Title of the invention: Polyethylene glycol modified product of hepatocyte growth factor or an active fragment thereof
Technical field
[0001]
　The present invention relates to a polyethylene glycol modified product of hepatocyte growth factor or an active fragment thereof.
Background technology
[0002]
　Hepatocyte growth factor is a growth factor having various physiological actions, and in addition to the first found hepatocyte growth factor, it has anti-apoptotic action, angiogenic action, angiogenic action, vasodilatory action, anti-organ fibrosis action, and anti. It is known to have epithelial-mesenchymal conversion action, and clinical application to various diseases is being attempted. However, since the half-life of hepatocyte growth factor in vivo is as short as about 30 minutes, it is necessary to frequently administer a large amount of hepatocyte growth factor in order to sustain its action (Non-Patent Document 1).
[0003]
　Further, as active fragments of hepatocyte growth factor, NK1 (Non-Patent Document 2) and NK2 (Non-Patent Document 3), which are natural splicing variants, and NK4 (Non-Patent Document 4) produced by recombinant technology are known. These have been shown to have bioactivity in vitro and in vivo.
[0004]
　Polyethylene glycol is a highly biocompatible polymer polymer, and is widely used as a protein modifier for the purpose of prolonging the in vivo half-life and reducing immunogenicity of protein drugs.
[0005]
　A polyethylene glycol modified product has also been reported for hepatocyte growth factor (Patent Document 1). In addition, a polyethylene glycol modified product of NK4, which is an antagonist fragment of hepatocyte growth factor, has also been reported (Patent Document 2).
Prior art literature
Patent documents
[0006]
Patent Document 1: US Pat. No. 5,977,310,
Japanese Patent Document 2: Japanese Patent Application Laid-Open No. 2010-174034
Non-patent literature
[0007]
Non-Patent Document 1: Liu K. X. Et al., American Journal of Physiology, 1998, Vol. 275, p. 835-842
Non-Patent Document 2: Jakubczak J. et al. L. Et al., Molecular and Cellular Biology, 1998, Vol. 18, No. 3, p. 1275-1283
Non-Patent Document 3: Otsuka T. et al. Et al., Molecular and Cellular Biology, 2000, Vol. 20, No. 6, p. 2025-2065
Non-Patent Document 4: Date K. Et al., FEBS Letters, 1997, Vol. 420, No. 1, p. 1-6
Outline of the invention
Problems to be solved by the invention
[0008]
　However, while the desirable effect of polyethylene glycol modification, it is known that in polyethylene glycol modification of a protein having physiological activity, the physiological activity decreases or disappears depending on the binding position of polyethylene glycol. For example, a modified product in which a plurality of polyethylene glycols are bound is described at random positions of hepatocyte growth factor, but the effect of prolonging the in vivo half-life is low, and further, the physiological activity is reduced by 30% or more. It is recognized (Patent Document 1).
[0009]
　Therefore, in the conventional technique, it is considered that the extension of the half-life in the living body and the maintenance of the physiological activity are contradictory by the polyethylene glycol modification, and a polyethylene glycol-modified product that simultaneously satisfies them is desired.
[0010]
　Therefore, an object of the present invention is to provide a polyethylene glycol-modified product of hepatocyte growth factor or an active fragment thereof, which has both the effect of extending the half-life in vivo and the retention of physiological activity by polyethylene glycol modification.
Means to solve problems
[0011]
　As a result of intensive studies to solve the above problems, the present inventors have achieved both the effect of prolonging the in vivo half-life and the retention of physiological activity by polyethylene glycol modification, and the polyethylene of hepatocyte growth factor or an active fragment thereof. A glycol-modified product was found, and the present invention was completed.
[0012]
　The present invention includes the following features.
　(1) Hepatocyte growth factor or its activity in which one molecule of fork-type polyethylene glycol is covalently bonded to the carboxyl-terminal region of each of two molecules of hepatocyte growth factor or its active fragment to form a homodimer. Fragment of polyethylene glycol modified product.
　(2) A polyethylene glycol modified product of the hepatocyte growth factor or an active fragment thereof according to the above (1), which is represented by the general formula (I).
[Chemical

formula 1] [In the formula, PEG represents a structural portion of fork-type polyethylene glycol, L represents a hydrolyzably stable branched portion, HGF represents hepatocyte growth factor or an active fragment thereof, and X Represents a binding moiety that provides a covalent bond of fork-type polyethylene glycol to hepatocyte growth factor or an active fragment thereof. ]
　(3) In formula (I), PEG is - (CH 2 CH 2 O) represents a structure including n- and the structure forms a linear structure or a branched structure, n is 2 to 2300, a polyethylene glycol modified product of the hepatocyte growth factor or an active fragment thereof according to (2) above.
　(4) A polyethylene glycol-modified form of the hepatocyte growth factor according to any one of (1) to (3) above or an active fragment thereof, which is represented by the general formula (II).
[Chemical

formula 2] [In the formula, X and HGF are as defined above. ]
　(5) The distance between the branched atom of the branched portion of the fork-type polyethylene glycol and the functional group that provides the covalent bond of the fork-type polyethylene glycol to the hepatocyte growth factor or an active fragment thereof is 20 Å or less. The polyethylene glycol modified product of the hepatocyte growth factor or the active fragment thereof according to any one of (1) to (4).
　(6) The polyethylene glycol modified product of the hepatocyte growth factor or the active fragment thereof according to any one of (1) to (5) above, wherein the active fragment of the hepatocyte growth factor is NK1.
　(7) The hepatocyte growth factor according to (6) above, wherein the NK1 comprises the amino acid sequence shown by SEQ ID NO: 2 in the sequence listing, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence. Polyethylene glycol modified product of the active fragment.
　(8) A medicament containing a polyethylene glycol-modified form of the hepatocyte growth factor or an active fragment thereof according to any one of (1) to (7) above as an active ingredient.
Effect of the invention
[0013]
　The polyethylene glycol modified product of the hepatocyte growth factor of the present invention or an active fragment thereof exhibits its medicinal effect with a lower administration frequency than the unmodified product because the in vivo half-life is extended and its physiological activity is maintained. It can be used as a medicine to be used.
A brief description of the drawing
[0014]
FIG. 1 is a diagram showing SDS electrophoresis images of His-Cys-added human NK1 and fork-type polyethylene glycol-modified NK1 dimer.
FIG. 2 is a diagram showing the physiological activities of heparin dimerized NK1, fork-type polyethylene glycol-modified NK1 dimer, and linear polyethylene glycol-modified NK1 dimer.
FIG. 3 is a diagram showing the time course of mouse serum concentration when His-Cys-added human NK1 (dotted line) or fork-type polyethylene glycol-modified NK1 dimer (solid line) was intravenously administered to the tail vein.
Mode for carrying out the invention
[0015]
　In the polyethylene glycol modified product of the hepatocyte growth factor or the active fragment thereof of the present invention, the hepatocyte growth factor or the active fragment thereof forms a homodimer, and the carboxyl of both monomers of the homodimer is obtained. It is characterized in that a dimer is formed by covalently bonding one molecule of fork-type polyethylene glycol to the terminal region.
[0016]

　Hepatocyte growth factor (hereinafter, also referred to as “HGF”) is a growth factor having various physiological activities. HGF consists of N domain, kringle 1, kringle 2, kringle 3, kringle 4 and SPH domain from the amino-terminal side, and N domain, kringle 1, kringle 2, kringle 3 and kringle 4 form an α chain, and the SPH domain. Consists of the β chain. HGF is biosynthesized as a single-stranded protease at the time of expression, but after the secretory signal sequence is removed when it is secreted, it is the 494th arginine residue and the 494th arginine residue counting from the starting methionine extracellularly. It is processed by a protease between the 495th valine residues to form a heterodimeric chain in which the α chain and β chain are bound by a disulfide bond and becomes the active form (Miyazawa K. et al., The Journal of Biological). Chemistry, 1996, Vol. 271, No. 7, p. 3615-3618). The HGF of the present invention means an active form of HGF having physiological activity.
