KR20170100936A - A long-acting conjugate of G protein-coupled receptor polypeptide ligand and a method for preparation the same - Google Patents

Abstract

The present invention relates to a G protein-coupled receptor (GPCR) polypeptide ligand; a protein conjugate in which a non-peptidyl polymer and an immunoglobulin Fc region are covalently linked, and a preparation method of the same; and a pharmaceutical composition comprising the same. The protein conjugate of the present invention shows a higher increase in biological half-life and retention time of the GPCR polypeptide ligand than any modified proteins previously reported, and thus has superior biological persistence and activity in vivo, and a negligible risk of producing immune responses, and thus may be beneficially used in the development of a long-acting preparation of the GPCR polypeptide ligand. Additionally, a long-acting preparation of a protein drug according to the present invention may reduce the pain in a patient due to frequent injections, and can maintain the effect of the drug reliably by maintaining the GPCR ligand concentration in blood. Further, the preparation method of the protein conjugate of the present invention has overcome the disadvantages of a production method of fusion protein by genetic modification such as the difficulty of establishing an expression system, glycosylation different from native forms, induction of immune responses, and limited orientation of protein fusion, as well as the problems of a chemical combination method such as low yield due to non-specificity of reactions, and toxicity of chemical materials used as conjugates, and thus can provide protein drugs having increased biological half-life and high activity in a convenient and economic manner.

Description

[0001] The present invention relates to a long-acting conjugate of a G protein-coupled receptor polypeptide ligand and a method for preparing the same,

The present invention relates to a G protein-coupled receptor (GPCR) polypeptide ligand, a non-peptide polymer, and a protein conjugate in which an immunoglobulin Fc region is covalently linked, a method for producing the same, and a pharmaceutical composition containing the same .

Peptides are generally poorly stable and are easily denatured and are degraded by the body's proteolytic enzymes to lose their activity. Since they are relatively small in size, they are readily removed through the kidneys. Therefore, the blood concentration of the peptide- Peptide drugs need to be frequently administered to patients to maintain their potency. However, most of the peptide drugs are administered to patients in the form of injections, and thus are injected frequently to maintain blood levels of the bioactive peptide, which causes great pain to the patient. Various attempts have been made to overcome these problems. One of them has been an attempt to increase the permeability of the drug of the peptide and to deliver the drug of the peptide into the body by inhalation through the oral or nasal cavity. However, this method is significantly lower in the delivery efficiency of the peptide than the injection, and thus it is difficult to maintain the activity of the peptide drug in the required conditions.

Efforts have been made to increase the blood stability of the peptide drug and to maintain the drug concentration in the blood for a long time to maximize the drug efficacy. The persistent preparation of such a peptide drug should increase the stability of the peptide drug, Do not induce an immune response in the patient.

The present invention relates to a protein conjugate in which a GPCR polypeptide ligand and an immunoglobulin Fc region are linked by polyethylene glycol (hereinafter abbreviated as "PEG ") so that the duration of physiological activity is longer than that in the native form, and its use .

On the other hand, G protein-coupled receptor (GPCR) exists extracellularly, cell membrane, and cell, and plays an important role in various physiological activities such as reproductive and metabolism, immunity, digestion and respiration. It is known to be involved.

Indeed, about 25% of drugs used to treat disease are known to be associated with GPCRs, and more than 40% of new drugs under development target GPCRs (Overington JP, et al., Nature Reviews Drug Discovery 5: 993-6 , 2006). To date, more than 375 GPCRs have been found in the human genome, of which about 250 ligands have been identified (Filmore D, Modern Drug Discovery 2004: 24-8, 2004). Its ligands range from low molecular weight substances to polypeptides and polymers. In particular, polypeptide ligands are recognized as important targets for the development of new drugs because they are involved in diseases with high social impact and cost, such as inflammation, metabolic diseases and cancer.

Because such GPCR polypeptide ligands are generally poorly stable, they are easily denatured and degraded by protein hydrolytic enzymes in the blood to be easily removed through the kidneys or liver. Thus, the plasma concentration of the GPCR polypeptide ligand- Protein drugs often need to be administered to patients to maintain their potency. However, in the case of protein medicines that are administered to patients in most injectable form, frequent injections to maintain blood levels of the GPCR polypeptide ligand cause tremendous pain to the patient. In order to solve such problems, efforts have been made to increase the stability of protein drugs in blood and to maintain the drug concentration in blood for a long time to maximize the drug efficacy. Such a sustained-release preparation of a protein drug should not only increase the stability of the protein drug but also maintain the titer of the drug itself sufficiently high and cause no immune response to the patient.

As a method for stabilizing a protein and suppressing contact with a protein hydrolyzing enzyme and inhibiting loss of kidney, a method of chemically adding a polymer having a high solubility such as PEG to a protein drug surface has been conventionally used. PEG is known to stabilize a protein by enhancing solubility by binding non-specifically to a specific site or various sites of a target protein, and is effective in preventing hydrolysis of proteins and does not cause any adverse side effects (Sada et al. Fermentation Bioengineering 71: 137-139, 1991). However, although the stability of the protein can be increased by the binding of PEG, the activity of the physiologically active protein is remarkably lowered, and as the molecular weight of PEG increases, the reactivity with the protein decreases and the yield decreases.

In recent years, it has been proposed to increase the activity by making a duplex with the same protein drug bound to both ends of PEG (U.S. Patent No. 5,738,846) or to bind two different types of protein drugs to both ends of PEG, (WO 92/16221) have also been developed, but they have not shown a significant effect in sustaining the activity of protein drugs.

Kintler et al. Reported that the fusion protein of granulocyte colony stimulating factor (G-CSF) and human albumin bound to PEG showed increased stability (Kinstler et al., Pharmaceutical Research 12 (12): 1883-1888, 1995). However, the modified drug of the above document having the G-CSF-PEG-albumin structure shows only about 4-fold increase in the residence time in the body compared to the case of administration of the natural drug alone, It has not been practically used as a sustained-release preparation.

As another method for enhancing the in vivo stability of physiologically active proteins, a protein gene having high stability in blood is recombined with a physiologically active protein gene by gene recombination, and animal cells transformed with the recombinant gene are then cultured to obtain a fusion protein A production method has been developed. For example, there has been reported a fusion protein produced by binding albumin or a fragment thereof, which has been known to be most effective for increasing the stability of a protein, to a desired physiologically active protein by gene recombination (International Publication Application WO 93 / 15199 and WO 93/15200, European published EP 413,622). In addition, the fusion protein of interferon alpha and albumin produced by Human Genome Science in yeast (Product Name: Albuferon ^™ ) increased the half-life of interferon from 5 hours to 93 hours in monkeys, but compared to unmodified interferon (Osborn et al., J. Phar. Exp. Ther. 303 (2): 540-548, 2002).

Meanwhile, immunoglobulin (Ig) is classified into Fab region having antigen binding site and Fc region having complement binding site. Interferon (Korean Patent Application No. 2003-9464) and interleukin- 4 receptor ligand, an interleukin-7 receptor ligand or an erythropoietic factor receptor ligand (Korean Patent No. 249572) in a form fused to the Fc region of an immunoglobulin is known in mammals, and International Patent Publication No. WO 01/03737 Discloses a fusion protein in which a cytokine or growth factor is bound to an Fc fragment of an immunoglobulin via a peptide linker.

