosylation can be prepared by conventional chemical synthesis or recombinant methods.
[90]
As used herein, the term “vector” refers to an expression means for expressing a target gene in a host cell, for example, a plasmid vector, a cosmid vector, and a bacteriophage vector, an adenoviral vector, a retroviral vector and It may be selected from the group consisting of viral vectors such as adeno-associated viral vectors, and the like. In one embodiment, the vector that can be used for the recombinant vector is a plasmid (eg, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1, M13, etc.) or virus (eg, SV40, etc.), but is not limited thereto.
[91]
In the recombinant vector, the nucleic acid molecule encoding the fusion polypeptide may be operably linked to a promoter. The term “operatively linked” refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter sequence) and another nucleic acid sequence. Such regulatory sequences may be "operatively linked" to control the transcription and/or translation of other nucleic acid sequences.
[92]
The recombinant vector can typically be constructed as a vector for cloning or an expression vector for expression. The expression vector may be a conventional one used in the art to express a foreign protein in plants, animals or microorganisms. The recombinant vector can be constructed through various methods known in the art.
[93]
The recombinant vector can be expressed using a eukaryotic cell as a host. When expressing a eukaryotic cell as a host, the recombinant vector contains the nucleic acid molecule to be expressed and the promoter, ribosome binding site, secretion signal sequence (see Patent Publication No. 2015-0125402) and/or transcription/ In addition to translation termination sequences, origins of replication operating in eukaryotic cells may include, but are not limited to, the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, and/or the BBV origin of replication. not. In addition, promoters derived from the genome of mammalian cells (eg, metallotionine promoter) or from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and HSV tk promoter) can be used, and any secretion signal sequence that is commonly used as a secretion signal sequence can be used. For example, the secretion signal sequence described in Patent Publication No. 2015-0125402 can be used, but It is not limited thereto, and may include a polyadenylation sequence as a transcription termination sequence.
[94]
The recombinant cell may be obtained by introducing the recombinant vector into an appropriate host cell (transformation or transfection). The host cell may be selected from all eukaryotic cells capable of stably and continuously cloning or expressing the recombinant vector. Eukaryotic cells that can be used as hosts include yeast ( Saccharomyces cerevisiae ), insect cells, plant cells, and animal cells, for example, mice (eg, COP, L, C127, Sp2/0, NS-0, NS-1, At20, or NIH3T3), rats (such as PC12, PC12h, GH3, or MtT), hamsters (such as BHK, CHO, GS defective CHO, or DHFR gene defective CHO), monkeys (such as COS ( COS1, COS3, COS7, etc.), CV1 or Vero), human (such as HeLa, HEK-293, retina-derived PER-C6, cells derived from diploid fibroblasts, myeloma cells or HepG2), other animal cells (such as , MDCK, etc.), insect cells (eg, Sf9 cells, Sf21 cells, Tn-368 cells, BTI-TN-5B1-4 cells, etc.), hybridomas, etc., but are not limited thereto.
[95]
By expressing the nucleic acid molecule encoding the fusion polypeptide provided herein in an appropriate host cell as described above, GDF15 or a fusion polypeptide comprising the same with improved in vivo stability compared to the unfused form can be prepared. The method for producing the fusion polypeptide may include culturing a recombinant cell containing the nucleic acid molecule. The culturing step may be performed under conventional culture conditions. In addition, the preparation method may further include isolating and/or purifying the fusion polypeptide from the culture after the culturing step.
[96]
Delivery (introduction) of the nucleic acid molecule or a recombinant vector containing the same into a host cell may use a delivery method well known in the art. The delivery method may include, but is not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and gene bombardment, for example, when the host cell is a eukaryotic cell. .
[97]
The method for selecting the transformed (recombinant vector introduction) host cell can be easily carried out according to a method well known in the art using the phenotype expressed by the selection marker. For example, when the selection marker is a specific antibiotic resistance gene, recombinant cells into which the recombinant vector is introduced can be easily selected by culturing in a medium containing the antibiotic.
[98]
The fusion polypeptide can be used for the prevention and/or treatment of any disease associated with GDF15 deficiency and/or dysfunction, or which can be treated, alleviated, or ameliorated by the activity of GDF15.
[99]
Accordingly, in one embodiment, a pharmaceutical comprising at least one selected from the group consisting of the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector A composition is provided. The pharmaceutical composition may be a pharmaceutical composition for preventing and/or treating a disease related to GDF15 deficiency and/or dysfunction or a disease in which GDF15 has a therapeutic and/or prophylactic effect included in the fusion protein.
[100]
Another example is the fusion protein comprising at least one selected from the group consisting of the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector GDF15 contained in the fusion protein, comprising administering to a patient in need of prophylaxis and/or treatment of a disease related to GDF15 deficiency and/or dysfunction or a disease in which the GDF15 has a therapeutic and/or prophylactic effect Provided is a method for preventing and/or treating a disease associated with deficiency and/or dysfunction or a disease in which the GDF15 has therapeutic and/or prophylactic effects. The method includes, prior to the step of administering, a patient in need of prevention and/or treatment of a disease related to GDF15 deficiency and/or dysfunction contained in the fusion protein or a disease in which GDF15 has a therapeutic and/or prophylactic effect. The step of confirming may be further included.