[0017]
　In the case of human hepatobiliary growth factor (HGF), it is biosynthesized and secreted as a secretory protein consisting of 728 amino acid residues (including a secretory signal sequence (31 amino acid residues from the starting methionine)) (GenBank accession number: M29145). At that time, it becomes a protein consisting of 697 amino acid residues from which the secretory signal sequence has been removed (SEQ ID NO: 1).
[0018]
　The above-mentioned hepatocyte growth factor (HGF) is not only one having the same amino acid sequence as the naturally occurring HGF (hereinafter, natural HGF) amino acid sequence, but also one or several in the natural HGF amino acid sequence. It contains an amino acid variant of HGF having an amino acid sequence in which an amino acid is deleted, substituted or added (or inserted) and has physiological activity as HGF, and further, the sugar chain portion of natural HGF is modified. It also includes HGF and HGF having no sugar chain moiety. As the HGF mutant, a mutant having 90% or more sequence identity with the amino acid sequence of natural HGF is preferable, a mutant having 95% or more sequence identity is more preferable, and 98% or more sequence identity is preferable. Variants having are more preferred. For example, defective HGF lacking 5 amino acid residues in kringle 1 (a naturally occurring mutant) (Kinosaki M. et al., FEBS Letters, 1998, Vol. 434, p.165-170). It has been reported that it has higher specific activity than native HGF in certain cell types.
[0019]
　As used herein, "sequence identity" refers to the identity between two sequences that can be determined using algorithms such as BLAST, FASTA, etc., and generally refers to the two sequences, including gaps or. When aligned for maximum coincidence without gaps, it can be calculated as a percentage of the number of matched amino acids to the total number of amino acids (including gaps) (Altschul S. et al., Journal of Algorithm). Biologic, 1990, Vol. 215, No. 3, p. 403-410; Altschul S. et al., Nuclear Acids Research, 1997, Vol. 25, No. 17, p. 3389-3402).
[0020]
　As used herein, "several" refers to integers from 2 to 10, i.e. 10, 9, 8, 7, 6, 5, 4, 3, 2.
[0021]
　The "variant" of the polypeptide used herein is by deletion, substitution or addition (or insertion) of one or more amino acids in the amino acid sequence of the native polypeptide (natural hepatocyte growth factor (HGF)). A polypeptide having an amino acid sequence different from the resulting sequence of the natural polypeptide is referred to as a variant of the natural polypeptide. Such mutants may occur naturally or may be artificially produced using common techniques such as genetic recombination techniques. When artificially producing a mutant, it is important not to impair the activity of the polypeptide. For that purpose, when preparing a mutant, for example, no mutation is introduced in the active site of the polypeptide and, if necessary, in the vicinity of the active site, and conservative amino acid substitution is performed when substituting. It should be noted that the HGF activity of the mutant is confirmed by the assay.
[0022]
　Conservative amino acid substitutions generally refer to substitutions between amino acids with similar chemical, electrical (or polar / hydrophobic) or structural properties. Such substitutions can suppress significant changes in the conformation of the polypeptide, so that the polypeptide can be retained without significantly impairing its activity and is higher than the native form. In some cases it can be active. Specific examples of such amino acid substitutions include substitutions between acidic amino acids (eg, aspartic acid (D) and glutamic acid (E)), substitutions between basic amino acids (eg, histidine (H), lysine (K) and arginine). (R)), substitutions between aromatic amino acids (eg, phenylalanine (F), tyrosine (Y) and tryptophan (W)), substitutions between hydrophilic amino acids (eg, cysteine ​​(C), aspartic acid (D), Substitution between glutamic acid (E) histidine (H), lysine (K), aspartic acid (N), glutamine (Q), arginine (R), serine (S) and threonine (T)), hydrophobic amino acids (eg, alanine) (A), phenylalanine (F), isoleucine (I), leucine (L), norleucine (Nle), methionine (M), valine (V), tryptophan (W) and tyrosine (Y)) and the like.
[0023]
　The above hepatocyte growth factor (HGF) also includes recombinant HGF produced by gene recombination technology based on the amino acid sequence or base sequence of natural HGF.
[0024]
　The receptor for hepatocyte growth factor (HGF) is known to be c-Met, and the diverse physiological activities of HGF are induced by the binding of HGF to the HGF receptor c-Met.
[0025]
　The above-mentioned active fragment of hepatocyte growth factor (HGF) is a protein that contains a part of the structure of HGF, binds to an HGF receptor, and exerts physiological activity (agonist activity) as HGF. In addition, the above-mentioned active fragment of HGF also includes a protein that contains a part of the structure of HGF, binds to an HGF receptor, and acts as an antagonist of HGF (exhibits antagonistic activity).
[0026]
　Examples of active fragments of hepatocyte growth factor (HGF) include NK1 and NK2, which are natural splicing variants of HGF. NK1 is composed of N-domain and kringle 1 on the amino-terminal side of HGF, and NK2 is composed of N-domain, kringle 1 and kringle 2 on the amino-terminal side of HGF. Each of these has been reported to act as an agonist or antagonist in the animal body (Jakubczak J. L. et al., Molecular and Cellular Biology, 1998, Vol. 18, No. 3, p. 1275-1283; Otsuka). T. et al., Molecular and Cellular Biology, 2000, Vol. 20, No. 6, p. 2055-2065).
[0027]
　In addition, examples of the active fragment of hepatocyte growth factor (HGF) include NK4 produced by a recombination technique. NK4 consists of the amino-terminal N-domain of HGF, kringle 1, kringle 2, kringle 3 and kringle 4, and has been reported to act as an antagonist (Date K. et al., FEBS Letters, 1997, No. 420). Volume 1, No. 1, p.1-6).
[0028]
　The above-mentioned active fragment of hepatocyte growth factor (HGF) is not only one having the same amino acid sequence as the amino acid sequence derived from the amino acid sequence of natural HGF, but also one or a number in the amino acid sequence derived from the amino acid sequence of natural HGF. A variant having an amino acid sequence in which individual amino acids are deleted, substituted or added, and having physiological activity (agonist activity) as HGF or antagonistic activity of HGF is included, and a sugar derived from natural HGF is further included. It also includes variants that have modified chain moieties or do not have sugar chain moieties derived from native HGF. As the mutant of the active fragment of HGF, a mutant having 90% or more sequence identity with the amino acid sequence of natural HGF is preferable, a mutant having 95% or more sequence identity is more preferable, and 98% or more is more preferable. Variants with sequence identity are more preferred. For example, as NK1 highly active mutants, 1K1 (Liesa D. et al., The EMBO Journal, 2001, Vol. 20, No. 20, p. 5543-5555) and M2.2 (Jones DS et al., Proceedings of the National Academic of Sciences of the United States of America, 2011, Vol. 108, No. 32, p. 13035-13040).
[0029]
　The above hepatocyte growth factor or an active fragment thereof may be hepatocyte growth factor (HGF) (including mutants), NK1 (including mutants), NK2 (including mutants) or NK4 (including mutants). Natural HGF, defective HGF (Kinosaki M. et al., FEBS Letters, 1998, Vol. 434, p.165-170), NK1 (including mutants), NK2 (including mutants). Alternatively, it is preferably NK4 (including a mutant), more preferably NK1 (including a mutant) or NK2 (including a mutant), and further preferably NK1 (including a mutant). Most preferably, it is human NK1 consisting of the amino acid sequence shown in SEQ ID NO: 2. In addition, SEQ ID NO: 2 does not include a human HGF-derived secretory signal sequence (MWVTKLLPALLQHVLLLHLLLPIAIPAEG: SEQ ID NO: 3).
[0030]
　The hepatocyte growth factor or an active fragment thereof includes an amino acid sequence derived from a mammal, and is preferably an amino acid sequence derived from a human, a cat or a dog, and more preferably a sequence derived from a human. ..