U.S. Patent No. 5,116,964 discloses a protein in which a lymphocyte cell surface glycoprotein (LHR) or CD4 protein is fused to the amino terminus or carboxy terminus of an immunoglobulin Fc region by a gene recombination method, and U.S. Patent No. 5,349,053 discloses an interleukin -2 is fused to an immunoglobulin Fc region. In addition, examples of Fc fusion proteins prepared by gene recombination include interferon-beta or derivatives thereof and fusion proteins of immunoglobulin Fc region (WO 00/23472), interleukin-5 receptor and immunoglobulin Fc region Fusion proteins (US Pat. No. 5,712,121), fusion proteins of the Fc region of interferon alpha and immunoglobulin G4 (US Patent No. 5,723,125), and fusion proteins of CD4 protein and Fc region of immunoglobulin G2 (US Patent No. 6,451,313 Lt; / RTI > In addition, U.S. Patent No. 5,605,690, in which an amino acid of an immunoglobulin Fc region is modified, specifically comprises TNFR-IgG1 produced by a recombinant method using Fc in which an amino acid of a complement binding site or a receptor binding site is modified in the immunoglobulin Fc region Fc fusion proteins are disclosed, and a method for producing recombinant fusion proteins using the Fc region of such modified immunoglobulin is disclosed in U.S. Patent Nos. 6,277,375, 6,410,008 and 6,444,792.

U.S. Patent No. 6,660,843 discloses a method of producing a recombinant construct in which an immunoglobulin Fc region and a target protein are fused using a linker by recombinant methods in E. coli. This method can be produced at a lower cost than the method using mammalian cells, and a conjugate can be obtained in a form in which sugar chains are removed. However, since the objective protein and the immunoglobulin Fc region are produced simultaneously in Escherichia coli, it is difficult to apply to a target protein having a sugar chain in a natural form, and a problem that a misfolding probability is very high due to the inclusion body .

The Fc fusion protein produced by such a recombinant method is capable of protein fusion only at a specific site of an immunoglobulin Fc region, that is, at an amino terminal or a carboxy terminal, and is expressed only in the form of a homodimer, There is a problem that fusion of glycosylated protein and non-glycosylated protein is impossible because fusion between glycosylated protein or non-glycosylated protein is possible. In addition, the newly generated amino acid sequence due to fusion can not only cause an immune response, but also increase the sensitivity of the protein hydrolase to the linker site.

Various methods for binding a polymer to a biologically active protein have been attempted. However, existing methods increase the stability of the polypeptide significantly or only increase the activity irrespective of the stability. Therefore, it is difficult to achieve the reduction of the activity of the GPCR polypeptide ligand, which has a great potential for development of new drugs, and the improvement of stability at the same time.

Accordingly, the inventors of the present invention have continued to develop a persistent GPCR polypeptide ligand drug that can attain minimization of activity reduction and stability at the same time, which has been considered to be difficult to achieve at the same time. As a result, immunoglobulin Fc region, And a GPCR polypeptide ligand are covalently linked to each other, the present inventors have accomplished the present invention by confirming that the protein complexes significantly increase the blood half-life of the GPCR ligand and maintain a higher potency than known protein drugs.

It is an object of the present invention to provide a protein conjugate in which a G protein-coupled receptor (GPCR) polypeptide ligand, a non-peptide polymer and an immunoglobulin Fc region are covalently linked.

It is another object of the present invention to provide a method for preparing the GPCR polypeptide ligand protein conjugate.

It is another object of the present invention to provide a pharmaceutical composition comprising the GPCR polypeptide ligand protein conjugate.

One aspect of the present invention for solving the above-mentioned problems is a protein binding body in which a G protein-coupled receptor (GPCR) polypeptide ligand, a non-peptide polymer, and an immunoglobulin Fc region are covalently linked.

In one embodiment, the non-peptide polymer has a reactive group at both ends and is covalently linked to the GPCR polypeptide ligand and the immunoglobulin Fc region through the corresponding two-terminal reactors.

In another embodiment, the one immunoglobulin Fc region is covalently linked to at least one linkage of the GPCR polypeptide ligand and the non-peptide polymer.

In another embodiment, the immunoglobulin Fc region is unchallenged.

In yet another embodiment, the immunoglobulin Fc region is characterized by a domain selected from one to four selected from the group consisting of CH1, CH2, CH3 and CH4 domains.

In yet another embodiment, the immunoglobulin Fc region further comprises a hinge region.

In yet another embodiment, the immunoglobulin Fc region is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and Fc regions of their hybrids.

In yet another embodiment, the immunoglobulin Fc region is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, combinations thereof, and Fc regions of hybrids thereof.

In another embodiment, the immunoglobulin Fc region is an IgG4 Fc region.

In yet another embodiment, the immunoglobulin Fc region is a human non-sugar chain IgG4 Fc region.

In another embodiment, the reactor of the non-peptide polymer is selected from the group consisting of an aldehyde group, a propionaldehyde group, a butylaldehyde group, a maleimide group and a succinimide derivative .

In still another embodiment, the succinimide derivative is succinimidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl or succinimidyl carbonate.

In yet another embodiment, the non-peptide polymer is characterized by having a reactor of reactive aldehyde groups at both ends.

In another embodiment, both ends of the non-peptide polymer are each bound to a glass reactor of an immunoglobulin Fc region and an amino terminus, lysine residue, histidine residue or cysteine residue of a GPCR polypeptide ligand.

In another embodiment, the non-peptide polymer is selected from the group consisting of polyethylene glycol homopolymer, polypropylene glycol homopolymer, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, poly Vinyl ethers, vinyl ethers, biodegradable polymers, lipid polymers, chitins, hyaluronic acid, and combinations thereof.

In another embodiment, the non-peptide polymer is polyethylene glycol.

In another embodiment, the GPCR polypeptide ligand is selected from physiologically active polypeptides such as hormones, cytokines, interleukins, neurotransmitters, and derivatives and analogs thereof.

In another embodiment, the GPCR polypeptide ligand is selected from the group consisting of angiotensin, apelin, gastrin-releasing peptide, bradykinin, chemerin, CCLs, complement, endothelin Ghrelin, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, thyroid stimulating hormone (TSH), kisspeptin, melanocyte Neurotensin, prolactin stimulating peptide, thrombin, cathepsin G, relaxin, cortistatin, neurokinin A (neurokinin A), neurokinin A ), Oxytocin, vasopressin, calcitonin, urocortin, Glucagon-like-pepetide (GLP-1), glucagon, parathyroid hormone.

In yet another embodiment, the GPCR polypeptide ligand is selected from the group consisting of octreotide, a cerebral natriuretic peptide (BNP), or calcitonin.

Another aspect of the present invention is a method of preparing a fusion protein comprising: (a) linking at least one non-peptide polymer having a reactor at both ends, at least one GPCR polypeptide ligand, and at least one immunoglobulin Fc region with a covalent bond; And (b) isolating a GPCR polypeptide ligand, a non-peptide polymer, and a protein conjugate essentially comprising an immunoglobulin Fc region linked by a covalent bond.

In one embodiment, the step (a)

(a1) linking an immunoglobulin Fc region or a GPCR polypeptide ligand to one end of an activated non-peptide polymer with a covalent bond;

(a2) isolating a ligand comprising an immunoglobulin Fc region or a GPCR polypeptide ligand linked to the non-peptide polymer from the reaction mixture; And

(a3) covalently linking the immunoglobulin Fc region or the GPCR polypeptide ligand to the other end of the non-peptide polymer of the separated linkage so that both ends of the non-peptide polymer are bound to the immunoglobulin Fc region and the GPCR polypeptide ligand Lt; RTI ID = 0.0 > of < / RTI >

In another embodiment, the reaction molar ratio of the GPCR polypeptide ligand to the non-peptide polymer in the step (a1) is 1: 2.5 to 1: 5.

In another embodiment, the reaction molar ratio of the immunoglobulin Fc region to the non-peptide polymer in the step (a1) is 1: 5 to 1:10.

In yet another embodiment, the reaction molar ratio of the linker: immunoglobulin Fc region or GPCR polypeptide ligand obtained in step (a3) in step (a3) is 1: 0.5 to 1:20.