[101]
Obesity, diabetes (type 1 diabetes, type 2 diabetes), cardiovascular disease, myocardium as examples of diseases (or symptoms) in which the GDF15 deficiency and/or dysfunction are related or in which the GDF15 has therapeutic and/or preventive effects Hypertrophy, liver disease (eg, nonalcoholic steatohepatitis (NASH), etc.), ischemic damage (ischemic brain damage, ischemic retinal damage), peripheral nerve damage, age-related sensory and/or motor nerve loss, renal tubules and / or renal interstitial injury and the like may be exemplified, but not limited thereto.
[102]
In another example, the pharmaceutical composition provided herein or a method comprising the step of administering the same is one or more effects selected from the group consisting of weight loss, diet control (reducing the amount of food), reducing body fat, conferring and/or enhancing glucose tolerance, and the like. may have, and in this case, the pharmaceutical composition or method may be applied for weight loss, body fat reduction, and/or use for imparting and/or enhancing glucose tolerance.
[103]
Accordingly, in one embodiment, a composition comprising at least one selected from the group consisting of the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector As such, it may be a pharmaceutical composition or a food composition (health functional food) for weight loss, dietary control (reducing the amount of food), reducing body fat, and imparting and/or enhancing glucose tolerance.
[104]
Another example is weight loss, diet control, at least one selected from the group consisting of the fusion polypeptide, a nucleic acid molecule encoding the fusion polypeptide, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the recombinant vector A method for weight loss, dietary control (meal reduction), body fat reduction, and conferring and/or enhancing glucose tolerance, comprising administering to a patient in need thereof to provide. The method may further include, prior to the administering step, identifying a patient in need of weight loss, dietary control (reduced amount of food), body fat reduction, and conferring and/or enhancing glucose tolerance.
[105]
The pharmaceutical composition may include at least one active ingredient selected from the group consisting of the fusion polypeptide, a fusion polypeptide dimer comprising the fusion polypeptide, a nucleic acid molecule, a recombinant vector, and a recombinant cell in a pharmaceutically effective amount. . The pharmaceutically effective amount means the content or dosage of an active ingredient capable of obtaining a desired effect. The content or dosage of the active ingredient in the pharmaceutical composition depends on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration interval, administration route, excretion rate, and response sensitivity. can be prescribed in a variety of ways. For example, the 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, or 0.01 to 1 mg/kg. It is not limited.
[106]
In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier in addition to the active ingredient. The carrier is commonly used in the formulation of drugs containing proteins, nucleic acids, or cells, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, It may be at least one selected from the group consisting of microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. However, the present invention is not limited thereto. The pharmaceutical composition may further include at least one selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, which are commonly used in the preparation of pharmaceutical compositions.
[107]
The administration target of the pharmaceutical composition may be a mammal including a human, a primate including a monkey, a mouse, a rodent including a rat, and the like, or a cell, tissue, cell culture or tissue culture derived therefrom.
[108]
The pharmaceutical composition may be administered by oral or parenteral administration, or administered by contacting cells, tissues, or body fluids. Specifically, in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. may be administered. When administered orally, the protein or peptide is digestible and therefore oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach.
[109]
In addition, the pharmaceutical composition may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be formulated in the form of an extract, powder, powder, granule, tablet or capsule, and a dispersion or stable for formulation. Additional topics may be included.
[110]
Effects of the Invention
[111]
GDF15 fused with the O-glycosylated polypeptide region provided herein has a long duration when administered in vivo, so that the administration interval can be increased and the dosage can be reduced through this, which is advantageous in terms of administration convenience and/or economics. and can be usefully applied to fields requiring GDF15 treatment.
[112]
Brief description of the drawing
[113]
1 schematically shows the amino acid sequence of GDF15.
[114]
Figure 2a schematically shows a fusion polypeptide comprising His Taq according to an embodiment.
[115]
Figure 2b schematically shows the structure of the fusion polypeptide according to an embodiment.
[116]
3 schematically shows the structures of various types of fusion polypeptides.
[117]
4 shows the results of SDS-PAGE analysis of the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 synthesized in one embodiment.
[118]
5 shows the results of Q-TOF Mass Spectrometry analysis of the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 synthesized in one example.
[119]
6 is a graph showing the change in body weight when the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 were administered to mice once, respectively.
[120]
FIG. 7 is a graph showing the results of the 4th day extracted from the results of FIG. 6 .
[121]
8 is a graph showing changes in feed intake of mice administered with fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15.
[122]
9 is a graph showing changes in body weight upon repeated administration of the fusion polypeptides HT-ID2-GDF15 and HT-ID3-GDF15 to mice.
[123]
10A is a graph showing the results of the 7th and 14th days extracted from the results of FIG. 9 .
[124]
10B is a graph showing the results of days 21 and 28 extracted from the results of FIG. 9 .
[125]
11 is a graph showing the cumulative feed intake up to day 7, 14, 21, and 28 when the fusion polypeptides HT-ID2-GDF15 and HT-ID3-GDF15 were repeatedly administered to mice, respectively.
[126]
12 is a graph showing changes in blood fusion polypeptide concentration with time when the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 are administered to SD Rat.
[127]
Modes for carrying out the invention
[128]
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
[129]
[130]
Example 1: Preparation of fusion polypeptides
[131]
1.1. Preparation of fusion polypeptides comprising GDF15
[132]
IgD hinge (ESPKAQA SS VP T AQPQAEGSLAKA TT APA TT RNT; SEQ ID NO: 1; underlined portion is O-Glycosylation possible site) or multiple (1, 2 or 3) IgD hinge combination is GDF15 (SEQ ID NO: 1) Fusion polypeptides IgD-GDF15 (ID1-GDF15), IgD-IgD-GDF15 (ID2-GDF15), IgD-IgD-IgD-GDF15 (ID3-GDF15) (see FIGS. 2A and 2B ) fused with No. 3, FIG. 1 ) ) was prepared. For the convenience of purification, a fusion polypeptide including a His-tag (SEQ ID NO: 15) and a TEV cleavage site (SEQ ID NO: 16) was also prepared. The amino acid sequence of each part included in the fusion polypeptide is summarized in Table 2 below.
