PEG-modified protein or PEG-modified hydrophobic substance, or method for producing the same

Abstract

To provide a PEG-modified protein having reduced immunogenicity.SOLUTION: A PEG-modified protein is represented by the formula (1) [where P is a protein residue, A is a group having a polyethylene glycol residue with a molecular weight of 1000-50000, L is a C1-10 alkylene group optionally containing an ether bond and / or an amide bond in the chain, X is a single bond or a divalent peptide residue comprising a divalent amino acid residue or 2-100 amino acids, Y is a single bond or -CH2-CO-NH-* or -CH2CH2-CO-NH-*, * is a point of bonding to a protein residue, and n is 1-100].SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a PEG-modified protein or PEG-modified hydrophobic substance, a method for producing the same, a pharmaceutical composition containing the same, and a method for PEG-modifying a protein or hydrophobic substance. [Background technology]

[0002] Polyethylene glycol (PEG) is a versatile polymer. In particular, PEG as a pharmaceutical is known to be a golden standard and highly effective means for improving the blood half-life of proteins and drug carriers and reducing the immunogenicity of proteins due to its hydration effect. However, in recent years, it has been discovered that PEG-modified pharmaceuticals can produce antibodies against PEG (PEG-specific antibodies) (Non-Patent Document 1). Therefore, there is a demand for the development of PEG-modified pharmaceuticals with reduced immunogenicity.

[0003] For protein formulations, the PEG modification of proteins has been conventionally and commonly performed using the NHS method, which involves reacting PEG having an NHS group at its terminal with the amino group of the protein. However, this method has raised the problem of the production of PEG-specific antibodies against uricase and asparaginase modified with PEG (Non-Patent Documents 1 to 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] RP Garay, et al., Expert Opin. Drug Deliv. (2012) 9(11):1319-1323 [Non-patent document 2] Lipsky et al., Arthritis Research & Therapy, 2014, 16:R60 [Non-patent document 3] Armstrong JK et al., Cancer (2007) 110 (1): 103-111 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a PEG-modified protein or PEG-modified hydrophobic substance with reduced immunogenicity, a method for producing the same, a pharmaceutical composition containing the same, and a method for PEG-modifying a protein or hydrophobic substance. [Means for solving the problem]

[0006] The present inventors have investigated various means for solving the above problems and have found that by forming a PEG-modified protein into a structure represented by formula (1), the immunogenicity can be reduced compared to conventional PEG-modified proteins, thereby completing the present invention.

[0007] That is, the gist of the present invention is as follows. [1] Formula (1): [ka] [In the formula, P is a protein residue, A is a group containing a polyethylene glycol residue having a molecular weight of 1,000 to 50,000, L is an alkylene group having 1 to 10 carbon atoms which may contain one or more bonds selected from an ether bond and an amide bond in the molecular chain, X is a single bond, a divalent amino acid residue, or a divalent peptide residue consisting of 2 to 100 amino acids; Y is a single bond or -CH2-CO-NH-* or -CH2CH2-CO-NH-*, * is the point of attachment to the protein residue n is 1 to 100. A PEG-modified protein represented by the formula: [2] The PEG-modified protein according to the above [1], wherein L is an alkylene group having 1 to 10 carbon atoms. [3] The PEG-modified protein according to [1] or [2] above, wherein X is a single bond. [4] The PEG-modified protein according to [1] or [2] above, wherein X is a divalent peptide residue, and the divalent peptide residue consists of 20 to 100 amino acids. [5] The PEG-modified protein according to [4] above, wherein the divalent peptide residue comprises an amino acid selected from aspartic acid and glutamic acid. [6] Formula (1): [ka] [In the formula, P is a protein residue, A is a group containing a polyethylene glycol residue having a molecular weight of 1,000 to 50,000, L is an alkylene group having 1 to 10 carbon atoms which may contain one or more bonds selected from an ether bond and an amide bond in the molecular chain, X is a single bond, a divalent amino acid residue, or a divalent peptide residue consisting of 2 to 100 amino acids; Y is a single bond or -CH2-CO-NH-* or -CH2CH2-CO-NH-*, * is the point of attachment to the protein residue n is 1 to 100. A method for producing a PEG-modified protein represented by the formula: (a) The following formula (2) AX-NH2(2) [In the formula, A and X have the same meanings as above.] The compound represented by the following formula (3): [ka] [In the formula, L has the same meaning as above.] in a solvent to obtain a compound represented by the following formula (4): [ka] [In the formula, A, L, and X have the same meanings as above.] forming a PEG maleimide derivative represented by (b) A PEG maleimide derivative represented by formula (4) in an excess amount is reacted with a compound represented by formula (5): [ka] [In the formula, P and n have the same meanings as above.] or a protein having a thiol group represented by the following formula (6): [ka] [In the formula, P and n are as defined above, m is 1 or 2.] in a solvent to obtain a PEG-modified protein represented by formula (1). [7] A method for PEG-modifying a protein, comprising the steps (a) and (b) described in [6] above. [8] A pharmaceutical composition comprising the PEG-modified protein according to any one of [1] to [5] above.