In another embodiment, the reaction of step (a1) and step (a3) is carried out in the presence of a reducing agent.

In yet one embodiment, it is characterized in that the reducing agent is sodium cyano borohydride selected from hydride (NaCNBH _3), sodium borohydride, dimethylamine borate and pyridine group consisting of borates.

Another aspect of the present invention is a pharmaceutical composition for increasing the in vivo persistence and stability of a GPCR polypeptide ligand, including the protein conjugate and a pharmaceutically acceptable carrier.

In one embodiment, the non-peptide polymer is covalently linked to the GPCR polypeptide ligand and immunoglobulin Fc region, respectively, through a two-terminal reactor.

In yet another embodiment, one immunoglobulin Fc region is covalently linked to one or more linkages of a GPCR polypeptide ligand and a non-peptide polymer.

In yet another embodiment, the immunoglobulin Fc region is non-glycosylated.

In yet another embodiment, the immunoglobulin Fc region is characterized by a domain selected from one to four selected from the group consisting of CH1, CH2, CH3 and CH4 domains.

In yet another embodiment, the immunoglobulin Fc region further comprises a hinge region.

In yet another embodiment, the immunoglobulin Fc region is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and Fc regions of their hybrids.

In yet another embodiment, the immunoglobulin Fc region is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, combinations thereof, and Fc regions of hybrids thereof.

In another embodiment, the immunoglobulin Fc region is an IgG4 Fc region.

In yet another embodiment, the immunoglobulin Fc region is a human non-sugar chain IgG4 Fc region.

In another embodiment, the reactor of the non-peptide polymer is selected from the group consisting of an aldehyde group, a propionaldehyde group, a butylaldehyde group, a maleimide group and a succinimide derivative .

In still another embodiment, the succinimide derivative is succinimidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl or succinimidyl carbonate.

In yet another embodiment, the non-peptide polymer is characterized by having a reactor of reactive aldehyde groups at both ends.

In another embodiment, both ends of the non-peptide polymer are each bound to a glass reactor of an immunoglobulin Fc region and an amino terminus, lysine residue, histidine residue or cysteine residue of a GPCR polypeptide ligand.

In another embodiment, the non-peptide polymer is selected from the group consisting of polyethylene glycol homopolymer, polypropylene glycol homopolymer, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, poly Vinyl ethers, vinyl ethers, biodegradable polymers, lipid polymers, chitins, hyaluronic acid, and combinations thereof.

In another embodiment, the non-peptide polymer is polyethylene glycol.

In another embodiment, the GPCR polypeptide ligand is selected from physiologically active polypeptides such as hormones, cytokines, interleukins, neurotransmitters, and derivatives and analogs thereof.

In another embodiment, the GPCR polypeptide ligand is selected from the group consisting of angiotensin, apelin, gastrin-releasing peptide, bradykinin, chemerin, CCLs, complement, endothelin Ghrelin, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, thyroid stimulating hormone (TSH), kisspeptin, melanocyte Neurotensin, prolactin stimulating peptide, thrombin, cathepsin G, relaxin, cortistatin, neurokinin A (neurokinin A), neurokinin A ), Oxytocin, vasopressin, calcitonin, urocortin, Glucagon-like-pepetide (GLP-1), glucagon, parathyroid hormone.

In another embodiment, the GPCR polypeptide ligand is octreotide, BNP, or calcitonin.

Since the protein binding complex of the present invention has higher blood half-life and residence time of the GPCR polypeptide ligand than any of the previously reported modified proteins, it not only has excellent blood persistence and bioactivity, but also poses no risk of inducing an immune response. And can be usefully used to develop a sustained-release preparation of a polypeptide ligand. In addition, the sustained-release preparation of the protein drug according to the present invention can reduce the patient's pain due to frequent injections and maintain the blood drug concentration of the GPCR ligand constantly and stably.

In addition, the method for producing the protein conjugate of the present invention is not limited to the disadvantages of the method for producing fusion proteins by gene manipulation such as difficulty in establishment of the expression system, different glycosylation from the natural type, induction of the immune response, restriction of the protein fusion direction, And the toxicity of the chemical used as the conjugate, overcoming the problems of the chemical coupling method, thereby providing the protein drug having an increased blood half-life and high activity in an easy and economical manner.

Figure 1 is a graph of pharmacokinetics and pharmacokinetics of Octreotide and sustained octreotide, one of the G protein-coupled receptor polypeptide ligands.
FIG. 2 is a graph showing the pharmacokinetic analysis of a cerebral sodium natriuretic peptide (BNP) and a sustained-type BNP, which are one of the G protein-coupled receptor polypeptide ligands.
Figure 3 is a graph of the pharmacokinetics of calcitonin and sustained calcitonin, one of the G protein-coupled receptor polypeptide ligands.

One embodiment of the present invention is directed to a G protein-couple receptor (GPCR) polypeptide ligand, a non-peptide polymer having a reactive group at both ends, and a protein wherein the immunoglobulin Fc region is linked by covalent bonds Lt; / RTI >

In the present invention, "protein complex" or "conjugate" includes at least one GPCR ligand, at least one non-peptide polymer having a reactive group at both ends and one or more immunoglobulin Fc regions, And are interconnected by a covalent bond. Further, in order to distinguish from the above-mentioned "conjugate ", a structure in which only two kinds of substance molecules selected by a GPCR ligand, a non-peptide polymer and an immunoglobulin Fc region are linked by a covalent bond is referred to as a" conjugate ".

The protein conjugate of the present invention is a variant for minimizing the decrease of physiological activity of a GPCR polypeptide ligand and enhancing persistence in vivo, and is characterized in that an immunoglobulin Fc region is bound in the present invention.

Since the immunoglobulin Fc region is a biodegradable polypeptide metabolized in vivo, it is safe for use as a carrier of drugs. In addition, since the immunoglobulin Fc region has a relatively small molecular weight as compared with the whole immunoglobulin molecule, it is not only advantageous in terms of preparation, purification and yield of the conjugate, but also because the amino acid sequence differs from antibody to antibody, It is expected that the homogeneity will be greatly increased and the possibility of inducing blood antigenicity will also be lowered.

In the present invention, the term "immunoglobulin Fc region" refers to a heavy chain constant region 2 (C _H 2) except for the light chain variable region of the heavy chain of the immunoglobulin, the heavy chain constant region 1 (C _H 1) and the light chain constant region 1 (C _L 1) And the heavy chain constant region 3 (C _H 3) portion, and may include a hinge portion in the heavy chain constant region. In addition, the immunoglobulin Fc region of the present invention may contain a part or all of the heavy chain constant region 1 (C _H 1) and / or the light chain (s) except for the heavy chain and the light chain variable region of the immunoglobulin as long as the immunoglobulin Fc region has substantially equivalent or improved effect And may be an extended Fc region including constant region 1 (C _L 1). It may also be a region in which some long amino acid sequences corresponding to C _H 2 and / or C _H 3 are removed. That is, the immunoglobulin Fc regions of the present invention is 1) C _H 1 domain, C _H 2 domain, C _H 3 domain, and C _H 4 domain, 2) C _H 1 domain and a C _H 2 domain, 3) C _H 1 domain And a C _H 3 domain, 4) a C _H 2 domain and a C _H 3 domain, 5) a combination of one or more domains with an immunoglobulin hinge region (or a portion of a hinge region), 6) And a light chain constant region dimer.

In addition, the immunoglobulin Fc region of the present invention includes a naturally occurring amino acid sequence as well as its sequence derivative (mutant). An amino acid sequence derivative means that at least one amino acid residue in the native amino acid sequence has a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322 or 327 to 331, which are known to be important for binding, can be used as sites suitable for modification.