[133]
[Table 2]
Amino acid sequence (N terminus → C terminus) SEQ ID NO:
Signal Peptide (SP7.2) MHRPEAMLLL LTLALLGGPT WA 14
Polypeptide of interest (GDF15) ARNGDHCPLG PGRCCRLHTV RASLEDLGWA DWVLSPREVQ VTMCIGACPS QFRAANMHAQ IKTSLHRLKP DTVPAPCCVP
ASYNPMVLIQ KTDTGVSLQT YDDLLAKDCH CI 3
Hinge region (ID) of immunoglobulin IgD ESPKAQASSV PTAQPQAEGS LAKATTAPAT TRNT One
His-Tag HHHHHHHHH 15
TEV Clean Site ENLYFQG 16
GS Linker GGGGSGGGGS GGGGSGGGGS 17
[134]
[135]
1.1.1. Preparation of Recombinant Expression Vector
[136]
1.1.1.1. Mature GDF15
[137]
To obtain a gene encoding Mature GDF15, a gene encoding Mature GDF15 (SEQ ID NO: 5) was synthesized with reference to the amino acid sequence information of UniprotKB Q99968 (Bioneer Co., Ltd.).
[138]
SEQ ID NO: 5 (339bp)
[139]
1 GCCCGGAACG GCGACCACTG CCCCCTGGGG CCCGGACGGT GCTGCCGGCT
[140]
51 GCACACCGTG CGGGCCTCCC TGGAGGACCT GGGCTGGGCC GACTGGGTGC
[141]
101 TGTCCCCAAG GGAGGTGCAA GTGACCATGT GCATCGGCGC CTGCCCATCT
[142]
151 CAGTTCCGGG CCGCCAACAT GCACGCTCAG ATCAAGACCA GCCTGCACCG
[143]
201 GCTGAAGCCC GACACCGTGC CCGCCCCCTG CTGCGTGCCC GCCTCCTACA
[144]
251 ACCCCATGGT GCTGATTCAG AAGACCGACA CCGGCGTGAG CCTGCAGACC
[145]
301 TACGACGACC TGCTGGCCAA GGACTGCCAC TGCATCTAA
[146]
[147]
1.1.1.2. IgD Hinge (ID)
[148]
To obtain a gene encoding Human IgD Hinge, a gene (SEQ ID NO: 6) encoding three Human IgD Hinges (hereinafter, denoted as 'ID3') was synthesized in Bioneer with reference to the amino acid sequence information of UniprotKB P01880.
[149]
SEQ ID NO: 6 (306bp)
[150]
1 GAGAGCCCTA AGGCTCAGGC CTCTAGCGTG CCAACAGCTC AGCCACAAGC
[151]
51 TGAAGGAAGC CTGGCCAAGG CTACAACCGC CCCTGCCACA ACACGGAATA
[152]
101 CA GAGTCCCC CAAGGCCCAG GCTAGCAGCG TGCCTACCGC CCAGCCTCAG
[153]
151 GCCGAGGGCT CCCTGGCTAA GGCCACAACC GCTCCCGCTA CAACCAGGAA
[154]
201 CACC GAGTCT CCAAAGGCAC AGGCCTCCTC CGTGCCCACT GCACAACCCC
[155]
251 AAGCAGAGGG CAGCCTCGCC AAGGCAACCA CAGCCCCAGC CACCACCCGG
[156]
301 AACACA
[157]
(1-102 polynucleotides (underlined), 103-204 polynucleotides (bold), and 205-306 polynucleotides (bold + underlined) encode IgD Hinge, respectively)
[158]
[159]
1.1.1.3. Preparation of expression vector
[160]
pDHDD-D1G1 (containing the promoter of KR10-1868139B1), a variant of pcDNA3.1(+) (Invitrogen, Cat. No. V790-20), was cut with BamHI and NotI, and the above genes (mature GDF15 encoding) Each recombinant vector was prepared by combining a gene and ID3 encoding gene) and inserting a gene designed to encode a fusion protein having the following structure (see FIG. 3 ).