[0031]
　The above-mentioned hepatocyte growth factor or an active fragment thereof may be one to which an artificial sequence such as a tag sequence is added for the purpose of purification of a protein or the like. Examples of the tag sequence include a 6 × His tag, a HAT tag, a c-Myc tag, a FLAG tag, a DYKDDDDK tag, a Strep tag, an HA tag, a GST tag, and an MBP tag. Furthermore, in order to remove these tag sequences after purification, it is also possible to further insert an artificial sequence such as a spacer sequence or a protease cleavage sequence between the above HGF or an active fragment thereof and the tag. In this case, the above-mentioned hepatocyte growth factor or an active fragment thereof may contain a cleavage fragment of an artificial sequence such as a spacer sequence or a protease cleavage sequence.
[0032]
　The above hepatocyte growth factor or active fragment thereof expresses extraction from tissue, protein synthesis using gene recombination technology, recombinant cells expressing hepatocyte growth factor or active fragment thereof (or hepatocyte growth factor). It can be obtained by using a known method such as biological production using natural cells). Further, as the HGF or an active fragment thereof, a commercially available hepatocyte growth factor or an active fragment thereof can also be used.
[0033]
　Regarding gene recombination technology, for example, Molecular Cloning, 2nd ed. , 1989, Cold Spring Harbor Laboratory. An example of such a technique will be described below.
　Prepare DNA encoding HGF or an active fragment thereof. The DNA can be obtained by selecting it from a cDNA library prepared from human tissues or cells, taking it out, and subjecting it to a DNA amplification method such as a PCR method. Alternatively, the DNA can be chemically synthesized, for example, using a DNA synthesizer utilizing the phosphoramidite method.
[0034]
　The above DNA is incorporated into an appropriate vector to prepare an expression vector. Such vectors contain elements such as regulatory sequences necessary to express (and secrete, if necessary) the DNA. Specific examples of elements include translation start codons and stop codons, promoters, enhancers, terminators, ribosome binding sites (or Shine dalgarno sequences), selectable marker sequences, signal sequences, etc., and the necessary elements are inserted into the vector. NS.
[0035]
　As for the promoter, a suitable promoter is selected according to the host cell. For example, the genus Escherichia (Escherichia) promoter suitable bacterial cell is a prokaryote, for example, trp promoter, lac promoter, recA promoter, .lambda.P L is promoter, a promoter suitable for the cell of Bacillus (Bacillus) bacteria, For example, SPO1 promoter, SPO2 promoter, penP promoter and the like. Suitable promoters for yeast cells are, for example, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX promoter and the like. Suitable promoters for plant cells are, for example, the cauliflower mosaic virus (CaMV) promoter and the like. Suitable promoters for insect cells are, for example, P10 promoter, polyhedrin promoter and the like. Suitable promoters for mammalian cells include, for example, Laus sarcoma virus, polyomer virus, chicken head virus, adenovirus, bovine papillomavirus, trisarcoma virus, cytomegalovirus (SMV), Simian virus 40 (SV40), vaccinia virus and the like. Virus promoter, melothionein promoter, heat shock promoter, etc.
[0036]
　Examples of the selection marker include HIS3 gene, LEU2 gene, TRP1 gene, URA3 gene, dihydrofolate reductase gene (methotrexate (MTX) resistance), ampicillin resistance gene, neomycin resistance gene, canamycin resistance gene and the like.
[0037]
　As the expression vector, a vector suitable for the host cell is selected, and for example, a plasmid, a phage, a cosmid, a viral vector, an artificial chromosome (for example, BAC, YAC, etc.) or the like is usually used. Examples of prokaryotic vectors include Escherichia coli-derived plasmids, such as pCR-based plasmids, pBR-based plasmids, and pUC-based plasmids, and Bacillus subtilis-derived plasmids, such as pUB110, pTP5, and pC194. The yeast vector is a yeast-derived plasmid, for example, a pSH-based plasmid or the like. The vector for plant cells is a binary vector or the like. For vectors for mammalian cells, commercially available vectors such as pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen, Stragene), viral vectors (eg, adenovirus, adeno-associated virus, poxvirus, simple herpesvirus, wrench) Viruses, Sendai virus, Wakusina virus, SV40 and the like.
[0038]
　Host cells are prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, plant cells, and animal cells (eg, mammalian cells, insect cells, etc.). When transforming or transfecting the above expression vector into a host cell, for example, known methods such as electroporation method, microinjection method, cell fusion method, DEAE dextran method, calcium phosphate method, particle gun method, and Agrobacterium method. Can be used.
[0039]
　Hepatic cell proliferation factors or active fragments thereof are known to be expressible using prokaryotic cells or eukaryotic cells, and the nucleic acid sequence (DNA) encoding the NK1 protein is transiently or stable in the cells. The recombinant protein can be expressed by introducing it into. The yeast expression system secretes and expresses recombinant proteins in the culture supernatant, and while it is also suitable for folding proteins having disulfide bonds, it can be cultured more easily than mammalian cells. Therefore, a yeast expression system is preferable for the expression of hepatocyte growth factor or an active fragment thereof having a plurality of disulfide bonds in the molecule.
[0040]
　As used herein, the term "homodimer" means a dimer formed by two molecules of proteins having the same amino acid sequence. Further, the term "monomer" as used herein means one of the proteins forming a dimer. Hepatocyte growth factor (HGF) is a homodimer of two molecules of HGF having the same amino acid sequence, which binds to the HGF receptor and expresses its physiological activity (Gherardi E. et al., Proceedings of the). National Academy of Sciences of the United States of America, 2006, Vol. 103, No. 11, p. 4046-4051). In addition, the active fragment of HGF is a homodimer of two active fragments having the same amino acid sequence in the presence of a heparin-like substance, which binds to the HGF receptor and exhibits its physiological activity (agonist activity). It is expressed (Chirgadze DY. et al., Nature Molecular Biology, 1999, Vol. 6, No. 1, p. 72-79). Alternatively, the active fragment of HGF binds to the HGF receptor as a monomer in the absence of a heparin-like substance and expresses antagonistic activity.
[0041]
　For example, according to the dimeric crystal structure of human NK1 (PDB ID: 3MKP) (Chirgadze DY. et al., Nature Structure Biology, 1999, Vol. 6, No. 1, p. 72-79), of human NK1. The two molecules form a dimer symmetrically, and the amino acid sequence in each klingle structure can bind to the sequence in the extracellular domain of the HGF receptor c-Met. It is shown.
[0042]

　Polyethylene glycol (hereinafter referred to as PEG) is a highly biocompatible polymer polymer, and by binding PEG to a protein, it has physical stability, thermal stability, and resistance to proteolytic enzymes. It is known that it brings about effects such as improvement of sex and solubility, reduction of distribution volume in vivo, and improvement of blood retention, and adds clinical usefulness (Inada et al., J. et al.). Bioact and Compact Polymers, 1990, Vol. 5, p. 343; Delgado et al., Critical Reviews in Therapetic Drugs, 1992, Vol. 9, p. 249; Volume 10, p.91).
[0043]
　PEG is compatible and contains water-soluble poly (ethylene oxide). Typically, PEG comprises a repeating unit structure "-(CH 2 CH 2 O) n-" and the structure of the end groups or the entire PEG moiety can change. For example, "-CH 2 CH 2- O (CH 2 CH 2 O) n-CH 2 CH 2- " and "-(OCH 2 CH 2 ) nO-" may be included depending on the presence or absence of oxygen substitution at the terminal . Commonly used PEG is end-capped PEG, in which end-capped PEG is an alkoxy group such as a group in which one end of the PEG is relatively inactive (typically methoxy (-OCH 3 )). ), Where the other end is an optionally chemically modified hydroxyl group or the like. PEG is available commercially or can be prepared by ring-opening polymerization of ethylene oxide according to known methods (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Volume 3, p. 138-161).