Also, various kinds of derivatives can be used, such as a site capable of forming a disulfide bond is removed, some amino acids at the N-terminus are removed from the native Fc, or a methionine residue is added at the N-terminus of the native Fc Do. In addition, the complement binding site, for example, the C1q binding site, or the antibody dependent cell mediated cytotoxicity (ADCC) site may be removed to eliminate the effector function. Techniques for producing the sequence derivatives of such immunoglobulin Fc regions are disclosed in International Patent Publication No. WO 97/34631, International Patent Publication No. WO 96/32478, and the like.

Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In some cases, the phosphorylation, phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, modification.

The Fc derivative described above exhibits the same biological activity as the Fc region of the present invention, and has an increased structural stability against heat, pH, etc. of the Fc region.

Such Fc region may also be obtained from natural forms isolated in vivo in animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, and may be obtained from transformed animal cells or microorganisms Recombinant or derivative thereof. Here, the method of obtaining from the native form may be a method of separating the whole immunoglobulin from the living body of human or animal, and then obtaining the protein by treating the proteolytic enzyme. When papain is treated, it is cleaved into Fab and Fc, and when pepsin is treated, it is cleaved into pF'c and F (ab) ₂ . Fc or pF'c can be isolated using size-exclusion chromatography or the like. Specifically, it is a recombinant immunoglobulin Fc region obtained from a microorganism from a human-derived Fc region.

In addition, the immunoglobulin Fc region may be a natural type sugar chain, an increased sugar chain as compared to the native type, and a reduced sugar chain or sugar chain as compared with the native type. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase or decrease immunoglobulin Fc sugar chains. Here, the immunoglobulin Fc region in which sugar chains are removed from Fc is significantly reduced in binding force with complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, resulting in unnecessary immune response in vivo Do not. In this regard, forms that are more consistent with the original purpose of the drug as a carrier will be referred to as immunoglobulin Fc regions where the sugar chain is removed or unglycosylated.

In the present invention, "Deglycosylation" refers to an Fc region in which sugar is removed by an enzyme, and aglycosylation refers to an Fc region produced in prokaryotes, specifically, Escherichia coli, and not glycosylated.

On the other hand, the immunoglobulin Fc region may be an animal origin such as human or bovine, goat, pig, mouse, rabbit, hamster, rat, guinea pig and the like, and may be human origin.

Also, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM or a combination thereof or a hybrid thereof. Specifically IgG derived from IgG or IgM most abundant in human blood, and most specifically IgG known to enhance half-life of binding protein.

In the present invention, the term " combination " means that when a dimer or a multimer is formed, a polypeptide encoding the same-originated short-chain immunoglobulin Fc region forms a bond with a short-chain polypeptide having a different origin. That is, it is possible to prepare a dimer or a multimer from two or more fragments selected from the group consisting of Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE.

In the present invention, the term "hybrid" means a sequence corresponding to two or more immunoglobulin Fc fragments of different origins in the short chain immunoglobulin Fc region. In the case of the present invention, various types of hybrids are possible. That is, hybrids of one to four domains from the group consisting of C _H 1, C _H 2, C _H 3 and C _H 4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc are possible , And a hinge.

On the other hand, IgG can be divided into subclasses of IgG1, IgG2, IgG3 and IgG4, and in the present invention, combinations thereof or hybridization thereof are also possible. Specifically, it is a subclass of IgG2 and IgG4, and most specifically, an Fc region of IgG4 having almost no effector function such as complement dependent cytotoxicity (CDC).

That is, the most preferable immunoglobulin Fc region for carrier of the drug of the present invention is a non-glycosylated Fc region derived from human IgG4. A human-derived Fc region is preferable to an Fc region derived from a non-human, which can act as an antigen in a human organism to cause an undesirable immune response such as generation of a new antibody thereto.

In the present invention, the immunoglobulin Fc region and the GPCR polypeptide ligand may be characterized by being linked to a non-peptide polymer.

The term "non-peptide polymer" in the present invention refers to a biocompatible polymer having two or more repeating units bonded together, and the repeating units are connected to each other through any covalent bond other than a peptide bond.

Non-peptide polymers usable in the present invention include, but are not limited to, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA A biodegradable polymer such as polylactic acid and PLGA (polylactic-glycolic acid), a lipid polymer, a chitin, a hyaluronic acid, and a combination thereof. Specific examples thereof include polyethylene Glycol. Derivatives thereof which are already known in the art and derivatives which can be easily prepared in the state of the art are included in the scope of the present invention. The nonpeptide polymer preferably has a molecular weight in the range of 1 to 100 kDa, specifically in the range of 1 to 20 kDa. In addition, a non-peptide polymer of the present invention that binds to the immunoglobulin Fc region may use not only one type of polymer but also a combination of different types of polymers.

The non-peptide polymers used in the present invention have an immunoglobulin Fc region and a reactor capable of binding GPCR polypeptide ligands.

The types of the both end reactors of the non-peptide polymer may be selected from the group consisting of reactive aldehyde groups, propionaldehyde groups, butylaldehyde groups, maleimide groups, and succinimide derivatives. As the succinimide derivative, succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate may be used. In particular, when the non-peptide polymer has a reactive group of reactive aldehyde groups at both ends, it is effective to bind the GPCR polypeptide ligand and the immunoglobulin Fc region at both ends of the non-peptide polymer, respectively, while minimizing the nonspecific reaction. The final products formed by reductive alkylation by aldehyde linkages are much more stable than those linked by amide linkages.

Both terminal reactors of the non-peptide polymer may be the same or different from each other. For example, it may have a maleimide group at one end and an aldehyde group, propionaldehyde group or butylaldehyde group at the other end. When polyethylene glycol (PEG) having a hydroxy group at both terminals is used as a non-peptide polymer, it is possible to activate the hydroxy group with the various reactors by a known chemical reaction, or to use a commercially available modified reactor The protein conjugate of the present invention can be prepared.

In the present invention, the term "G protein-coupled receptor (GPCR) ligand", "GPCR polypeptide ligand" or "GPCR protein ligand" refers to a polypeptide or protein that binds to GPCRs involved in physiological phenomena in vivo Can be used interchangeably.

Such protein drugs are disadvantageous in that they can not continue their physiological activity for a long time because they are easily denatured or decomposed by proteolytic enzymes present in vivo. However, in the case of the conjugate in which the immunoglobulin Fc region of the present invention is bound to the polypeptide, the structural stability of the drug is increased and the half-life of the drug is increased. However, the decrease in the physiological activity of the polypeptide by binding of the Fc region will be very slight compared to other known polypeptide drug formulations. Therefore, the binding capacity of GPCR polypeptide ligand and immunoglobulin Fc region of the present invention is remarkably increased in bioavailability compared to the bioavailability of existing polypeptide drugs. As is clearly disclosed in the following Examples of the present invention, the immunoglobulin Fc region-conjugated octreotide, brain natriuretic peptide (B-type natriuretic peptide), BNP, It can be confirmed that the blood half-life is increased in conventional preparations in which only PEG such as calcitonin or PEG and albumin are combined (Example 1, Example 2, and Example 3).

On the other hand, the binding of the immunoglobulin Fc region of the present invention to the protein is not a fusion by a conventional recombinant method. The form in which the immunoglobulin Fc region and the GPCR polypeptide ligand used as the drug are fused by a recombinant method is a form in which the GPCR polypeptide ligand is linked to the N-terminal or C-terminal of the Fc region by a peptide bond, Lt; RTI ID = 0.0 > protein. ≪ / RTI >

Based on the fact that the activity of the protein as a physiological functional body is determined by the structure, this leads to a sharp decrease in the activity of the fusion protein. Thus, when a GPCR polypeptide ligand is fused to an immunoglobulin Fc region by a recombinant method, it is ineffective in terms of bioavailability even though the structural stability is increased. In addition, such fusion proteins tend to be misfolded and expressed in the form of aggregates, which is not economical in terms of the yield of protein production and separation. In addition, when the active polypeptide is glycosylated, it has to be expressed in eukaryotic cells. In this case, since the Fc region is also glycosylated, it may cause an inappropriate immune response in vivo.