[161]
pGDF15
[162]
'(N-terminal)-[BamHI restriction site (GGATCC)-signal peptide (SEQ ID NO: 14)-Mature GDF15 (SEQ ID NO: 3)-NotI restriction site (GCGGCCGC)]-(C-terminal)'
[163]
pHT-GDF15
[164]
'(N-terminal)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-His-Taq (SEQ ID NO: 15)-TEV Cleavage Site (SEQ ID NO: 16)-Mature GDF15 (SEQ ID NO: 3)-NotI restriction site] -(C-terminal)'
[165]
pID1-GDF15
[166]
'(N-terminal)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17)-Mature GDF15 (SEQ ID NO: 3)-NotI restriction site]-( C-terminus)'
[167]
pHT-ID1-GDF15
[168]
'(N-terminus)-[BamHI restriction site-Signal peptide (SEQ ID NO: 14)-His-Taq (SEQ ID NO: 15)-TEV Cleavage Site (SEQ ID NO: 16)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 1) No. 17)-GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminus)'
[169]
pID2-GDF15
[170]
'(N-terminal)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17)-GDF15 (SEQ ID NO: 3) -NotI restriction site]-(C-terminal)'
[171]
pHT-ID2-GDF15
[172]
'(N-terminal)-[BamHI restriction site-Signal peptide (SEQ ID NO: 14)- His-Taq (SEQ ID NO: 15)-TEV Cleavage Site (SEQ ID NO: 16)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1) No. 1)-GS Linker (SEQ ID NO: 17)-GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminal)'
[173]
pID3-GDF15
[174]
'(N-terminal)-[BamHI restriction site-signal peptide (SEQ ID NO: 14)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17 )-GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminus)'
[175]
pHT-ID3-GDF15
[176]
'(N-terminal)-[BamHI restriction site-Signal peptide (SEQ ID NO: 14)-His-Taq (SEQ ID NO: 15)-TEV Cleavage Site (SEQ ID NO: 16)-IgD Hinge (SEQ ID NO: 1)-IgD Hinge (SEQ ID NO: 1) No. 1)-IgD Hinge (SEQ ID NO: 1)-GS Linker (SEQ ID NO: 17)-GDF15 (SEQ ID NO: 3)-NotI restriction site]-(C-terminal)'
[177]
[178]
1.1.2. Expression of the fusion polypeptide
[179]
The above-prepared recombinant expression vectors pGDF15, pHT-GDF15, pID1-GDF15, pHT-ID1-GDF15, pID2-GDF15, pHT-ID2-GDF15, pID3-GDF15, and pHT-ID3-GDF15 were transferred to ExpiCHO-S TM cells (Thermo Fisher Scientific) and cultured in ExpiCHO Expression Medium (Thermo Fisher Scientific; 400 mL) for 12 days (Fed-Batch Culture; Day 1 & Day 5 Feeding), and the fusion polypeptides GDF15, HT-GDF15, ID1-GDF15 , HT-ID1-GDF15, ID2-GDF15, HT-ID2-GDF15, ID3-GDF15, and HT-ID3-GDF15 were expressed.
[180]
[181]
1.1.3. Purification of the fusion polypeptide
[182]
The fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 produced through the expression of the recombinant vector were purified, and O-Glycan site occupancy was measured using Sialic Acid content analysis and Q-TOF Mass Spectrometry. analyzed.
[183]
Specifically, the fusion polypeptide was purified by successively performing ultrafiltration / diafiltration (Ultrafiltration / Diafiltration), metal ion affinity chromatography (IMAC, Immobilized Metal Affinity Chromatography), and anion exchange chromatography (AEX, Anion Exchange Chromatography). First, the culture solution of the fusion protein from which cells were removed was filtered with a 0.22 μm filter. The filtrate was concentrated using the TFF system, and then buffer exchange was performed with a tromethamine buffer solution. A column packed with HiTrap TM Chelating HP (GE Healthcare Life Sciences) resin was mounted on AKTA TM Pure (GE Healthcare Life Sciences) and an equilibration buffer (20 mM Tris pH 8.0, 0.5 M NaCl, 5 mM Imidazole) was flowed through the column. was equalized. After the process solution, which had previously been completed ultrafiltration/diafiltration, was injected into the column, the equilibration buffer was again flowed to wash the column. After the column was washed, an elution buffer (20 mM Tris pH 8.0, 0.5 M NaCl, 0.5 M Imidazole) was flowed through the column to elute the target protein.
[184]
The resulting eluate was concentrated using an Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge, and then the buffer was exchanged with tromethamine buffer. The process solution thus prepared was injected into the equilibrated anion exchange column, and an equilibration buffer (20 mM Tris pH 8.0) was flowed to wash the column. After the column was washed, an elution buffer (20 mM Tris pH 8.0, 0.5 M NaCl) was flowed through the column under gradient conditions to elute the target protein. Among the eluted fractions, fractions having a high concentration of the fusion polypeptide and high purity were collected and stored frozen.
[185]
For animal experiments, samples were concentrated and buffered with Phosphate Buffered Saline (PBS, 10 mM Sodium Phosphate, 150 mM NaCl pH 7.4) using Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge.
[186]
Quantitative analysis of the fusion polypeptide was performed by measuring the absorbance at 280 nm and 340 nm in a UV Spectrophotometer (G113A, Agilent Technologies) and using the following formula. The extinction coefficient of each material was theoretically calculated using the amino acid sequence.
[187]

[188]
*Extinction coefficient (0.1%): Assuming that the protein concentration is 0.1% (1g/L) and all cysteines in the primary sequence are oxidized to form disulfide bonds, this is the theoretical absorbance at 280nm. Calculated via ProtParam tool (https://web.expasy.org/protparam/).
[189]
[Table 3] Extinction coefficient of fusion polypeptide
Sample name Extinction coefficient (0.1%, 1 mg/mL)
HT-ID1-GDF15 0.833
HT-ID2-GDF15 0.706
HT-ID3-GDF15 0.612
[190]
Purified fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 were analyzed for sialic acid content and reduced, and then O-Glyan site occupancy was analyzed using Q-TOF Mass Spectrometry. Fusion Poly The results of analyzing the peptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 by SDS-PAGE are shown in FIG. 4, and the results of analyzing by Q-TOF Mass Spectrometry are shown in FIG. 5 and Table 4. In addition, the sialic acid content is also shown in Table 4.