[0044]
　The PEG for producing a PEG-modified form of the above-mentioned hepatocyte growth factor or an active fragment thereof is a folk-type PEG. Fork-type PEG is known in the art. Typically, fork-type PEG has a branched portion and two hydroxyl groups linked to the branched portion (which can later undergo chemical modification) at one end of the PEG. For example, fork-type PEG is described in International Patent Publication WO 99/45964. The PEG structural portion of the fork-type PEG may be linear or branched. A particularly preferred fork-type PEG is a branched type.
[0045]
　The molecular weight of the fork-type PEG of the PEG-modified form of the hepatocyte growth factor or the active fragment thereof is preferably 5,000 or more and 240,000 or less, more preferably 10,000 or more and 80,000 or less, and 20,000 or more and 45. More preferably 000 or less, and most preferably 20,000. The effect of PEG modification on extending the half-life in vivo is known to correlate with the molecular weight of PEG (Sundqvist T. et al., Computers and Biomedical Research, 1988, Vol. 21, Vol. 2, p. 110-116). ), If it is 20,000 or more, a sufficient extension effect of the in vivo half-life can be expected. On the other hand, 20,000 is most preferable because it is known that high molecular weight PEG modification reduces tissue transferability.
[0046]
　In PEG, one molecule is composed of a large number of repeating unit structures "-(CH 2 CH 2 O) n-", and the molecular weight of PEG generally differs depending on each molecule. It is represented by the molecular weight. Therefore, the molecular weight of PEG as used herein means the average molecular weight.
[0047]
　Depending on the method of binding the hepatic cell growth factor or its active fragment to PEG, it is necessary to activate the terminal of PEG used in the covalent bond reaction, but the terminal is N-hydroxysuccinimide ester or nitrobenzenesulfonate ester. , Maleimide, orthopyridyl disulfide, vinyl sulfone, iodoacetamide, carboxylic acid, azide, phosphine or amine structure activated PEG can be used, and these activated PEGs can be synthesized by known methods. It can also be obtained as a commercial product. For example, as the activated fork-type PEG, SUNBRIGHT (registered trademark) PTE2 Series of Yuka Sangyo Co., Ltd. can be mentioned. In the present specification, the functional group added to the PEG terminal to show the binding reactivity with the protein is simply a "functional group" or "PEG functional group", and the PEG having these terminal structures is "activated by the functional group". Described as "PEG", "activated PEG" or "PEG containing functional groups".
[0048]
　To covalently attach fork-type PEG to the carboxyl-terminal region of the above hepatocyte growth factor or an active fragment thereof, fork-type PEG activated by two functional groups is used. The functional group may be either a homofunctionality having two identical functional groups or a heterofunctionality having two different functional groups. The fork-type PEG described above is preferably a fork-type PEG activated by two identical functional groups. More specifically, the fork-type PEG activated by two identical functional groups is preferably represented by the following formula (III).
[Chemical 3]

[0049]
　In the formula, "-" represents a bond and "n" generally ranges from 2 to about 2300. "N" can be appropriately determined depending on the desired PEG molecular weight.
[0050]
　Here, binding PEG to a protein is also referred to as chemically modifying or modifying the protein with PEG, or PEGylation, and is a covalent conjugate in which PEG is covalently bound to hepatocyte growth factor or an active fragment thereof. Is also referred to as a hepatocyte growth factor or an active fragment thereof chemically modified with PEG, or a PEG-modified form of the hepatocyte growth factor or an active fragment thereof.
[0051]
　In order to covalently bind PEG to the carboxyl-terminal region of the above hepatocyte growth factor or its active fragment, hepatocyte growth factor or an amino acid contained in the active fragment thereof, or a hepatocyte growth factor using a known gene recombination technique. Alternatively, an amino group (-NH 2 ), a thiol group (-SH) and a carboxyl group (-COOH) of cysteine ​​or an unnatural amino acid artificially introduced into the active fragment thereof can be used. For example, in the case of human hepatocyte growth factor (HGF), the amino group is present in the side chain of the lysine residue at the 4th and 10th residues from the carboxyl terminal amino acid, and N-hydroxysuccinimide (NHS) as the PEG functional group. Selective modification using an ester group is possible. Further, for example, the thiol group exists in the side chain of the cysteine ​​residue artificially inserted into the carboxyl terminal region, and the thiol group in the side chain does not form an intramolecular or intermolecular disulfide bond. , Selective modification using a maleimide group as the PEG functional group is possible. Further, for example, in the case of human NK1, the carboxyl group is the side chain of aspartic acid at the 8th residue from the carboxyl-terminal amino acid, the side chain of glutamate at the 2nd residue from the carboxyl-terminal amino acid, or glutamic acid which is the carboxyl-terminal amino acid. It exists in the side chain and the carboxyl terminal and can be modified using an amino group or the like. Further, as a method for inserting an unnatural amino acid into the carboxyl-terminal region of hepatocyte growth factor or an active fragment thereof, a method for introducing azidophenylalanine, azido-Z-lysine, etc. by codon modification has been reported (Japanese Patent Laid-Open No. 2009). -207490 (No. 207490), the azide group contained in these unnatural amino acids can be selectively modified with triallylphosphine.
[0052]
　In order to covalently bond PEG to the carboxyl-terminal region of the above HGF or its active fragment, an amino acid exhibiting selective reactivity can be artificially inserted into the carboxyl-terminal region of the above hepatocyte growth factor or its active fragment. preferable. In particular, it is more preferable to artificially insert a cysteine ​​residue, azidophenylalanine residue or azido-Z-lysine residue into the carboxyl-terminal region of the above hepatocyte growth factor or an active fragment thereof, and artificially insert the cysteine ​​residue. It is more preferable to insert it into. In this case, it is preferable to use activated PEG in which the PEG functional group is a maleimide group, whereby site-selective PEG to a cysteine ​​residue inserted into the carboxyl-terminal region of hepatocyte growth factor or an active fragment thereof. Modification is possible.
[0053]
The PEG-modified form of the above-mentioned hepatocyte growth factor or its active fragment is each monomer forming a homodimer (that is, one molecule of hepatocyte). A dimer is formed by covalently binding one molecule of fork-type PEG to the carboxyl-terminal region of (growth factor or an active fragment thereof). The amino ends of each monomer forming a homodimer of the hepatocyte growth factor or its active fragment are close to each other on the cell membrane side where the hepatocyte growth factor (HGF) receptor is present. On the other hand, the carboxyl ends of each monomer are close to each other on the intercellular side. Therefore, in the PEG-modified form of the above-mentioned hepatocyte growth factor or its active fragment, since PEG is covalently bonded to the carboxyl-terminal region of the hepatocyte growth factor or its active fragment, the physiology of the hepatocyte growth factor or its active fragment Can retain activity. PEG modification of hepatocyte growth factor or its active fragment to or near the amino terminus does not produce the desired effect.
[0054]
　The carboxyl-terminal region of hepatocyte growth factor or an active fragment thereof to which PEG is covalently bound includes the carboxyl-terminal amino acid of hepatocyte growth factor or an active fragment thereof or its vicinity. That is, it contains amino acid residues from the carboxyl-terminal amino acid to the 10th residue. For example, in the case of human hepatocellular proliferation factor (HGF) (GenBank accession number: M29145 (SEQ ID NO: 5 (base sequence), SEQ ID NO: 6 (amino acid sequence)), the starting methionine is the first in the amino acid sequence of SEQ ID NO: 6. It corresponds to the region of the amino acid residue from the 718th to the 728th (carboxyl terminal amino acid). For example, in the human NK1 (GenBank accession number: M29145, the 32nd amino acid sequence of SEQ ID NO: 6). In the case of (pointing from glutamine to the 210th glutamic acid), in the amino acid sequence of SEQ ID NO: 6, the starting methionine is set as the first, and the region of the amino acid residue from the 200th to the 210th (carboxyl terminal amino acid) Applicable. The amino acid residue in the carboxyl-terminal region may be an amino acid originally contained in the hepatocellular growth factor or an active fragment thereof (including a variant) or an artificially introduced amino acid, preferably. The carboxyl-terminal region of the hepatocellular proliferation factor or an active fragment thereof is preferably from the carboxyl-terminal amino acid to the 10th residue, more preferably from the carboxyl-terminal amino acid to the 4th residue, and most preferably the carboxyl-terminal amino acid.