That is, only by the present invention, a glycosylated active GPCR polypeptide ligand can be linked to an unglycosylated immunoglobulin Fc region, and it is possible to prepare and separate each of them in the best system, The above problems can be overcome.

GPCR polypeptide ligands applicable to the protein conjugate of the present invention include physiologically active polypeptides such as hormones, cytokines, interleukins, and neurotransmitters, and derivatives and analogues thereof.

Specifically, the GPCR polypeptide ligand may be selected from the group consisting of angiotensin, apelin, gastrin-releasing peptide, bradykinin, chemerin, CCLs, complement, endothelin-1, (Ghrelin), follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, thyroid stimulating hormone (TSH), kisspeptin, melanocyte stimulating hormone, motilin neurotensin, prolactin stimulating peptide, thrombin, cathepsin G, relaxin, cortistatin, neurokinin A, oxytocin (also known as oxytocin), motilin, neurotensin, ), Vasopressin, calcitonin, urocortin, Glucagon-like-pepetide (GLP-1), glucagon, parathyroid hormone and the like. More specifically, the GPCR polypeptide ligand may be octreotide, BNP, or calcitonin.

Particularly, it is recommended that the GPCR polypeptide ligand of the present invention, when administered to a human body for the purpose of treatment or prevention of disease, has a high dose rate. Also, any derivatives or derivatives thereof are included within the scope of the GPCR polypeptide ligands of the present invention, so long as they have substantially the same or increased function, structure, activity or stability as the native form of the GPCR polypeptide ligand.

In the present invention, the antibody fragment may be Fab, Fab ', F (ab') ₂ , Fd or scFv having the ability to bind to a specific antigen, preferably Fab '. The Fab fragment comprises a variable domain (V _L ) and a constant domain (C _L ) of the light chain and a variable domain (V _H ) and a first constant domain (C _H 1) of the heavy chain. Fab fragments are distinguished from Fab fragments in that several amino acid residues are added at the carboxyl terminus of the C _H 1 domain, including one or more cysteines from the hinge region. The Fd fragment is a fragment consisting of only the V _H and C _H 1 domains, and F (ab ') ₂ is the Fab' fragment of two molecules bound through a disulfide bond or a chemical reaction. scFv is a single polypeptide chain in which only the VL and VH domains are linked by a peptide linker.

On the other hand, when the immunoglobulin Fc region and the GPCR polypeptide ligand bind through the non-peptide polymer, the binding site of the immunoglobulin Fc region includes at least one of the glass reactors of the amino acid residues existing in the hinge region or the constant region. Specifically, it is preferred that the immunoglobulin Fc constant region and the amino terminal of the protein drug, the amino residue of the lysine, the amino residue of the histidine or the free cysteine residue are linked to the covalent bond with the terminal reactor of the non-peptide polymer.

The protein conjugate of the present invention may comprise one or more unit structures of [GPCR polypeptide ligand-non-peptide polymer-immunoglobulin Fc region], wherein all components are linearly linked by covalent bonds. The non-peptide polymer may have a reactor at both ends, through which a GPCR polypeptide ligand and an immunoglobulin Fc region may each be covalently linked. That is, one or more linkages of a non-peptide polymer linked to a GPCR polypeptide ligand are covalently linked to one immunoglobulin Fc region, thereby linking an immunoglobulin Fc region as a GPCR polypeptide ligand monomer, It is possible to form a multimer, whereby the in vivo activity and stability can be more effectively achieved.

In the protein conjugate of the present invention, the GPCR polypeptide ligand and the immunoglobulin Fc region can be combined at various molar ratios.

In addition, as is known in the art, when two proteins are linked through an oligopeptide, there is a risk that the newly formed protein sequence due to the linkage region may induce an immune response, And C-terminal, whereas the protein conjugate of the present invention is mediated by a non-peptide polymer having biocompatibility, and thus has no side effects such as toxicity or induction of an immune response, and various protein conjugates There is an advantage that can be provided.

In addition, a method of directly fusing an immunoglobulin Fc region and an active protein by conventional gene recombination can be fused only at a terminal sequence of an immunoglobulin Fc region used as a fusion partner, and is dependent on an animal cell culture, In addition, the activity can be reduced due to the unnatural glycosylation of the active protein, accurate folding can be achieved, a homodimer-type fusion protein can be produced, and in particular, the production of an Escherichia coli-derived conjugate There is a problem in that it is very difficult to remove the pentacene insoluble superabsorbent. However, the protein conjugate of the present invention can attain higher continuity and stability without causing such problems, is preferable in terms of maintaining the activity of the GPCR polypeptide ligand, and is useful as a combination of a glycosylated therapeutic protein and an unglycosylated Fc Can be manufactured.

On the other hand, when a low molecular weight chemical binding agent such as carbodiimide or glutaraldehyde is used, the chemical binding agent binds to various sites of the protein at the same time to denature the protein, or makes it difficult to control the binding site by nonspecific binding , And it is difficult to purify the bound protein. On the other hand, the protein conjugate of the present invention has an advantage that the binding site can be easily controlled, the nonspecific reaction is minimized, and the purification is easy since the non-peptide polymer is used.

The GPCR polypeptide ligand-PEG-Fc region of the present invention, in which a GPCR polypeptide ligand and an immunoglobulin Fc region are bonded at both ends of PEG, ) Exhibits a better blood half-life than the GPCR polypeptide ligand-PEG linkage or GPCR polypeptide ligand. Pharmacokinetic analysis showed that the plasma half-life of sustained octreotide was increased by about 120-fold compared to the native form and that the plasma concentration of IGF-1, a pharmacodynamic marker, decreased more than 144 hours 1). In addition, the same results were obtained when BNP and calcitonin were used instead of octreotide as a target protein. Compared with the case where the protein conjugate of the present invention conjugated with the PEG-Fc region was bound to a native protein or PEG The mean residence time (MRT) and the half-life of blood increased by about 10 to 100 times (see FIGS. 1, 2, and 3).

As such, the protein conjugate of the present invention is applied to GPCR polypeptide ligands including octreotide, BNP, or calcitonin, and exhibits excellent blood half-life and mean residence time (MRT), so that the persistence of various kinds of GPCR polypeptide ligands And can be used for development of mold formulation.

As another aspect of the present invention,

(a) reacting a non-peptide polymer, a GPCR polypeptide ligand and an immunoglobulin Fc region having a reactor at both ends, and linking them by covalent bonds; And

(b) separating a conjugate in which the GPCR polypeptide ligand and the immunoglobulin Fc region are linked by covalent bonds, respectively, at both ends of the non-peptide polymer, and a method for producing a protein conjugate having increased persistence and stability in vivo to provide.

In step (a), the covalent bonding of the three components can occur sequentially or simultaneously. For example, when the GPCR polypeptide ligand and the immunoglobulin Fc region are bound to both ends of the non-peptide polymer, either the GPCR polypeptide ligand or the immunoglobulin Fc region is first bound to one end of the non-peptide polymer After the binding, the reaction proceeds sequentially in such a manner that the remaining components are bound to the other end of the non-peptide polymer. This is advantageous in minimizing the production of by-products other than the desired protein-bound product.