[0008] Furthermore, the present inventors have investigated various means for solving the above-mentioned problems and have found that by forming a PEG-modified hydrophobic substance into a structure represented by the following formula (7), immunogenicity can be reduced compared to conventional PEG-modified hydrophobic substances, thereby completing the present invention.

[0009] The following formula (7): [ka] [In the formula, P' is a hydrophobe residue, A' is a group containing a polyethylene glycol residue having a molecular weight of 1,000 to 50,000, L' is an alkylene group having 1 to 10 carbon atoms which may contain one or more bonds selected from an ether bond and an amide bond in the molecular chain, X' is a single bond, a divalent amino acid residue, or a divalent peptide residue consisting of 2 to 100 amino acids; Y' is -CH2CH2-NH-Z-**, Z is a divalent group derived from a hydrophobic substance, ** denotes the point of attachment to the hydrophobic substance residue, n' is 1 to 100. A PEG-modified hydrophobic substance represented by the formula: [Effects of the Invention]

[0010] The PEG-modified protein or PEG-modified hydrophobic substance according to the present invention has reduced immunogenicity compared to conventional PEG-modified proteins or PEG-modified hydrophobic substances. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing the results of evaluation of anti-PEG IgM production in an animal experiment. [Figure 2] FIG. 2 is a graph showing the results of evaluation of anti-PEG IgM production in an animal experiment. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention relates to a compound represented by the following formula (1): [ka] [In the formula, P is a protein residue, A is a group containing a polyethylene glycol residue having a molecular weight of 1,000 to 50,000, L is an alkylene group having 1 to 10 carbon atoms which may contain one or more bonds selected from an ether bond and an amide bond in the molecular chain, X is a single bond, a divalent amino acid residue, or a divalent peptide residue consisting of 2 to 100 amino acids; Y is a single bond or -CH2-CO-NH-* or -CH2CH2-CO-NH-*, * is the point of attachment to the protein residue n is 1 to 100. The present invention relates to a PEG-modified protein represented by the formula (I). The PEG-modified protein of the present invention has reduced immunogenicity compared to conventional PEG-modified proteins, particularly proteins PEG-modified by the commonly used NHS method. The present inventors have found that immunogenicity can be reduced by linking a protein to PEG with a linker having the above-mentioned specific structure. PEG-specific antibodies are antibodies induced by PEG, but are thought to bind weakly to PEG itself but to bind cooperatively to the protein bound to PEG. Without being bound by theory, it is thought that the physical distance between the protein and PEG is increased by the above-mentioned specific linker, thereby preventing the binding of PEG-specific antibodies to the PEG-modified protein.

[0013] As used herein, the term "polyethylene glycol residue" refers to -(CH2CH2O) a - [wherein a is a positive number].

[0014] As used herein, the term "amino acid residue" refers to a group in which some atoms or groups have been removed from an amino acid, such as a group in which a hydroxy group in a carboxyl group or a hydrogen atom or amino group in an amino group has been removed from an amino acid.

[0015] As used herein, the term "peptide residue" refers to a group in which some atoms or groups have been removed from a peptide, such as a group in which a hydroxy group in a carboxyl group, a hydrogen atom in an amino group, or an N-terminal amino group has been removed from a peptide.

[0016] As used herein, the term "protein residue" refers to a residue in which some atoms or groups have been removed from a protein.

[0017] As used herein, the term "hydrophobic substance" refers to lipids composed of fatty acids, compounds composed of hydrophobic amino acids, and hydrophobic polymers. Also, as used herein, the term "hydrophobic substance residue" refers to a group in which some atoms or groups have been removed from a hydrophobic substance.