[0055]
　A known method can be used to covalently bond PEG to the carboxyl-terminal region of the above HGF or an active fragment thereof. For example, a method of covalently bonding PEG to an amino group of a protein is described in US Pat. No. 4,917,888 and WO 1987/00056. A method for covalently bonding PEG to a thiol group of a protein is described in WO 1999/55377. Methods for covalently linking PEG to unnatural amino acids introduced into proteins are described in Bioorganic & Medical Chemistry Letters, 2004, Vol. 14, p. 5734-5745.
[0056]
　Purification or concentration of the PEG-modified form of the above-mentioned hepatocyte growth factor or its active fragment can be carried out by using a known method after the covalent reaction between the hepatocyte growth factor or its active fragment and the fork-type PEG. For example, unreacted hepatocyte growth factor or an active fragment thereof, fork-type PEG, and by-products are removed by methods such as ion exchange, gel filtration, chromatogram using a hydrophobic or affinity carrier, or a combination thereof. The PEG-modified form of hepatocyte growth factor or an active fragment thereof can be purified or concentrated.
[0057]
　The PEG-modified form of the above-mentioned hepatocyte growth factor or an active fragment thereof is specifically represented by the following formula (I).
[Chemical 4]

[0058]
　In the formula, "-" represents a bond, "PEG" represents a structural portion of fork-type polyethylene glycol, "L" represents a hydrolyzably stable bifurcated portion, and "X" represents hepatocyte growth factor or a hepatocellular growth factor thereof. Representing a binding moiety that provides a covalent bond of fork-type polyethylene glycol to an active fragment, "HGF" represents hepatocyte growth factor or an active fragment thereof. The bond (X) between hepatocyte growth factor or an active fragment thereof and fork-type polyethylene glycol is hydrolyzically stable.
[0059]
　"PEG" is a structural part of fork-type polyethylene glycol, and forms a branched structure even if the structure containing the polyethylene glycol repeating unit "-(CH 2 CH 2 O) n-" forms a linear structure. You may be doing it. Examples of those having a branched structure include a two-chain branched type, a three-chain branched type, and a four-chain branched type, and the number of branches includes a larger number, but the two-chain branched type is preferable. The branch atom (branch point) of the branch structure may be included in "L".
[0060]
　"L" may represent a single group such as "-CH-", or an even longer atomic chain (eg, alkylene bond (-CH 2- ), ether bond on the "PEG" side. (-O-), ester bonds (-O-CO- or -CO-O-), amide bonds (-CONH- or -NHCO-) ​​or combinations thereof) may be included. For example, the "L" group includes lysine, glycerol, pentaerythritol or sorbitol. A typical specific branch atom (atom serving as a branch point) in the branch portion is a carbon atom.
[0061]
　“X” is a bond portion that provides a covalent bond of fork-type polyethylene glycol to a hepatocellular proliferation factor or an active fragment thereof, provided that the distance between the branched atom and the functional group is 20 Å or less, and the above formula (III) is used. ) May represent only the atoms derived from the functional group, or an atom chain (for example, an alkylene bond (-CH 2- )) longer on the "L" side in addition to the atom derived from the functional group . It may contain an ether bond (-O-), an ester bond (-O-CO- or -CO-O-), an amide bond (-CONH- or -NHCO-) ​​or a combination thereof). The choice of an appropriate functional group depends on the binding site with hepatocyte growth factor or an active fragment thereof, as described above. The corresponding "X" in the PEG-modified form of the resulting hepatocyte growth factor or active fragment thereof is preferably the reaction of the activated fork-type PEG with the appropriate reaction site of the hepatocyte growth factor or the active fragment thereof. It is caused by the result of. For example, if the activated fork-type PEG contains an activating ester such as N-hydroxysuccinimide ester, the amine site-mediated binding of hepatocyte growth factor or an active fragment thereof forms the corresponding amide bond. ..
[0062]
　As used herein, a "functional group" is a reactive group at the end of a fork-type polyethylene glycol or the end of an atomic chain to provide a covalent bond with hepatocyte growth factor or an active fragment thereof.
[0063]
　More specifically, the PEG-modified form of the above-mentioned hepatocyte growth factor or an active fragment thereof is preferably represented by the following formula (II).
[Chemical 5]

[0064]
　The definitions of X and HGF in the formula are the same as those in the above formula (I). The n number of "-(CH 2 CH 2 O) n-" can be appropriately determined so as to have a desired PEG molecular weight (above).
[0065]
　A "hydrolyzably stable" bond is a chemical bond that is substantially stable in water (ie, does not hydrolyze under physiological conditions to the extent perceptible over a long period of time) (typically). Refers to covalent bond). Examples of hydrolyzably stable bonds include carbon-carbon bonds (eg, within aliphatic chains), ether bonds, amide bonds, urethane bonds and the like. Generally, a hydrolysis-stable bond is one that exhibits a hydrolysis rate of less than about 1-2% per day under physiological conditions.
[0066]
　A functional group (eg, for example) that provides a covalent bond between the branched atom of fork-type PEG of the PEG-modified form of the hepatocyte growth factor or the active fragment thereof and the fork-type polyethylene glycol to the hepatocyte growth factor or the active fragment thereof. The distance from the fork-type PEG functional group) is not limited as long as the PEG-modified form of hepatocyte growth factor or an active fragment thereof retains its physiological activity, but is preferably 20 Å or less, and is preferably 1.5 Å or less. It is more preferably ~ 19 Å, further preferably 1.5 to 8 Å, and most preferably 7.2 Å.
[0067]
　The branched atom of PEG is an atom serving as a branching point in "L" in the above formula (I), that is, a branched atom. As the branched atom, a carbon atom is preferable.
[0068]
　The distance between the branched atom of PEG and the PEG functional group means the distance from the above-mentioned branched atom to the binding site of the PEG functional group (for example, the 5-membered ring nitrogen atom of the maleimide group). That is, the distance from the branched atom in "L" to the binding site of the functional group in "X" in the above formula (I) is shown. The distance between the branched atom of PEG and the PEG functional group can be calculated, for example, by structural simulation. From the viewpoint of the PEG-modified form of hepatocyte growth factor or its active fragment, the distance between the branched atom of PEG and the PEG functional group is the PEG-modified form (homodimer) of hepatocyte growth factor or its active fragment. ) Means half the distance between the carboxyl ends of each monomer (ie, one molecule of hepatocyte growth factor or an active fragment thereof). For example, when the distance between the branched atom of PEG and the PEG functional group is 20 Å, the distance between the carboxyl terminus of each monomer is 40 Å.
[0069]
　In order for the PEG-modified form of the above-mentioned hepatocyte growth factor or its active fragment forming a homodimer by PEG modification to retain its physiological activity, each monomer forming the dimer ( That is, it is desirable that the distance between the carboxyl ends of one molecule of hepatocyte growth factor or an active fragment thereof reproduces the dimeric crystal structure of the hepatocyte growth factor or an active fragment thereof.
[0070]
　In human NK1, the 206th cysteine ​​residue (corresponding to the 175th cysteine ​​residue of the amino acid sequence of SEQ ID NO: 2) counting from the 1st starting methionine in the amino acid sequence of SEQ ID NO: 6 is intramolecular. From the 207th serine residue to the 210th glutamate (corresponding to the 176th serine residue to the 179th glutamate in the amino acid sequence of SEQ ID NO: 2). Human NK1 dimer crystal structure (PDB ID: 3MKP) (Cysteine ​​DY et al., Nature Structural Biology, 1999, Vol. 6, No. 1, p. Based on 72-79), using Molecular Operating Environment (version 2013.08) of Chemical Computing Group, from the 207th serine residue to the 210th glutamic acid counting from the first starting methionine. A structural simulation of the distance between the carboxyl ends of each monomer (that is, one molecule of human NK1) when human NK1 formed a dimer when it was flexibly moved showed a minimum distance of 3 Å and a maximum distance of 38 Å. There was (ie, the permissible range of distance between the PEG branched atom and the PEG functional group is a minimum of 1.5 Å and a maximum of 19 Å).