Thus, step (a)

(a1) linking an immunoglobulin Fc region or a GPCR polypeptide ligand to one end of the non-peptide polymer by covalent bonding;

(a2) separating the immunoglobulin Fc region or the GPCR polypeptide ligand conjugate bound to the non-peptide polymer from the reaction mixture; And

(a3) An immunoglobulin Fc region or a GPCR polypeptide ligand is covalently linked to the other end of the non-peptide polymer of the above-mentioned ligated body so that both ends of the non-peptide polymer are bound to the immunoglobulin Fc region and the GPCR To produce a protein conjugate linked to a polypeptide ligand.

In the above step (a1), the optimum reaction molar ratio of the GPCR polypeptide ligand to the non-peptide polymer is 1: 2.5 to 1: 5, and the optimal reaction molar ratio of the immunoglobulin Fc region to the non- 10.

On the other hand, in step (a3), the reaction molar ratio of the linker: immunoglobulin Fc region or GPCR polypeptide ligand obtained in step (a2) may range from 1: 0.5 to 1:20, .

The reaction of step (a1) and step (a3) can be carried out in the presence of a reducing agent, if necessary, in consideration of the kind of both terminal reactors of the non-peptide polymer participating in the reaction. As a preferable reducing agent, sodium cyanoborohydride (NaCNBH ₃ ), sodium borohydride, dimethylamine borate or pyridine borate and the like can be used.

Herein, steps (a2) and (b) can be appropriately selected and carried out according to needs, among the usual methods used for protein separation, in consideration of characteristics such as required purity and molecular weight and charge amount of the resultant product . For example, a variety of known methods including size exclusion chromatography or ion exchange chromatography can be applied, and if necessary, a plurality of different methods can be used in combination to purify at higher purity.

As another embodiment of the present invention, the present invention provides a pharmaceutical composition of a GPCR polypeptide ligand having increased persistence and stability in vivo, comprising a protein conjugate of the present invention as an active ingredient and a pharmaceutically acceptable carrier do.

In the present invention, "administration" means introducing a predetermined substance into a patient in any appropriate manner, and the administration route of the conjugate can be administered through any conventional route so long as the drug can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal. However, when orally administered, since the peptide is digested, it is preferable to formulate the oral composition so as to coat the active agent or protect it from decomposition at the top. Preferably in the form of an injection. In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell.

The pharmaceutical composition comprising the GPCR polypeptide ligand protein conjugate of the present invention is useful as a composition for preventing, improving or treating metabolic syndrome, endocrinol disease, oncology and inflammation related diseases. .

The pharmaceutical composition comprising the conjugate of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like in the case of oral administration. A solubilizing agent, an isotonic agent, a stabilizer and the like may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other, solutions, suspensions, tablets, pills, capsules, sustained-release preparations and the like.

Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. In addition, it may further include a first agent, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The actual dosage of the drug in which the Fc fragment is used as a carrier in the present invention is determined by the kind of the active ingredient, such as the type of the active ingredient, together with various related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, . Since the pharmaceutical composition of the present invention is highly persistent in vivo, the frequency and frequency of administration of the pharmaceutical preparation of the present invention can be remarkably reduced.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  1: G protein coupled receptor ( GPCR ) Polypeptide  Preparation of a continuous conjugate of a ligand

The present inventors have investigated the pharmacokinetics and pharmacokinetics of the G protein-coupled receptor (GPCR) polypeptide ligand conjugated with the immunoglobulin Fc region of the present invention.

A continuous type conjugate was prepared by selecting octreotide, brain natriuretic peptide (BNP), or calcitonin as a representative G protein-coupled receptor polypeptide ligand. .

1-1. Octreotide  Preparation of continuous type conjugate

3.4K PropionALD (2) In order to pegylate PEG (PEG having two propionaldehyde groups, NOF, Japan) at the N terminus of octreotide, the molar ratio of octreotide to 3.4K PropionALD (2) 2, and the protein concentration was 1 mg / ml at 4 to 8 ° C for about 1 hour. At this time, the reaction was carried out in an environment containing 20 mM sodium cyanoborohydride (NaCNBH 3) as a reducing agent in 0.1 M sodium acetate buffer (pH 5.2). After the reaction was completed, the reaction solution was applied to SP Sepharose High Performance (GE, USA) using a gradient of 20 mM sodium citrate (pH 2.0) and a 1 M sodium chloride gradient to obtain a mono-pegylated (Monopegylated) octreotide was purified.

Next, the molar ratio of purified mono-pegylated octreotide to immunoglobulin Fc was 1: 4 and the protein concentration was adjusted to 65.6 mg / ml at about 4 ° C for about 19 hours. The reaction solution was carried out in an environment containing 20 mM sodium cyanoborohydride (NaCNBH3) as a reducing agent in 0.1 M potassium phosphate buffer (pH 6.0). Thereafter, the reaction solution was applied to Source 15Q (GE, USA) using a gradient of 20 mM Tris (pH 7.5) and 1 M sodium chloride, and octreotide was added to the immunoglobulin Fc by covalent bonding Was purified.

1-2. thunder Sodium diuretic peptide (Brain natriuretic  peptide, BNP) continuous type conjugate

3.4K PropionALD (2) In order to pegylate PEG (PEG having 2 propionaldehyde groups, NOF, Japan) at the N terminus of BNP, the molar ratio of BNP and 3.4K PropionALD (2) PEG was 1:10, And the reaction was allowed to proceed at 4 to 8 ° C for about 1 hour at a concentration of 3 mg / ml. At this time, the reaction was carried out in an environment containing 20 mM sodium cyanoborohydride (NaCNBH3) as a reducing agent in 0.1 M sodium acetate buffer (pH 4.0). After the reaction was completed, the reaction solution was applied to Source 15S (GE, USA) using a gradient of 20 mM sodium phosphate (pH 7.5) and a 1 M sodium chloride gradient to obtain monopegylated ) BNP was purified.

Next, the molar ratio of the purified mono-pegylated BNP to immunoglobulin Fc was 1: 5 and the protein concentration was 70 mg / ml, and the mixture was reacted at about 4 ° C for about 16 hours. The reaction solution was carried out in an environment containing 20 mM sodium cyanoborohydride (NaCNBH3) as a reducing agent in 0.1 M potassium phosphate buffer (pH 6.0). After the reaction was completed, the reaction solution was applied to Source 15S (GE, USA) using a gradient of 20 mM sodium phosphate (pH 8.0) and a 1 M sodium chloride gradient, and BNP was added to the immunoglobulin Fc The conjugate linked by covalent bond by PEG was purified.

1-3. Calcitonin  Preparation of continuous type conjugate

3.4K PropionALD (2) In order to pegylate PEG (PEG having 2 propionaldehyde groups, NOF, Japan) at the N terminus of calcitonin, the molar ratio of calcitonin to 3.4K PropionALD (2) PEG was 1: And the reaction was allowed to proceed at 4 to 8 ° C for about 1 hour at a concentration of 3 mg / ml. At this time, the reaction was carried out in an environment containing 20 mM sodium cyanoborohydride (NaCNBH 3) as a reducing agent in 0.1 M sodium acetate buffer (pH 5.2). After the reaction was completed, the reaction solution was applied to SP Sepharose High Performance (GE, USA) using a concentration gradient of 20 mM sodium citrate (pH 6.0) and 1 M sodium chloride, Monopegylated calcitonin was purified.

Next, the molar ratio of the purified mono-pegylated calcitonin to immunoglobulin Fc was 1: 4, and the concentration of the protein was 50 mg / ml. The reaction was carried out at about 4 ° C for about 18 hours. The reaction solution was carried out in an environment containing 20 mM sodium cyanoborohydride (NaCNBH3) as a reducing agent in 0.1 M potassium phosphate buffer (pH 6.0). Subsequently, the reaction solution was applied to Source 15Q (GE, USA) using a concentration gradient of 20 mM Tris (pH 7.5) and 1 M sodium chloride so that the calcitonin in the immunoglobulin Fc was covalently bound to PEG The ligated conjugate was purified.