[0018] As used herein, the term "PEG-modified protein" refers to a protein in which a group containing a polyethylene glycol residue is bound to a certain atom or group in the protein directly or via an arbitrary or specific linker.

[0019] As used herein, the term "PEG maleimide derivative" refers to a compound in which a group containing a polyethylene glycol residue is bound to a maleimide group directly or via an arbitrary or specific linker.

[0020] As used herein, the term "thiolated protein" refers to a protein into which a thiol group has been introduced, such as a protein into which a thiol group has been introduced at a primary amino group using a thiol-adding reagent such as N-succinimidyl S-acetylthioacetate (SATA) or N-succinimidyl 3-(acetylthio)propionate (SATP).

[0021] As used herein, the term "protein having a thiol group" includes not only proteins that themselves contain thiol groups, but also proteins in which thiol groups have been generated, such as proteins in which thiol groups have been generated by cleaving disulfide bonds in the protein with a reducing agent such as dithiothreitol (DTT) or 2-mercaptoethylamine (2-MEA).

[0022] As used herein, "reduced immunogenicity" means that the PEG-modified protein of the present invention exhibits lower immunogenicity compared to the target protein itself or PEG-modified proteins other than the PEG-modified protein of the present invention, particularly PEG-modified proteins obtained by the conventional NHS method. Here, reduced immunogenicity can be determined by a decrease in the ability to produce anti-PEG antibodies, suppression of inflammatory responses, and acute responses such as complement activation and anaphylactic responses.

[0023] In formula (1), P is a protein residue. Target proteins include polypeptides, enzymes, peptides, and the like. Proteins are preferably, but not limited to, those with physiological or pharmacological activity. Furthermore, proteins are preferably immunogenic in themselves or those that become immunogenic through PEG modification.

[0024] Proteins, polypeptides, and peptides of interest include, but are not limited to, serum proteins such as hemoglobin, blood factors such as Factor VII, Factor VIII, and Factor IX; immunoglobulins; cytokines, such as interleukins (IL-1 through IL-13), α-, β-, and γ-interferons; colony-stimulating factors, such as granulocyte colony-stimulating factor, platelet-derived growth factor, and phospholipase-activating protein (PLAP). Other proteins of general biological or therapeutic interest include insulin, plant proteins, such as lectins and ricin, tumor necrosis factor and related proteins, growth factors such as transforming growth factors (TGFα or TGFβ) and epidermal growth factor, hormones, somatomedins, erythropoietin, chromohormones, hypothalamic-releasing factor, antidiuretic hormone, prolactin, chorionic gonadotropin, follicle-stimulating hormone, thyroid-stimulating hormone, tissue plasminogen activator, and the like. Immunoglobulins of interest include IgG, IgE, IgM, IgA, IgD, and fragments thereof.

[0025] Enzymes of interest include carbohydrate-specific enzymes, proteolytic enzymes, oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Specific, non-limiting examples include asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, and bilirubin oxidase, glucose oxidase, glucidase, galactosidase, glucocerebrosidase, glucuronidase, and functional derivatives or fragments thereof.

[0026] In formula (1), A is a group containing a polyethylene glycol residue having a molecular weight of 1,000 to 50,000. In this specification, molecular weight means an average molecular weight. The molecular weight of the polyethylene glycol residue is not particularly limited as long as it exhibits the desired effect on proteins, such as the hydration effect due to PEG modification, but is, for example, 2,000 to 20,000, or even 5,000 to 15,000.

[0027] Examples of groups containing a polyethylene glycol residue include, but are not limited to, the following: [ka] [In the formula, a is the degree of polymerization; J is hydrogen or a capping group. The capping group is selected from the group consisting of -NH2, -SH, -CO2H, C 1-6 Any alkyl group, or other PEG end group (such groups will be understood by those skilled in the art) may be selected, but those that are water soluble are preferred.

[0028] The degree of polymerization (a) represents the number of repeating units in the polyethylene glycol residue, and may be 20-1200, preferably 40-500, and more preferably 100-350, depending on the molecular weight of the polyethylene glycol residue.