[0071]
　When an artificial sequence such as a tag sequence or a spacer sequence is added to the carboxyl terminus of the above hepatocyte growth factor or its active fragment, when the above hepatocyte growth factor or its active fragment forms a dimer. The distance between the carboxyl terminus of each monomer of the above may be further increased. For example, when a 6 × His tag is added to the carboxyl terminus of human NK1, glutamic acid at positions 176 to 179 of the serine residue of the amino acid sequence of SEQ ID NO: 2 of human NK1 (the first in the amino acid sequence of SEQ ID NO: 6). Assuming that the 207th serine residue to the 210th glutamic acid) and the 6 × His tag portion can be flexibly moved from the 207th starting methionine, a structural simulation similar to the above was performed. The distance between the carboxyl terminus of the NK1 dimer was a minimum distance of 3 Å and a maximum distance of 88 Å (that is, the allowable range of the distance between the branched atom of PEG and the PEG functional group was a minimum of 1.5 Å and a maximum of 44 Å. Become).
[0072]
　Therefore, in the PEG-modified form of the hepatocyte growth factor or the active fragment thereof, the distance between the carboxyl terminus of each monomer when forming a dimer is more preferably 3 to 38 Å. However, as described above, the distance between the carboxyl terminus of each monomer can be substantially extended when an artificial sequence is inserted into the carboxyl terminus of hepatocyte growth factor or an active fragment thereof. An example of an artificial sequence is a peptide having 2 to 30 amino acids consisting of an arbitrary amino acid, for example, a peptide containing the above tag sequence, for example, a peptide of SEQ ID NO: 4 (HHHHHHC).
[0073]
　When linear PEG in which both ends of PEG are activated by two functional groups is used to PEG-modify hepatocyte growth factor or an active fragment thereof to dimerize, the space between the functional groups is It is desirable that the PEG chain length is 40 Å or less, but in this case, the PEG molecular weight is 1,000 or less, so that the effect of sufficiently extending the in vivo half-life cannot be obtained. On the other hand, in the case of the present invention using fork-type PEG activated by two functional groups, the PEG molecular weight is high even if the distance between the branched atom of PEG and the PEG functional group is 20 Å or less, which is a preferable range. Not restricted. In addition, if hepatocyte growth factor or an active fragment thereof is modified with fork-type PEG, between the carboxyl ends of each monomer forming a dimer (that is, one molecule of hepatocyte growth factor or an active fragment thereof). The distance can be controlled, and the physiological activity of hepatocyte growth factor or an active fragment thereof can be maintained even after PEG modification.
[0074]
　A preferred embodiment of the PEG-modified form of hepatocyte growth factor or an active fragment thereof is that the hepatocyte growth factor or an active fragment thereof forms a homodimer and both monomers of the homodimer. A single fork-type PEG having a distance of 7.2 Å and a PEG molecular weight of 20,000 is covalently bonded to a cysteine ​​residue artificially inserted into the carboxyl terminal region of PEG. It is a PEG-modified form of hepatocyte growth factor or an active fragment thereof forming a dimer. The PEG-modified form of this hepatocyte growth factor or an active fragment thereof retains its physiological activity because the distance between the carboxyl terminus of each monomer when forming a dimer is controlled, and further has a molecular weight. Since it is modified with 20,000 PEG, it has the effect of prolonging the in vivo half-life.
[0075]
　A more preferred embodiment of the PEG-modified form of hepatocyte growth factor or an active fragment thereof is represented by the following formula (IV).
[Chemical

　formula 6] The binding between hepatocyte growth factor or an active fragment thereof (“HGF” in the formula) and a maleimide group (functional group) was artificially inserted into the carboxyl-terminal region of hepatocyte growth factor or an active fragment thereof. Cysteine ​​residues are bound to form an "-S-" bond. The n number of "-(CH 2 CH 2 O) n-" is determined by the PEG molecular weight (above).
[0076]

　The PEG-modified form of the above-mentioned hepatocyte growth factor or its active fragment retains the physiological activity originally possessed by the hepatocyte growth factor or its active fragment, and further has the effect of prolonging the in vivo half-life by PEG modification. Since it has, it can be used as an active ingredient of a medicine (which can be replaced with a "therapeutic agent" or a "pharmaceutical composition").
[0077]
　The physiological activity of the PEG-modified form of the above-mentioned hepatocyte growth factor or an active fragment thereof can be easily measured using cultured cells. For example, c-Met phosphorylation-inducing action, cell proliferation action, cell migration action, anti-apoptotic action, etc., which are actions of natural hepatocyte growth factor, can be used as indicators (Rubin JS et al., The Journal of). Biological Chemistry, 2001, Vol. 276, No. 35, p. 32977-32983; Lietha D. et al., The EMBO Journal, 2001, Vol. 20, No. 20, p. 5543-5555; Liu Y, American Journal. of Phosphorylation, 1999, Vol. 277, No. 4, p. 624-633).
[0078]
　The in vivo half-life of the PEG-modified form of the above-mentioned hepatocyte growth factor or its active fragment can be determined by measuring the blood concentration when intravenously, intraperitoneally, subcutaneously or intradermally administered to a model animal. Can be calculated. In particular, simple measurement is possible by radiolabeling the PEG-modified form of the above-mentioned hepatocyte growth factor or an active fragment thereof.
[0079]
　The medicine containing the PEG-modified form of the hepatocyte growth factor or the active fragment thereof as an active ingredient can be used for the treatment of various diseases utilizing the physiological activity of the hepatocyte growth factor or the active fragment thereof. For example, acute inflammatory disease, chronic inflammatory disease, acute ischemic disease, chronic ischemic disease, muscular atrophic lateral sclerosis, organ fibrosis, diabetes, spinal cord injury, peritoneal adhesions, transplantation treatment, wound healing, neuropathic It can be used for the treatment of sexual pain, etc.
[0080]
　The above-mentioned medicines can be used as useful therapeutic agents for mammals (eg, mice, rats, hamsters, rabbits, dogs, cats, monkeys, cows, sheep or humans), especially humans. When the above-mentioned drug is used as a useful therapeutic agent for humans, the above-mentioned hepatocyte growth factor or an active fragment thereof is preferably based on an amino acid sequence derived from human hepatocyte growth factor.
[0081]
　As the administration form of the above-mentioned pharmaceuticals, the PEG-modified form of the above-mentioned hepatocyte growth factor or an active fragment thereof, which is an active ingredient, is orally or parenterally administered as it is or in combination with a pharmaceutically acceptable carrier. can do. It is preferably administered by subcutaneous, intramuscular or intravenous injection.
[0082]
　Dosage forms for oral administration of the above-mentioned pharmaceuticals include, for example, tablets, pills, capsules, granules, syrups, emulsions or suspensions, which can be produced by known methods. It can contain carriers or excipients commonly used in the pharmaceutical field and, if necessary, additives. Carriers and excipients for tablets include, for example, lactose, maltose, saccharose, starch or magnesium stearate. Examples of the additive include a binder, a disintegrant, a preservative, a delayed release agent, a colorant, a flavoring agent, a stabilizer, a dissolving agent, a thickener, an emulsifier and the like.
[0083]
　Dosage forms for parenteral administration of the above drugs include, for example, injections, eye drops, ointments, poultices, suppositories, nasal absorbents, pulmonary absorbents, transdermal absorbents or topical sustained release agents. These can be produced by known methods. In the solution preparation, for example, the PEG-modified form of the above-mentioned hepatocyte growth factor or an active fragment thereof, which is an active ingredient, is dissolved in a sterile aqueous solution used for an injection, suspended in an extract, emulsified, and encapsulated in liposomes. It can be prepared in a buried state. The solid preparation is prepared as a lyophilized product by adding mannitol, trehalose, sorbitol, lactose, glucose or the like as an excipient to the PEG-modified form of the above-mentioned hepatocyte growth factor or an active fragment thereof, which is an active ingredient. Can be done. Further, this can be powdered and used. Further, these powders can be mixed with polylactic acid, glycolic acid or the like to be solidified and used. The gelling agent dissolves, for example, a PEG-modified form of the above-mentioned hepatocyte growth factor or an active fragment thereof, which is an active ingredient, in a thickener such as glycerin, PEG, methyl cellulose, carboxymethyl cellulose, hyaluronic acid or chondroitin sulfate, or a polysaccharide. Can be prepared. In addition, the above additives can be added to the formulation, if necessary.