Example  2: Octreotide  Drugs of the persistent conjugate kinetics  And Pharmacokinetics  Research

The pharmacokinetics and pharmacokinetics of the octreotide continuous conjugate prepared in Example 1-1 were compared with that of natural octreotide, and blood half-life and activity were confirmed.

Specifically, the blood pharmacokinetic parameters of the natural type octreotide (Octreotide, control group) and the continuous type octreotide (octreotide-PEG-immunoglobulin Fc domain conjugate) prepared in the SD rats and the drug The dynamics were compared.

The control group (naturally occluded octreotide) and the test group (continuous octreotide conjugate) were subcutaneously injected at 98.1 nmol / kg, respectively, and the control group was collected at 0.25, 0.5, 1, 2, 3 and 5 hours after the injection , And the test group was collected after 0.5, 1, 2, 5, 10, 24, 30, 48, 72, 96, 120, 168, 216, 264, 336 and 384 hours after the injection (see A in FIG.

The collected blood sample was stored in a tube containing heparin to prevent coagulation, and cells were removed by centrifugation for 5 minutes in an Eppendorf high-speed microcentrifuge. Plasma protein levels were measured by ELISA using antibodies to octreotide. The pharmacokinetic analysis results are shown in Table 1 below.

Octreotide Octreotide continuous binder Dose 98.1 nmol / kg 98.1 nmol / kg Tmax (hr) 0.3 36.0 T1 / 2 (hr) 1.7 63.7 MRT (hr) 1.7 121.6

In the above table, T _max means the time to reach the highest drug concentration, T _1/2 means the blood half-life of the drug, and Mean resis- tence time (MRT) means the mean residence time of the drug.

As can be seen from the above results, the octreotide-PEG-Fc domain conjugate of the GPCR receptor polypeptide ligand conjugate of the present invention has a half-life of about 37 times and a residence time of about 72 times higher than that of native octreotide Respectively.

Next, IGF-1, a pharmacodynamic marker of octreotide, was measured for pharmacodynamic comparison of octreotide-continuous conjugates. Specifically, the same concentrations of the natural octreotide and the octreotide continuous conjugate were administered at the same dose, and the control group was collected after 0, 0.5, 2, 5, 10, and 24 hours. The test group was collected at 0, 10, 24, 72, 96, and 168 hours after the injection. Cells were removed by centrifugation and the antibody to plasma IGF-1 was measured by ELISA.

As a result, as shown in FIG. 1B, in the case of the octreotide continuous conjugate of the present invention, the amount of plasma IGF-1 showed the lowest concentration at 24 hours and was maintained to be decreased until 168 hours have.

Example  3: BNP-PEG-immunoglobulin Fc  Area ( Fc ) Combination drugs kinetics  Research

BNP-PEG conjugate prepared in Example 1-2 was compared with BNP-PEG conjugate, and pharmacokinetics and pharmacokinetics were confirmed to confirm blood half-life and potency.

Specifically, blood pharmacokinetic coefficients of the natural type BNP-PEG linkage (control group) and the BNP persistent conjugate (BNP-PEG-immunoglobulin Fc domain linkage) of the present invention were compared in SD rats. Control group and BNP persistent conjugate (test group) were subcutaneously injected at 100 ㎍ / kg each, respectively, and blood was collected at specific time. Blood was collected at 0.5, 1, 2, 4, and 8 hours in the control group, and at 0.5, 1, 5, 10, 24, 48, 72, and 96 hours in the test group.

The collected blood samples were stored in a tube containing heparin to prevent coagulation, and the cells were removed by centrifugation for 5 minutes in an Eppendorf high-speed microcentrifuge. Plasma protein levels were measured by ELISA using antibodies against BNP. The pharmacokinetic analysis results are summarized in Table 2 below.

BNP-PEG conjugate BNP continuous bond Dose 100 [mu] g / kg 100 [mu] g / kg Tmax (hr) One 24 T1 / 2 (hr) 2.41 21.12 MRT (hr) 4.5 39.7

In the above table, T _max means the time to reach the highest drug concentration, T _1/2 means the blood half-life of the drug, and Mean resis- tence time (MRT) means the mean residence time of the drug.

As can be seen from the above results, the BNP-PEG-Fc domain conjugate of the GPCR receptor polypeptide continuous ligand of the present invention has a half-life and a residence time of about 9 times higher than that of the BNP-PEG conjugate.

Example  4: Calcitonin -PEG-immunoglobulin Fc  Area ( Fc ) Combination drugs kinetics  Research

The pharmacokinetics and pharmacokinetics of the calcitonin-type conjugate prepared in Example 1-3 were compared with that of the calcitonin-PEG conjugate to confirm the half-life and potency of the blood.

Specifically, natural calcitonin (Calcitonin, control group) and the sustained calcitonin conjugate (calcitonin-PEG-immunoglobulin Fc domain conjugate) prepared above were subcutaneously injected into SD rats at a dose of 28.9 nmol / kg, Blood was collected at a specific time. Blood was collected at 0.25, 0.5, 1, 2, 3, and 5 hours in the control group and 0.5, 1, 2, 5, 10, 48, 72, 96, 120, 168, and 216 hours.

The collected blood samples were stored in a tube containing heparin to prevent coagulation, and the cells were removed by centrifugation for 5 minutes in an Eppendorf high-speed microcentrifuge. Plasma protein levels were measured by ELISA using antibodies against calcitonin. The pharmacokinetic analysis results are summarized in Table 3 below.

Calcitonin Calcitonin persistent conjugate Dose 28.9 nmol / kg 28.9 nmol / kg Tmax (hr) One 28 T1 / 2 (hr) 2.3 41.8 MRT (hr) 2.7 64.2

In the above table, T _max means the time to reach the highest drug concentration, T _1/2 means the blood half-life of the drug, and Mean resis- tence time (MRT) means the mean residence time of the drug.

As can be seen from the above results, the calcitonin-PEG-Fc domain conjugate, which is a persistent conjugate of the GPCR receptor polypeptide ligand of the present invention, has a half-life of 18 times and a residence time of about 24 times greater than that of the calcitonin-PEG conjugate.

As can be seen from the above results, when the GPCR receptor polypeptide ligand was prepared as a continuous conjugate of the present invention, it was confirmed that the half-life of the GPCR receptor was increased from at least 9 times to at most 37 times, and at least 9 times to 72 times Respectively. In addition, the activity of octreotide was measured by the amount of IGF-1. As a result, it was confirmed that the activity of the sustained conjugate of the present invention remained effective for a long period of time.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (45)