[0029] In formula (1), L is an alkylene group having 1 to 10 carbon atoms, which may contain one or more ether bonds and amide bonds in the molecular chain. Examples of alkylene groups having 1 to 10 carbon atoms include, but are not limited to, methylene, ethylene, trimethylene, 1-methyltrimethylene, 2-methyltrimethylene, 2,2-dimethyltrimethylene, tetramethylene, 2-methyltetramethylene, 2,3-dimethyltetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, and decamethylene. Among these, alkylene groups having 1 to 7 carbon atoms are preferred, and alkylene groups having 1 to 5 carbon atoms are more preferred, from the viewpoints of maintaining the desired effects of PEG modification, such as the hydration effect, and ensuring a physical distance between the protein and PEG that is appropriate for reducing binding of PEG-specific antibodies to the PEG-modified protein. Furthermore, the alkylene group having 1 to 10 carbon atoms may contain one or more ether bonds and amide bonds in its molecular chain, and the number and positions of the ether bonds and / or amide bonds in the molecular chain are not particularly limited.

[0030] In formula (1), X is a single bond, a divalent amino acid residue, or a divalent peptide residue consisting of 2 to 100 amino acids. When X is a divalent amino acid residue or a divalent peptide residue consisting of amino acids, the amino acid can be selected from any known natural L-amino acid (e.g., alanine, valine, leucine, isoleucine, glycine, serine, threonine, methionine, cysteine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, proline, and / or combinations thereof). However, from the viewpoints of avoiding interactions with biological substances and ensuring the water solubility of the PEG-modified protein, anionic aspartic acid and glutamic acid are preferred. From the same viewpoint, the divalent peptide residue preferably contains an amino acid selected from anionic aspartic acid and glutamic acid, and more preferably consists of an amino acid selected from aspartic acid and glutamic acid. When the divalent peptide residue contains amino acids other than aspartic acid and / or glutamic acid, the other amino acids are preferably highly water-soluble amino acids such as glycine. Furthermore, the amino acids may be derivatives and analogs of natural amino acids, as well as various unnatural amino acids (D or L) known in the art. When X is a divalent peptide residue, the number of amino acids contained can be appropriately adjusted, particularly in relation to the L group, from the viewpoint of maintaining the desired effects of PEG modification, such as the hydration effect, and ensuring a physical distance between the protein and PEG that is appropriate for reducing binding of PEG-specific antibodies to the PEG-modified protein. For example, the number can be 20 to 100, or even 30 to 60.

[0031] In formula (1), Y is a single bond, or -CH2-CO-NH-* or -CH2CH2-CO-NH-*, where * is the point of attachment to the protein residue. When the target protein is a protein having a thiol group, Y is a single bond, and the sulfur atom adjacent to P in formula (1) is derived from the thiol group. When the target protein is a protein thiolated using N-succinimidyl S-acetylthioacetate (SATA) as a thiol-adding reagent, Y is -CH2-CO-NH-*, and the nitrogen atom adjacent to P in formula (1) is derived from a primary amine of the protein. When the target protein is a protein thiolated using N-succinimidyl 3-(acetylthio)propionate (SATP) as a thiol-adding reagent, Y is -CH2CH2-CO-NH-*, and the nitrogen atom adjacent to P in formula (1) is derived from a primary amine of the protein.

[0032] In formula (1), n corresponds to the number of groups containing polyethylene glycol residues to be introduced into the protein and is 1 to 100. The preferred range of n varies depending on the target protein, and can be appropriately selected by those skilled in the art. For example, when the target protein is uricase, n is preferably 1 to 50, more preferably 1 to 20. When the target protein is asparaginase, n is preferably 1 to 50, more preferably 1 to 20.

[0033] The method for producing a PEG-modified protein and the method for PEG-modifying a protein of the present invention include the steps of: The following formula (2) AX-NH2(2) [In the formula, A and X have the same meanings as above.] The compound represented by the following formula (3): [ka] [In the formula, L has the same meaning as above.] in a solvent to obtain a compound represented by the following formula (4): [ka] [In the formula, A, L, and X have the same meanings as above.] The method includes producing a PEG maleimide derivative represented by

[0034] The above step (a) can be carried out by appropriately adopting general conditions for reacting an amino group with an NHS ester group to form an amide group. Those skilled in the art can appropriately determine the reaction conditions depending on the specific reactants. For example, the reaction can be carried out at room temperature in an aprotic polar solvent such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, or N-methyl-2-pyrrolidone. Compounds represented by formulas (2) and (3) can be prepared by appropriately adopting organic synthesis methods generally known to those skilled in the art, or commercially available products can be used. For example, when X in the compound represented by AX-NH2 of formula (2) is a divalent amino acid residue or a divalent peptide residue, the compound can be synthesized with reference to Yokoyama, M et al., Bioconjugate Chem. 1992, 3, 295-301. In this case, the amino group of the amino acid constituting the amino acid residue or peptide residue can be used as the terminal -NH2 group of the compound represented by AX-NH2 of formula (2). Preferred embodiments of each substituent are as described above for the PEG-modified protein of the present invention.