[0084]
　The above-mentioned medicine is appropriately determined according to the age, body weight, target disease, symptom, administration form, administration route, molecular weight of PEG, etc. of the patient, but is generally 0.001 mg to 100 mg / kg / dose. It can be administered in the range of 0.01 mg to 10 mg / kg / dose once / month to once a day, preferably once a month to once a week.
　The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention shall not be limited by the examples provided as examples.
Example
[0085]
(Example 1)
　Expression of human NK1 protein Expression of recombinant human NK1 protein was carried out using a commercially available yeast expression kit, Multi-Copy Pichia Expression Kit (Invitrogen).
[0086]
　A human NK1 protein in which a 6 × His tag and a cysteine ​​residue are sequentially added (that is, HHHHHHC (SEQ ID NO: 4) is added) to the carboxyl terminus of the human NK1 protein from which the secretory signal sequence has been removed (hereinafter, His-Cys-added human NK1). The DNA encoding the above was inserted into the pPIC9K expression vector attached to the Multi-Copy Pichia Expression Kit using an In-Fusion HD cloning kit (Takara Bio). The prepared expression plasmid was linearized with a restriction enzyme, and then the gene was introduced into the GS115 strain attached to the above kit by an electroporation method.
[0087]
　After the cells after gene transfer were selectively cultured in the first stage on a histidine-deficient agar medium, the grown colonies were selectively cultured in the second stage on an agar medium containing 4 mg / mL Yeastin (registered trademark) (Roche). A yeast strain into which the gene was introduced in multiple copies was isolated. The isolated strain is pre-cultured in a BMGY medium in a constant temperature chamber at 30 ° C., then transferred to a BMMY medium containing 0.5% methanol, and main-cultured in a constant temperature chamber at 20 ° C. for 3 days to obtain His, which is the target protein. -Cys-added human NK1 expression was induced.
[0088]
(Example 2) Purification of human NK1 protein The
　yeast culture supernatant obtained in Example 1 was centrifuged, and then His-Cys-added human NK1 in the supernatant was purified using nickel resin and heparin resin.
[0089]
　Heparin resin (GE Healthcare) equilibrated with PBS (−) in advance was passed through a yeast culture supernatant that had been centrifuged to bind His-Cys-added human NK1 to the heparin resin. Subsequently, a buffer in which the NaCl concentration contained in PBS (−) was changed in the concentration range of 150 mM to 2000 mM was passed in ascending order of the NaCl concentration to obtain each eluted fraction. Each eluted fraction was subjected to SDS electrophoresis to confirm the His-Cys-added human NK1 eluted fraction.
[0090]
　Next, the His-Cys-added human NK1 elution fraction obtained by heparin resin purification was passed through a compacte His-Tag Purification Resin (Roche) previously equilibrated with 300 mM NaCl-containing PBS (-) to allow the resin to become His. -Cys-added human NK1 was bound. Subsequently, a buffer in which imidazole (concentration range of 0 mM to 500 mM) was added to PBS (−) containing 300 mM NaCl was passed in ascending order of imidazole concentration to obtain each eluted fraction. Each eluted fraction was subjected to SDS electrophoresis to confirm the His-Cys-added human NK1 eluted fraction. The obtained His-Cys-added human NK1 elution fraction was concentrated using Amicon Ultra-15 (MWCO = 10,000; Merck Millipore) to obtain the target protein, His-Cys-added human NK1.
[0091]
(Example 3) Synthesis of PEG-modified NK1 dimer NK1 dimerized
　by covalently bonding one molecule of PEG to the carboxyl end of two molecules of His-Cys-added human NK1 (hereinafter, PEG-modified NK12). The metric) was synthesized by the following method.
[0092]
　38 mg in a solution prepared in Example 2 in which His-Cys-added human NK1 was adjusted to 1.0 mg / mL (hereinafter, NK1 solution) (composition: 1.0 mol / L NaCl-containing PBS (-), pH 7.4). A 0.07-fold amount of a / mL 2-mercaptoethylamine (2-MEA) solution was added, and the mixture was incubated at 37 ° C. for 30 minutes. A gel filtration column (Zeba desert spin colon) in which the incubated NK1 solution was equilibrated with a phosphate buffer solution (0.3 mol / L NaCl, 0.002 mol / L EDTA, 0.1 mol / L phosphate (pH 6.0)). It was passed through 7KDa; Thermo Fisher Scientific) to remove 2-MEA from the solution.
[0093]
　Fork-type PEG (SUNBRIGHT PTE2-200MA2, 2 functional groups, PEG functional group = maleimide group, distance between PEG branched atom and PEG functional group = 7.2 Å, molecular weight, in NK1 solution after 2-MEA removal treatment 20,000; Yuka Sangyo Co., Ltd.) or linear PEG (SUNBRIGHT DE-100MA, 2 functional groups, PEG functional group = maleimide group, distance between PEG functional groups = about 400 Å, molecular weight 10,000; Yuka Sangyo Co., Ltd.) was added so that the reaction molar ratio was 5: 1 (PEG: His-Cys-added human NK1). The reaction was carried out at 25 ° C. for 16 to 18 hours to prepare a PEG-modified NK1 dimer. The PEG-modified NK1 dimer modified with fork-type PEG is called a fork-type PEG-modified NK1 dimer, and the PEG-modified NK1 dimer modified with linear PEG is referred to as a linear PEG-modified NK1 dimer. Call.
[0094]
　The post-reaction solution containing the PEG-modified NK1 dimer was diluted 10-fold with NaCl-free PBS (-), passed through an SP carrier (SP-Sepharose6 Fast Flow; GE Healthcare), and not from the solution. The PEG reagent of the reaction was removed. The PEG-modified NK1 dimer adsorbed on the SP carrier was eluted with 1.0 mol / L NaCl-containing PBS (−) and then concentrated with Amicon Ultra-30 (MWCO = 30,000; Merck Millipore).
[0095]
　FIG. 1 shows SDS electrophoresis images of His-Cys-added human NK1 and fork-type PEG-modified NK1 dimer.
[0096]
　Fork-type PEG-modified NK1 dimer or linear PEG-modified NK1 dimer has one molecule of PEG shared by two molecules of His-Cys-added human NK1 at the carboxyl terminus of the artificially added cysteine ​​residue. It is combined and dimerized. The fork-type PEG-modified NK1 dimer is represented by the following formula (V), and the linear PEG-modified NK1 dimer is represented by the following formula (VI)
[Chemical formula 7].

[0097]
　In the formula, "Cys-6 x His-NK1" indicates a His-Cys-added human NK1, and a thiol group (-SH) and a maleimide group (functional group) of a cysteine ​​residue artificially inserted into the human NK1 carboxyl end. Is combined with "-S-". The n number of "-(CH 2 CH 2 O) n-" is determined by the PEG molecular weight.
[Chemical 8]

[0098]
　In the formula, "Cys-6 x His-NK1" and "NK1-6 x His-Cys" indicate His-Cys-added human NK1, and the thiol group of the cysteine ​​residue artificially inserted at the terminus of the human NK1 carboxyl (Cysteine ​​residue). -SH) and the maleimide group (functional group) are bonded to form an "-S-" bond. The n number of "-(CH 2 CH 2 O) n-" is determined by the PEG molecular weight.