[191]
[Table 4] Average number of O-Glycan and Sialic Acid content
sample Theoretical
number of O-Glycans (One Chain) Average
number of O-Glycans O-Glycan distribution (main) Silic Acid content (mol/mol)
HT-ID1-GDF15 7 5.4 2-7 (6) 14.4
HT-ID2-GDF15 14 9.7 3-15 * (10) 16.3
HT-ID3-GDF15 21 13.5 5-21 (13) 18.9
[192]
* More than the theoretical number of O-Glycans, it is estimated that O-Glycans are attached to the GS Linker (Spahr et al., 2014, mAbs, 6: 904)
[193]
[194]
Example 2. Pharmacological effect of fusion polypeptide (in vivo)
[195]
2.1. single dose
[196]
2.1.1 Examination process
[197]
The pharmacological effects of the fusion polypeptide produced and purified in Example 1 were tested in mouse (C57BL/6J, 6 weeks old, male, 100 mice; Raon Bio Co., Ltd.).
[198]
In this example, the DIO mouse model (Mouse, C57BL/6J-DIO, male, 100 mice, 14 weeks old (obesity feed for 8 weeks) induced obesity by feeding the high-fat diet to the C57BL/6J mice for 8 weeks )) was used. The DIO mouse model is an animal model widely used to evaluate the efficacy of improving diabetes and insulin resistance because it shows clinical features of type 2 diabetes such as hyperlipidemia, insulin resistance, and hyperglycemia, and for the study of metabolic diseases such as obesity, diabetes and hyperlipidemia Since many comparable basic data have been accumulated, this model was selected as appropriate for the pharmacological effect test of this example.
[199]
The mouse model fed with the obesity feed for 8 weeks was subjected to a quarantine and acclimatization period of 2 weeks, and during this period, general symptoms were observed once a day, and healthy animals were selected by confirming whether they were healthy and suitable for carrying out the experiment. . During the acclimatization period, the animals were individually marked with a red oil pen at the time of acquisition, and a temporary individual identification card (test name, individual number, delivery time) was attached to the breeding box during the quarantine and acclimatization period. At the time of group separation, individuals were marked with a black oil pen on the tail of the animals, and an individual identification card (test name, group information, individual number, gender, time of stocking, administration period) was attached to each cage.
[200]
In order to minimize the stress experienced by the experimental animals due to the subcutaneous administration of the test substance (fusion polypeptide), 200 uL/head of sterile distilled saline was administered subcutaneously to all animals using a 1 mL syringe from 3 days before the administration of the test substance. Pre-adaptation training for subcutaneous administration was performed.
[201]
For healthy animals with no abnormalities found during quarantine and acclimatization period, body weight and feed intake were measured for all individuals after the acclimatization period.
[202]
Body weight and feed intake were measured, and group separation was performed so that the averages of the two measured values ​​were similar between groups based on body weight. Test substance administration was started from the day after group separation. Remaining animals that were not selected were excluded from the test system after group separation was terminated.
[203]
Information of the high fat feed (obesity feed; High fat diet (HFD)) fed to the C57BL/6J -DIO is as follows:
[204]
5.24 kcal/g, 60% fat, 20% protein, and 20% carbohydrate-derived calories; Research Diet Inc., USA; Product No. High fat diet (Fat 60 kcal%, D12492).
[205]
The feed was fed in a free feeding (acclimatization and feeding during the test period) mode.
[206]
In the drinking water method, tap water was filtered with a filter oil-water sterilizer, irradiated with ultraviolet rays, and freely ingested using a polycarbonate drinking water bottle (250 mL).
[207]
The test substances HT-ID1-GDF15, HT-ID2-GDF15, HT-ID3-GDF15 and the control substance Semaglutide (Bachem) were administered from the next day after group separation, and the administration time was performed at 9 am every day. Both the control and test substances were administered subcutaneously. For the administration route of the control substance and the test substance, the subcutaneous route was selected according to the clinically planned administration route.
[208]
The dose for both control and test substances was 5 mL/kg, and the dose for each individual was calculated based on the recently measured body weight, and was administered by subcutaneous injection once on the day of the start of the test using a disposable syringe (1 mL). The test substance was administered only once on the test start date. For comparison, for comparison, a control group to which the control substance Semaglutide was administered was prepared, and in the case of the control group to which Semaglutide was administered, it was administered once a day every day, and all administrations were carried out from 9:00 am.
[209]
The composition and dosage of the test group are summarized in Table 5 below:
[210]
[Table 5] Composition of fusion polypeptide administration group
High Fat Diet test substance route of administration Dosage (nmol/kg) Administration volume (mL/kg) number of animals
O Vehicle, qw subcutaneously - 5 5
O Semaglutide, qd subcutaneously 3 5 5
O HT-ID1-GDF15 subcutaneously 10 5 5
O HT-ID2-GDF15 subcutaneously 10 5 5
0 HT-ID3-GDF15 subcutaneously 10 5 5
[211]
As for the observation, measurement, and test schedule for the test group, the start date of administration was Day 0, and 7 days from the start of administration were one week of administration. The test schedule is summarized in Table 6:
[212]
[Table 6] Inspection Schedule
observation items acclimatization period
(week) Duration (day)
One 2 0 One 2 3 4 5 6 7 8 9
high fat feed ● ● ● ● ● ● ● ● ● ● ● ●
Adaptation for oral and subcutaneous administration ●
administration ●
weighing ● ● ● ● ● ● ● ● ● ● ●
Feed intake measurement ● ● ● ● ● ● ● ● ● ●
[213]
General clinical symptoms were observed once a day for all animals, and the presence or absence of moribund and dead animals was checked twice a day, and these observations were conducted from the 1st day of administration to the end of administration. Only when there were abnormal symptoms during observation, it was recorded on the recording sheet. The body weight of each mouse was measured on the start date of administration of the test substance (before administration), and thereafter, the body weight was measured every day (measured up to 9 days). The dose of the test substance was determined based on the most recently measured body weight.