[0035] The method for producing a PEG-modified protein and the method for PEG-modifying a protein of the present invention include the steps of: An excess amount of a PEG maleimide derivative represented by formula (4) is reacted with a compound represented by formula (5): [ka] [In the formula, P and n have the same meanings as above.] or a protein having a thiol group represented by the following formula (6): [ka] [In the formula, P and n are as defined above, m is 1 or 2.] in a solvent with a thiolated protein represented by formula (1) to obtain a PEG-modified protein represented by formula (1). The obtained PEG-modified protein can be purified by any of known purification methods, either alone or in combination.

[0036] The above step (b) can be carried out by appropriately adopting general conditions for reacting a maleimide group with an SH group of a protein to form a thioether group. Those skilled in the art can appropriately determine the reaction conditions depending on the specific reactants. For example, the solvent used is not particularly limited as long as it is an aqueous solution under general physiological conditions in terms of pH, salt concentration, etc., and those skilled in the art can appropriately select it. The reaction temperature is preferably room temperature. The thiolated protein represented by formula (6) can be synthesized by a known method using a thiol-adding reagent, N-succinimidyl S-acetylthioacetate (SATA) or N-succinimidyl 3-(acetylthio)propionate (SATP), on a primary amine of a protein, such as a primary amine of lysine. Furthermore, the above description of the PEG-modified protein of the present invention is to be cited for preferred embodiments of each substituent.

[0037] The composition of a pharmaceutical composition containing a PEG-modified protein of the present invention is determined by considering several factors, including, but not limited to, the nature of the protein, the concentration of the protein, the desired pH range, how the pharmaceutical composition will be stored (e.g., temperature), the duration of storage of the pharmaceutical composition, and how the formulation will be administered to a patient.

[0038] The pharmaceutical compositions of the present invention are also intended for oral and / or parenteral administration. Examples of dosage forms for oral administration of pharmaceutical compositions containing the PEG-modified proteins of the present invention as an active ingredient include tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules and microcapsules), syrups, emulsions, and suspensions. Examples of dosage forms for parenteral administration of pharmaceutical compositions containing the PEG-modified proteins of the present invention as an active ingredient include injections, infusions, drip infusions, suppositories, liniments, and patches.

[0039] Preparations of the above dosage forms can be prepared according to known manufacturing methods commonly used in the pharmaceutical field. In this case, if necessary, excipients, binders, lubricants, disintegrants, sweeteners, surfactants, suspending agents, emulsifiers, etc. commonly used in the pharmaceutical field can be added.

[0040] Mammals to which the pharmaceutical composition of the present invention can be applied include, for example, primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs), pets (e.g., dogs, cats, rabbits), working animals or livestock (e.g., cows, horses, pigs, sheep, goats), and from the viewpoint of clinical application, humans are preferred. [Example]

[0041] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0042] [1. Preparation of PEG-modified proteins] [Reference Example 1] Preparation of thiolated uricase (corresponding to the compound represented by formula (6)) Thiolated uricase was prepared by conjugation of N-succinimidyl S-acetylthioacetate (SATA) to uricase. Uricase (Merck, trade name Uricase from Candida sp. recombinant, expressed in E. coli) (18.6 mg, 0.572 μmol) was dissolved in 0.1 M PBS (2.33 mL, 8.0 mg / mL), and SATA (2.9 mg, 6.3 μmol) was added and stirred at room temperature for 1 hour. The solution was filtered and washed using a centrifugal ultrafiltration (AmiconUltra4, molecular weight cutoff = 10kJ) to remove excess SATA, and the solution was recovered. 0.5 M NH2OH·HCl (150 μL) was added to the resulting solution, and the solution was stirred at room temperature for 1 hour to perform deacetylation. The solution was filtered and washed using a centrifugal ultrafiltration (AmiconUltra4, molecular weight cutoff = 10kJ) to remove excess NH2OH·HCl, and the solution was recovered. The resulting solution containing thiolated uricase was subjected to quantitative analysis of SH groups in the uricase using Ellman's reagent and cysteine as a standard substance, confirming the introduction of eight SH groups per uricase unit.