[0099]
(Example 4) Biological activity of
　PEG-modified NK1 dimer The physiological activity of PEG-modified NK1 dimer is used as an index of the phosphorylation-inducing activity of the HGF receptor present on the cell surface of human lung epithelial cell line A549. -Evaluated by the Cell ELISA method. In addition, His-Cys-added human NK1, which is a comparative control of physiological activity, induces heparin-dependent dimerization by mixing with 1 μg / mL porcine purified heparin (sigma) -containing MEM medium in advance, and dimerizes. A body was formed (hereinafter, heparin dimerized NK1).
[0100]
　A549 cells were suspended in MEM medium containing 10% FCS , seeded on a 96-well plate for imaging (Becton Dickinson) at a density of 1.5 × 10 4 / well, and cultured overnight. After the cells became about 70% confluent, the medium was replaced with a serum-free MEM medium, and the cells were cultured for 16 hours or more to obtain a serum starvation state. Heparin dimerized NK1, fork-type PEG-modified NK1 dimer or linear PEG-modified NK1 dimer (10 or 250 ng / mL) is added to serum-starved cells at 37 ° C. for 10 minutes. It was reacted.
[0101]
　The cells were fixed with PBS (-) containing 4% formalin, and then the cell membrane was permeated with PBS (-) containing 0.3% Triton-X and 0.6% hydrogen peroxide, and then the cells were blocked with 10% BSA. Processed. Anti-phosphorylated c-Met antibody, which is a primary antibody, and HRP-labeled secondary antibody, which is a secondary antibody, were added to the cells after the blocking treatment and reacted, and then 1 × QuantaRed Enhanced Chemifluorescent HRP Substrate (Thermofisher Scientific). ) Was added. After incubation at room temperature, the fluorescence intensity (RFU) at a wavelength of 590 nm was measured using a plate reader (PerkinElmer) and used as the amount of HGF receptor phosphorylation induced.
[0102]
　The result is shown in FIG. The vertical axis of FIG. 2 shows the fluorescence intensity (RFU) representing the amount of HGF receptor phosphorylation induced, and the horizontal axis shows heparin dimerized NK1, fork-type PEG-modified NK1 dimer or linear PEG-modified NK1 dimer. The processing concentration of the body (ng / mL) is shown.
[0103]
　The fork-type PEG-modified NK1 dimer retained the same physiological activity as the heparin dimerized NK1, but the linear PEG-modified NK1 dimer had a higher physiological activity than the heparin dimerized NK1. It was greatly weakened. Therefore, it was shown that it is important to use fork-type PEG that can control the distance between the carboxyl terminus of NK1 in order to dimerize it by PEG modification while maintaining the physiological activity of NK1.
[0104]
(Example 5) Blood dynamics of
　fork-type PEG-modified NK1 dimer The blood dynamics of fork-type PEG-modified NK1 dimer was measured by [125I] -labeled fork-type PEG-modified NK1 dimer in the tail vein of mice. It was evaluated by administration. As a comparative control of blood kinetics, His-Cys-added human NK1 was used.
[0105]
[125I] labeling of 　His-Cys-added human NK1 or fork-type PEG-modified NK1 dimer was performed by the IODO-BEADS method using Na [ 125I ] (iodine-125 Radionuclide; PerkinElmer). The radioactivity of the supernatant and the sediment after TCA precipitation was measured, and it was confirmed that the [125I] labeling rate of the protein used for administration was 90% or more.
[0106]
　 [125I] Labeled His-Cys-added human NK1 or fork-type PEG-modified NK1 dimer was intravenously administered to Crlj: CD1 (ICR) male mice (25 μg / 0.5 MBq / kg) to increase radioactivity. As an index, the serum concentration was measured for 5 minutes to 24 hours after administration.
[0107]
　The result is shown in FIG. The vertical axis of FIG. 3 shows the mouse serum concentration (ng eq./mL), and the horizontal axis shows the time (h) after administration. The dotted line shows the result of [125I] labeled His-Cys-added human NK1, and the solid line shows the result of [125I] labeled fork-type polyethylene glycol-modified NK1 dimer.
[0108]
　The folk-type PEG-modified NK1 dimer showed almost the same concentration transition as the His-Cys-added human NK1 up to 1 hour after the administration, but after 4 hours after the administration, the serum was compared with the His-Cys-added human NK1. The decrease in medium concentration was alleviated. Furthermore, 24 hours after administration, a fork-type PEG-modified NK1 dimer having a concentration of about 4.2 times was observed in the serum as compared with the His-Cys-added human NK1. The half-life (t1 / 2) was 7.9 hours with His-Cys-added human NK1 administration, but was extended to 15.4 hours with the fork-type PEG-modified NK1 dimer, which was about twice as long. Therefore, it was shown that the fork-type PEG-modified NK1 dimer also exhibits the effect of extending the in vivo half-life, which is the effect of PEG modification.
Industrial applicability
[0109]
　The polyethylene glycol modified product of the hepatocyte growth factor of the present invention or an active fragment thereof exhibits its medicinal effect with a lower administration frequency than the unmodified product because the in vivo half-life is extended and its physiological activity is maintained. It can be used as a medicine to be used.
[0110]
　SEQ ID NO: 1: Amino acid sequence of human HGF not containing a secretory signal sequence
　SEQ ID NO: 2: Amino acid sequence of human NK1 not containing
　a secretory signal sequence
　SEQ ID NO: 3: Secretory signal sequence derived from human HGF SEQ ID NO: 4: Carboxynia of human NK1 protein Amino acid sequence of 6 × His tag and cysteine ​​residue added to the end
　SEQ ID NO: 5: Nucleotide sequence of human HGF
　SEQ ID NO: 6: Amino acid sequence of human HGF
The scope of the claims
[Claim 1]
　Polyethylene of hepatocyte growth factor or active fragment thereof in which one molecule of fork-type polyethylene glycol is covalently bonded to each carboxyl terminal region of two molecules of hepatocyte growth factor or active fragment thereof to form a homodimer. Glycol modified product.
[Claim 2]
　The polyethylene glycol modified product of the hepatocyte growth factor according to claim 1 or an active fragment thereof, which is represented by the general formula (I).
[Chemical

formula 1] [In the formula, PEG represents a structural portion of fork-type polyethylene glycol, L represents a hydrolyzably stable branched portion, HGF represents hepatocyte growth factor or an active fragment thereof, and X Represents a binding moiety that provides a covalent bond of fork-type polyethylene glycol to hepatocyte growth factor or an active fragment thereof. ]
[Claim 3]
　In the general formula (I), PEG represents a structure containing- (CH 2 CH 2 O) n-, the structure forming a linear structure or a branched structure, where n is 2 to 2300. The polyethylene glycol modified product of the hepatocyte growth factor or the active fragment thereof according to claim 2.
[Claim 4]
　A polyethylene glycol modified product of the hepatocyte growth factor or an active fragment thereof according to any one of claims 1 to 3, which is represented by the general formula (II).
[Chemical

formula 2] [In the formula, X and HGF are as defined above. ]
[Claim 5]
　The distance between the branched atom of the branched portion of the fork-type polyethylene glycol and the functional group that provides a covalent bond of the fork-type polyethylene glycol to the hepatocyte growth factor or an active fragment thereof is 20 Å or less, claims 1 to 1. 4. A polyethylene glycol-modified form of the hepatocyte growth factor or an active fragment thereof according to any one of 4.
[Claim 6]
　The polyethylene glycol modified product of the hepatocyte growth factor or the active fragment thereof according to any one of claims 1 to 5, wherein the active fragment of the hepatocyte growth factor is NK1.
[Claim 7]
　The hepatocyte growth factor or an active fragment thereof according to claim 6, wherein the NK1 comprises the amino acid sequence shown by SEQ ID NO: 2 in the sequence listing, or an amino acid sequence having 90% or more sequence identity with the amino acid sequence. Polyethylene glycol modified product.
[Claim 8]
　A medicament containing a polyethylene glycol modified product of the hepatocyte growth factor according to any one of claims 1 to 7 or an active fragment thereof as an active ingredient.