G protein-coupled receptor (GPCR) polypeptide ligand, non-peptide polymer, and immunoglobulin protein binding covalently linked Fc region.
The protein conjugate according to claim 1, wherein the non-peptide polymer has a reactive group at both ends and is covalently linked to a GPCR polypeptide ligand and an immunoglobulin Fc region through a corresponding two-terminal reactor.
3. The protein conjugate according to claim 2, wherein one immunoglobulin Fc region is linked with at least one covalent bond of a GPCR polypeptide ligand and a non-peptide polymer.
2. The protein conjugate according to claim 1, wherein the immunoglobulin Fc region is unglycosylated.
2. The protein conjugate according to claim 1, wherein the immunoglobulin Fc region comprises one to four selected from the group consisting of C _H 1, C _H 2, C _H 3 and C _H 4 domains.
6. The protein conjugate of claim 5, wherein the immunoglobulin Fc region further comprises a hinge region.
2. The protein conjugate of claim 1, wherein the immunoglobulin Fc region is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and Fc regions of their hybrids.
8. The protein conjugate according to claim 7, wherein the immunoglobulin Fc region is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, combinations thereof, and Fc regions of hybrids thereof.
9. The protein conjugate according to claim 8, wherein the immunoglobulin Fc region is an IgG4 Fc region.
10. The protein conjugate of claim 9, wherein the immunoglobulin Fc region is a human non-glycosylated IgG4 Fc region.
3. The method of claim 2, wherein the reactor of the non-peptide polymer is selected from the group consisting of an aldehyde group, a propionaldehyde group, a butylaldehyde group, a maleimide group, and a succinimide derivative .
12. The protein conjugate of claim 11, wherein the succinimide derivative is succinimidyl propionate, succinimidyl carboxymethyl, hydroxysuccinimidyl or succinimidyl carbonate.
13. The protein conjugate according to claim 12, wherein the non-peptide polymer has a reactive aldehyde group reactor at both terminals.
The protein conjugate of claim 1, wherein both ends of the non-peptide polymer are bound to a free reactor of an amino terminal, lysine residue, histidine residue or cysteine residue of an immunoglobulin Fc region and a GPCR polypeptide ligand, respectively.
The composition of claim 1, wherein the non-peptide polymer is selected from the group consisting of polyethylene glycol homopolymer, polypropylene glycol homopolymer, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl Ethyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid, and combinations thereof.
16. The protein conjugate according to claim 15, wherein the non-peptide polymer is polyethylene glycol.
The protein conjugate according to claim 1, wherein the GPCR polypeptide ligand is selected from physiologically active polypeptides such as hormones, cytokines, interleukins, neurotransmitters, and derivatives and analogues thereof.
18. The method of claim 17, wherein the GPCR polypeptide ligand is selected from the group consisting of angiotensin, apelin, gastrin releasing peptide, bradykinin, chemerin, CCLs, complement, endothelin- 1), annexin, galanin, ghrelin, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, thyroid stimulating hormone (TSH), kisspeptin, melanocyte stimulation Neurotensin, prolactin stimulating peptide, thrombin, cathepsin G, relaxin, cortistatin, neurokinin A, neurokinin A, A protein conjugate selected from the group consisting of oxytocin, vasopressin, calcitonin, urocortin, Glucagon-like-pepetide (GLP-1), glucagon, parathyroid hormone.
18. The protein conjugate of claim 17, wherein the GPCR polypeptide ligand is octreotide, a brain natriuretic peptide (B-type natriuretic peptide, BNP), or a calcitonin.
(a) linking at least one non-peptide polymer, at least one GPCR polypeptide ligand, and at least one immunoglobulin Fc region having a covalent bond at both ends with a covalent bond; And
(b) separating a GPCR polypeptide ligand, a non-peptide polymer, and a protein conjugate essentially comprising an immunoglobulin Fc region linked by a covalent bond.
21. The method of claim 20, wherein step (a)
(a1) linking an immunoglobulin Fc region or a GPCR polypeptide ligand to one end of an activated non-peptide polymer with a covalent bond;
(a2) isolating a ligand comprising an immunoglobulin Fc region or a GPCR polypeptide ligand linked to the non-peptide polymer from the reaction mixture; And
(a3) covalently linking the immunoglobulin Fc region or the GPCR polypeptide ligand to the other end of the non-peptide polymer of the separated linkage so that both ends of the non-peptide polymer are bound to the immunoglobulin Fc region and the GPCR polypeptide ligand Lt; RTI ID = 0.0 > of: < / RTI >
22. The method of claim 21, wherein the reaction molar ratio of the GPCR polypeptide ligand to the non-peptide polymer in step (a1) is from 1: 2.5 to 1: 5.
22. The method according to claim 21, wherein the reaction molar ratio of the immunoglobulin Fc region to the non-peptide polymer in step (a1) is from 1: 5 to 1:10.
The process according to claim 21, wherein the reaction molar ratio of the linker: immunoglobulin Fc region or GPCR polypeptide ligand obtained in step (a2) in step (a3) is 1: 0.5 to 1:20.
22. The process according to claim 21, wherein the reaction of step (a1) and step (a3) is carried out in the presence of a reducing agent.
26. The method of claim 25, a method in which the reducing agent is sodium cyano borohydride (NaCNBH _3), selected from sodium borohydride, dimethylamine borate and pyridine borate group consisting of.
A pharmaceutical composition for increasing the in vivo persistence and stability of a GPCR polypeptide ligand, comprising the protein conjugate of claim 1 and a pharmaceutically acceptable carrier.
28. The pharmaceutical composition of claim 27, wherein the non-peptide polymer is covalently linked to the GPCR polypeptide ligand and the immunoglobulin Fc region, respectively, via a two-terminal reactor.
29. The pharmaceutical composition according to claim 28, wherein one immunoglobulin Fc region is linked with at least one covalent bond of a GPCR polypeptide ligand and a non-peptide polymer.
28. The pharmaceutical composition according to claim 27, wherein the immunoglobulin Fc region is unglycosylated.
28. The pharmaceutical composition according to claim 27, wherein the immunoglobulin Fc region comprises one to four selected from the group consisting of C _H 1, C _H 2, C _H 3 and C _H 4 domains.
32. The pharmaceutical composition of claim 31, wherein the immunoglobulin Fc region further comprises a hinge region.
28. The pharmaceutical composition according to claim 27, wherein the immunoglobulin Fc region is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and Fc regions of their hybrids.
34. The pharmaceutical composition of claim 33, wherein the immunoglobulin Fc region is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, combinations thereof and Fc regions of hybrids thereof.
35. The pharmaceutical composition of claim 34, wherein the immunoglobulin Fc region is an IgG4 Fc region.
36. The pharmaceutical composition according to claim 35, wherein the immunoglobulin Fc region is a human non-glycosylated IgG4 Fc region.
The pharmaceutical composition according to claim 28, wherein the reactor of the non-peptide polymer is selected from the group consisting of an aldehyde group, a propionaldehyde group, a butylaldehyde group, a maleimide group and a succinimide derivative. Composition.
38. The pharmaceutical composition according to claim 37, wherein the succinimide derivative is succinimidyl propionate, succinimidyl carboxymethyl, hydroxysuccinimidyl or succinimidyl carbonate.
29. The pharmaceutical composition of claim 28, wherein the non-peptide polymer has a reactive aldehyde group reactor at both ends.
28. The pharmaceutical composition according to claim 27, wherein both ends of the non-peptide polymer are bound to a free reactor of an immunoglobulin Fc region and an amino terminus, a lysine residue, a histidine residue or a cysteine residue of a GPCR polypeptide ligand, respectively.
28. The composition of claim 27, wherein the non-peptide polymer is selected from the group consisting of polyethylene glycol homopolymers, polypropylene glycol homopolymers, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohols, polysaccharides, Ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, and combinations thereof.
42. The pharmaceutical composition of claim 41, wherein the non-peptide polymer is polyethylene glycol.
28. The pharmaceutical composition of claim 27, wherein the GPCR polypeptide ligand is selected from physiologically active polypeptides such as hormones, cytokines, interleukins, neurotransmitters, and derivatives and analogs thereof.
44. The method of claim 43, wherein said GPCR polypeptide ligand is selected from the group consisting of angiotensin, apelin, a gastrin releasing peptide, bradykinin, chemerin, CCLs, complement, endothelin- 1), annexin, galanin, ghrelin, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, thyroid stimulating hormone (TSH), kisspeptin, melanocyte stimulation Neurotensin, prolactin stimulating peptide, thrombin, cathepsin G, relaxin, cortistatin, neurokinin A, neurokinin A, Wherein the pharmaceutical composition is selected from oxytocin, vasopressin, calcitonin, urocortin, Glucagon-like-pepetide (GLP-1), glucagon, parathyroid hormone.
44. The pharmaceutical composition of claim 43, wherein said GPCR polypeptide ligand is octreotide, BNP, or calcitonin.

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