[0043] [Reference Example 2] Preparation of PEG (molecular weight 12k)-poly(benzyl L-aspartate) (PEG-PBLA) PEG-PBLA was synthesized as follows. [ka] [wherein a is the number of repeating units in the polyethylene glycol residue, corresponding to a polyethylene glycol residue with a molecular weight of 12,000, and b is 50.] The synthesis was carried out according to the methods described in Shiraishi et al., Journal of Controlled Release, 165 (2013) 183-90 and Yokoyama, M et al., Bioconjugate Chem. 1992, 3, 295-301. Specifically, PEG-PBLA was obtained by ring-opening polymerization of N-carboxyanhydride of β-benzyl-L-aspartate (BLA-NCA) using PEG-NH2 (Yuka Sangyo Co., Ltd., trade name: SUNBRIGHT MEPA-12T) as an initiator in dehydrated DMF at 35-40°C, followed by reprecipitation of the resulting DMF solution into ice-cooled diethyl ether. 58 equivalents of BLA-NCA were used. The resulting white powder was dissolved in CDCl3. 1 The polymerization degree (b) of BLA was determined by the peak ratio of the OCH2CH2 peak of PEG to the CH2 peak of the benzyl group using H NMR (400 MHz). δ / ppm: 2.69 (CH2H), 3.11 (CH2H), 3.38 (terminal -OCH3), 3.64 (PEG OCH2CH2), 4.28 (CH2H), 5.05 (benzyl CH2), 7.26 (PhH), 8.84 (NH).

[0044] [Reference example 3] PEG-P(Asp) 50 Preparation of —NH2 (corresponding to the compound represented by formula (2)) PEG-P(Asp) as shown below 50 -NH2 was synthesized. [ka] [wherein a is the number of repeating units in the polyethylene glycol residue, corresponding to a polyethylene glycol residue with a molecular weight of 12,000, and b is 50.] The synthesis was carried out according to the method described in Yokoyama, M et al., Bioconjugate Chem. 1992, 3, 295-301. Specifically, the PEG-PBLA prepared in Reference Example 2 was dissolved in 0.5N NaOH, deprotected, and neutralized with 6N hydrochloric acid. The solution was dialyzed against water using a dialysis membrane to remove leaving group components, and then freeze-dried. The resulting white powder was dissolved in DO+NaOD. 1 The polymerization degree (b) of P(Asp) was determined by the peak ratio of the OCH2CH2 of PEG to the CH2 of the aspartic acid group using H NMR (400 MHz). δ / ppm: 2.78 (H of CH2), 3.39 (terminal -OCH3), 3.71 (OCH2CH2 of PEG), 4.47, 4.67 (H of CH).

[0045] [Example 1] The following PEG-modified uricases were synthesized: [ka] [wherein a is the number of repeating units in the polyethylene glycol residue, corresponding to a polyethylene glycol residue with a molecular weight of 12,000, and P is a uricase residue.]

[0046] (a) Synthesis of PEG maleimide derivative (corresponding to the compound represented by formula (4)) The chemical structure of the spacer molecule used (corresponding to the compound represented by formula (3)) is shown below. [ka]

[0047] PEG-NH2 (Yuka Sangyo Co., Ltd., trade name: SUNBRIGHT MEPA-12T) (200.0 mg, 0.0167 mmol) (corresponding to the compound represented by formula (2)) was mixed with 50 equivalents of spacer molecule 1 (Tokyo Chemical Industry Co., Ltd.) (188.9 mg, 0.84 mmol) in dehydrated DMF (5.0 mL) and stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was transferred to a dialysis membrane (SpectraPor6, molecular weight cutoff = 1,000) and dialyzed by solvent exchange against DMSO three times and against methanol three times. After dialysis, the methanol solution was recovered, the methanol was concentrated, and reprecipitation from the methanol solution into diethyl ether at 0 °C was carried out. The white precipitate was recovered using a Kiriyama funnel (5C) and dried under reduced pressure (yield: 166.8 mg). The spacer introduction rate for PEG was determined by the addition of 100% of the spacer to the PEG in CDCl3. 1 The resulting white powder was identified by H NMR and confirmed to have an incorporation rate of 85% or more. 1 H NMR (400 MHz) was measured. The introduction rate was determined from the peak ratio of the terminal OCH3 of PEG to the 2CH of the maleimide group. δ / ppm: 2.29 (CH2), 3.21 (terminal -OCH3), 3.49 (PEG OCH2CH2), 6.93 (2H of CH).