[214]
In addition, after administration of the test substance to the mice, daily feed intake was measured, and the daily feed intake was calculated by measuring the remaining amount after measuring the feeding amount using an electronic scale for each breeding box. In the case of an individual that gnawed heavily on feed, it was excluded from the measurement.
[215]
All experimental results obtained in this Example were expressed as mean ± standard error, and were tested using Prism5 (version 5.01). One-way analysis of variance (ANOVA) was performed on all data, and if significance was observed, Dunnett's test was performed to find out the test group with a significant difference from the control group (significance level: two-sided 5% and 1%, 0.1 %).
[216]
[217]
2.1.2. Weight Loss Test Results
[218]
The weight change measured in Example 2.1.1 is shown in FIGS. 6 and 7, and Table 7 (Body Weight (Group, % of initial)).
[219]
[Table 7]
Group Day 0 One 2 3 4 5 6 7 8 9
DIO Vehicle Control
　(Daily Inj.) Mean 100 100 100 99 100 100 100 100 101 101
SE 0 0 One 0 0 0 0 One 0 One
DIO Semaglutide
3 nmol/kg
　(Daily Inj.) Mean 100 94 91 90 87 87 85 86 83 85
SE 0 0 One 2 2 3 3 3 3 3
HT-ID1-GDF15
10 nmol/kg
　(Single Inj.) Mean 100 99 98 97 96 95 95 96 96 97
SE 0 0 0 One One One One One One One
HT-ID2-GDF15
10 nmol/kg
　(Single Inj.) Mean 100 98 97 96 94 94 94 93 93 94
SE 0 0 One One One 2 2 2 2 2
HT-ID3-GDF15
10 nmol/kg
　(Single Inj.) Mean 100 98 97 96 95 94 95 95 95 96
SE 0 0 0 One 0 One One One One One
[220]
6 and Table 7 show the change in body weight when the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 were administered once, respectively, in a negative control group (vehicle administration group) and positive control group (Semaglutide daily administration group) is shown in comparison with In addition, FIG. 7 is a graph showing the results of the 4th day extracted from the results of FIG. 6 . As shown in the above results, in the case of the negative control group (vehicle administration group), there was little change in body weight, whereas in the case of the positive control group (Semaglutide daily administration group), it was confirmed that the weight loss effect appeared continuously from the first day after administration. In addition, in the case of the fusion polypeptide in which GDF15 is fused with IgD Hinge, it can be confirmed that the weight loss effect was immediately observed after one administration on Day 0, and the weight loss effect did not decrease until 3-4 days. .
[221]
[222]
2.1.3. Dietary intake test results
[223]
Changes in feed intake measured in Example 2.1.1 are shown in Table 8 and FIG. 8 (cumulative intake up to day 6), respectively.
[224]
[Table 8]
Group Day 0 One 2 3 4 5 6 7 8 9
DIO Vehicle Control
(Daily Inj.) Mean 3.3 2.7 3.0 3.0 3.1 3.5 3.0 3.5 3.3 3.4
SE 0.1 0.1 0.2 0.1 0.1 0.2 0.2 0.1 0.1 0.2
DIO Semaglutide 3
nmol/kg (Daily Inj.) Mean 3.7 2.1 3.3 2.8 2.3 2.9 2.7 3.2 2.4 3.7
SE 0.3 0.7 1.4 0.6 0.4 0.9 0.6 0.3 0.4 0.1
HT-ID1-GDF15
10 nmol/kg (Single Inj.) Mean 3.3 2.2 2.9 2.3 2.8 3.1 3.1 3.7 3.4 3.5
SE 0.2 0.3 0.2 0.5 0.2 0.3 0.1 0.3 0.3 0.5
HT-ID2-GDF15
10 nmol/kg (Single Inj.) Mean 2.7 1.7 2.3 2.2 2.6 2.4 2.8 3.1 2.7 3.3
SE 0.2 0.2 0.1 0.1 0.3 0.2 0.1 0.1 0.2 0.2
HT-ID3-GDF15
10 nmol/kg (Single Inj.) Mean 3.2 2.0 2.5 2.3 2.3 2.7 2.8 3.3 3.3 3.6
SE 0.2 0.2 0.3 0.2 0.2 0.3 0.2 0.3 0.2 0.2
[225]
As shown in the above results, in the case of the fusion polypeptide administration group in which GDF15 is fused with IgD Hinge, compared to the negative control (vehicle administration group) administration group, the effect of reducing feed intake up to the 6th day according to the fusion polypeptide is shown, and this fusion The effect of reducing the feed intake of the polypeptide can be said to be comparable to that of the positive control, Semaglutide, administered once a day throughout the test period.
[226]
[227]
2.2. repeat dosing
[228]
2.2.1 Examination process
[229]
Except for the dose, the number of animals and the administration cycle, most of the test procedures were the same as in Example 2.1.1.
[230]
The test substance was administered twice a week (days 0, 4, 7, 11, 14, 18, 21, 25) for a total of 8 administrations. For comparison, a control group to which the control substance Semaglutide was administered daily was prepared, and in the case of the control group to which Semaglutide was administered every day, it was administered once a day, and all administrations were carried out from 9:00 am.
[231]
The composition and dosage of the test group are summarized in Table 9 below:
[232]
[Table 9] Composition of fusion polypeptide administration group
High Fat Diet test substance route of administration dosing cycle Dosage (nmol/kg) Administration volume (mL/kg) number of animals
O Lean Vehicle Control subcutaneously Once a week - 5 4
O DIO Vehicle Control subcutaneously Once a week - 5 8
O Semaglutide subcutaneously once a day 3 5 8
O HT-ID2-GDF15 subcutaneously Twice a week 3 5 8
O HT-ID2-GDF15 subcutaneously Twice a week 10 5 8
O HT-ID2-GDF15 subcutaneously Twice a week 30 5 8
O HT-ID3-GDF15 subcutaneously Twice a week 3 5 8
0 HT-ID3-GDF15 subcutaneously Twice a week 10 5 8
0 HT-ID3-GDF15 subcutaneously Twice a week 30 5 8
[233]
[234]
2.2.2. Weight Loss Test Results
[235]
Changes in body weight measured in Example 2.2.1 are shown in FIGS. 9, 10A, 10B, and Table 10 (Body Weight (Group, % of initial)).
[236]
[Table 10]
[237]

[238]
9 and Table 10 show the weight change of the fusion proteins HT-ID2-GDF15 and HT-ID3-GDF15 after repeated administration (Table 9) compared to a negative control group (vehicle administration group) and a positive control group (Semaglutide daily administration group). . Also, FIG. 10A is a graph showing the results of the 7th and 14th days among the results of FIG. 9 , and FIG. 10B is a graph showing the results of the 21st and 28th days extracted from the results of FIG. 9 .
[239]
As shown in the above results, in the case of the negative control group (vehicle administration group), there was little change in body weight, whereas in the case of the positive control group (Semaglutide daily administration group), it was confirmed that the weight loss effect appeared continuously from the first day after administration. In addition, in the case of the fusion polypeptide in which GDF15 is fused with IgD Hinge, the weight loss effect was immediately observed after one administration on Day 0, and the weight loss effect did not decrease during the test period and appeared continuously, and the weight loss effect was concentration-dependent. It can be confirmed that
[240]
[241]
2.2.3. Dietary intake test results
[242]
Changes in feed intake measured in Example 2.2.1 are shown in Table 11 and FIG. 11 (cumulative intake up to day 7, 14, 21, and 28), respectively.
[243]
[Table 11]
[244]

[245]
[246]
As shown in the above results, in the case of the fusion polypeptide administration group in which GDF15 is fused with IgD Hinge, compared to the negative control (vehicle administration group) administration group, the effect of reducing feed intake throughout the test period according to the fusion polypeptide was exhibited, and a concentration-dependent trend showed
[247]
[248]
Example 3. Pharmacokinetic testing of fusion polypeptides
[249]
3.1. Preparation of test and control sera
[250]
In order to evaluate the pharmacokinetic properties of each fusion polypeptide when administered subcutaneously to rats, the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 were added to SD Rat (Coatec, male, 7 weeks old, About 250 g; each n=3; test group) was administered subcutaneously at an amount of 2 mg/kg, respectively, and about 200 μl of blood was collected through the caudal vein at a predetermined time. Blood collection time was performed before and 1, 2, 4, 8, 24, 48, 72, 96, 168, 240 and 336 hours after administration of the fusion polypeptide. As a control for comparison of pharmacokinetic properties, a GDF15 administration group was prepared by subcutaneously administering GDF15 (R&D Systems) at an amount of 2 mg/kg in the same manner as above.
[251]
After administration to SD Rat as described above, serum was obtained by centrifuging the blood collected for each time-point, and ELISA was performed using Human GDF15 Immunoassay (SGD150, R&D Systems), and serum according to time of each polypeptide My concentration was measured. Using this data, values ​​of parameters including AUC (area under the curve) were obtained using software for PK analysis (WinNonlin (Certara LP), etc.).
[252]
[253]
3.2 Pharmacokinetic study results
[254]
The pharmacokinetic parameters of the obtained fusion polypeptide are shown in Table 12, and the concentration change of the fusion polypeptide with time is shown in FIG. 12 .
[255]
[Table 12]
PK paramper Group 1 Group 2 Group 3 Group 4
rhGDF15 HT-ID1-GDF15 HT-ID2-GDF15 HT-ID3-GDF15
C max (ug/mL) 0.443 2.09 0.945 1.11
T max (hr) One 8 24 24
AUC last (ug*hr/mL) 4.86 81.2 53.5 76.2
AUC inf (ug*hr/mL) 4.88 81.3 53.5 76.4
t 1/2 (hr) 19.0 24.7 22.3 26.2
AUC extp (%) 0.463 0.0700 0.100 0.287
[256]
(C max : peak plasma concentration, T max : time to reach peak blood concentration, AUC inf : area under the blood concentration-time curve calculated by extrapolating from the last measurable blood sampling time to infinity time, AUC last : the last measurable blood sampling time Area under the blood concentration-time curve to, T 1/2 : elimination half-life, AUC Extp (%): [(AUC inf -AUC last )/AUC inf ]*100) As shown in the above results, GDF15 (half-life : 19 hours), it was confirmed that the half-life of the fusion polypeptide fused with IgD Hinge was increased, and in particular, AUC last increased up to 16.7 times compared to GDF15.
[257]
[258]
From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.
Claims
[Claim 1]
GDF15 (Growth/differentiation factor 15), and a total of 1 to 10 O-glycosylated polypeptide regions bound to the N-terminus of GDF15, wherein the 1 to 10 O-glycosylated polypeptide regions are is a polypeptide comprising 3 to 10 amino acid residues capable of O-glycosylation, respectively.
[Claim 2]
The fusion polypeptide according to claim 1, which is represented by the formula: N'-(Z)nY-C' wherein N' is the N-terminus of the fusion polypeptide and C' is the C of the fusion polypeptide -terminus, Y is GDF15, Z is an O-glycosylated polypeptide region, and n is the number of O-glycosylated polypeptide regions bound to the N-terminus of GDF15, which is an integer from 0 to 10.
[Claim 3]
The method according to claim 1, wherein the 1 to 10 O-glycosylated polypeptide region comprises 1 to 10 immunoglobulin hinge regions or 3 to 10 amino acid residues capable of O-glycosylation among proteins of SEQ ID NOs: 23 to 113 A fusion polypeptide comprising a polypeptide region comprising at least 10 contiguous amino acids comprising
[Claim 4]
The fusion polypeptide according to claim 3, wherein the hinge region of 1 to 10 immunoglobulins is a hinge region of immunoglobulin D (IgD).
[Claim 5]
The fusion polypeptide according to claim 4, wherein the hinge regions of the 1 to 10 immunoglobulins are each independently selected from the group consisting of: (1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, (2) ) a polypeptide comprising 5 or more consecutive amino acids including 3 to 7 O-glycosylation residues in the amino acid sequence of SEQ ID NO: 1, and (3) the polypeptide of (1) or (2) in IgD A polypeptide comprising at least 34 consecutive amino acids comprising
[Claim 6]
The fusion polypeptide according to claim 4, wherein the hinge regions of the 1 to 10 immunoglobulins are each independently selected from the group consisting of: (1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, (2) ) a polypeptide comprising at least 5 consecutive amino acids comprising SEQ ID NO: 9 in the amino acid sequence of SEQ ID NO: 1 or at least 7 consecutive amino acids comprising SEQ ID NO: 10, and (3) above (1) in IgD or A polypeptide comprising at least 34 consecutive amino acids comprising the polypeptide of (2).
[Claim 7]
[Claim 7] The area under the blood concentration-time curve until the last blood collection point measurable upon in vivo administration of GDF15 bound to an O-glycosylated polypeptide region in the fusion polypeptide according to any one of claims 1 to 6 (AUC last ) is increased at least 2-fold compared to GDF15 not associated with an O-glycosylated polypeptide region.
[Claim 8]
A nucleic acid molecule encoding the fusion polypeptide of any one of claims 1-6.
[Claim 9]
A recombinant vector comprising the nucleic acid molecule of claim 8 .
[Claim 10]
A recombinant cell comprising the recombinant vector of claim 9 .
[Claim 11]
A method for producing the fusion polypeptide of any one of claims 1 to 6, comprising the step of culturing the recombinant cell of claim 10, comprising GDF15 and an O-glycosylated polypeptide region.
[Claim 12]
linking a total of 1 to 10 O-glycosylated polypeptide regions to the N-terminus of GDF15 in vitro, wherein each of the 1 to 10 O-glycosylated polypeptide regions is an O-glycosylated amino acid A method for enhancing in vivo stability of GDF15, which is a polypeptide comprising 3 to 10 residues.
[Claim 13]
13. The method of claim 12, wherein the 1 to 10 O-glycosylated polypeptide region comprises 1 to 10 hinge regions of immunoglobulin or 3 to 10 O-glycosylation residues in the protein of SEQ ID NOs: 23 to 113. A method for enhancing in vivo stability of GDF15, which is a polypeptide region comprising at least 10 consecutive amino acids.
[Claim 14]
The method of claim 13, wherein the hinge regions of 1 to 10 immunoglobulins are hinge regions of immunoglobulin D (IgD).
[Claim 15]
15. The method according to claim 14, wherein the hinge regions of the 1 to 10 immunoglobulins are each independently selected from the group consisting of: (1) Polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , (2) a polypeptide comprising 5 or more consecutive amino acids comprising 3 to 7 O-glycosylation residues in the amino acid sequence of SEQ ID NO: 1, and (3) above (1) or (2) in IgD A polypeptide comprising at least 34 consecutive amino acids comprising a polypeptide of
[Claim 16]
15. The method according to claim 14, wherein the hinge regions of the 1 to 10 immunoglobulins are each independently selected from the group consisting of: (1) Polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , (2) a polypeptide comprising 5 or more consecutive amino acids comprising SEQ ID NO: 9 in the amino acid sequence of SEQ ID NO: 1 or 7 or more consecutive amino acids comprising SEQ ID NO: 10, and (3) the above in IgD ( A polypeptide comprising at least 34 consecutive amino acids comprising the polypeptide of 1) or (2).
[Claim 17]
A fusion polypeptide dimer comprising two fusion polypeptides according to any one of claims 1 to 6.
[Claim 18]
The fusion polypeptide dimer of claim 17 , wherein the GDF15 of each fusion polypeptide binds to each other to form a dimer.
[Claim 19]
The fusion polypeptide dimer of claim 17 , wherein the dimer is a homodimer.
[Claim 20]
The fusion polypeptide of any one of claims 1 to 6 or a pharmaceutical composition for preventing or treating diseases related to GDF15 deficiency or dysfunction, comprising a fusion polypeptide dimer in which two fusion polypeptides are linked to each other in GDF15 .