[0048] (b) Synthesis of PEG-modified uricase (corresponding to the PEG-modified protein represented by formula (1)) by reaction of PEG maleimide derivative with thiolated uricase The solution containing the thiolated uricase prepared in Reference Example 1 was reacted with an excess amount of PEG maleimide derivative in Dulbecco's phosphate-buffered saline (D-PBS) (600 μL) for 2 hours. The reaction solution was subjected to centrifugal ultrafiltration using an AmiconUltra4 (molecular weight cutoff = 100kJ). The reaction solution was then washed with D-PBS by HPLC (Toso G4000PWXL, eluent = D-PBS, flow rate = 1.0 mL / min, detection = RI / UV @ 280 nm) until the excess amount of PEG maleimide derivative (retention time = 9.9 min) disappeared. The solution was then sterilized by filtration through a 0.45 μm PVDF filter to obtain PEG-modified uricase. The amount of SH groups was quantified using the same method as in Reference Example 1, confirming the absence of SH groups. A portion of the resulting PEG-modified uricase solution was freeze-dried, and the solution concentration was determined from the weight of the resulting white solid. The resulting white solid was dissolved in DO and sterilized. 1 H NMR (400 MHz) was measured. A calibration curve in DO was prepared using PEG of the same molecular weight as used as the initiator in Reference Example 2 as the standard, and the PEG concentration in the PEG-modified uricase solution was determined. δ / ppm: 3.36 (terminal -OCH3), 3.72 (PEG OCH2CH2). The molecular weight of the PEG-modified uricase solution was determined by GPC in aqueous solution using PEG as the standard. The GPC elution curve showed a single peak, without peaks corresponding to the raw uricase (molecular weight 35,000) or PEG (molecular weight 12,000). The number-average molecular weight of the PEG-modified uricase solution was 181,000, and the weight-average molecular weight was 238,000. A calibration curve was prepared at the uricase absorption wavelength of 280 nm, and the uricase concentration in the PEG-modified uricase solution was determined. From these results, the number of PEGs modified per uricase tetramer, n, was calculated to be 7.4 from the PEG concentration and uricase concentration in the solution.

[0049] [Example 2] The following PEG-modified uricase was synthesized in the same manner as in Example 1, except that spacer molecule 2 was used instead of spacer molecule 1. [ka] [wherein a is the number of repeating units in the polyethylene glycol residue, corresponding to a polyethylene glycol residue with a molecular weight of 12,000, and P is a uricase residue.] [ka]

[0050] PEG-NH2 (Yuka Sangyo Co., Ltd., trade name: SUNBRIGHT MEPA-12T) (330 mg, 0.0276 mmol) (corresponding to the compound represented by formula (2)) was mixed with 33 equivalents of spacer molecule 2 (Tokyo Chemical Industry Co., Ltd.) (234.5 mg, 0.93 mmol) in dehydrated DMF (5.5 mL) and stirred at room temperature for 2.5 hours. After completion of the reaction, purification was carried out in the same manner as in Example 1 (yield: 302.2 mg). The spacer introduction rate for PEG was 1 The product was identified by H NMR (400 MHz), and the introduction rate was determined from the peak ratio of the terminal OCH3 of PEG to the 2CH of the maleimide group. δ / ppm: 2.29 (CH2), 3.38 (terminal -OCH3), 3.65 (OCH2CH2 of PEG), 6.77 (2H of CH). The reaction with thiolated uricase was carried out in the same manner as in Example 1. A portion of the obtained PEG-modified uricase solution was freeze-dried, and the solution concentration was calculated from the weight of the resulting white solid. The obtained white solid was dissolved in D2O and 1 H NMR (400 MHz) was measured. A calibration curve in DO was prepared using PEG of the same molecular weight as used as the initiator in Reference Example 2 as a standard substance, and the PEG concentration in the PEG-modified uricase solution was determined. δ / ppm: 3.39 (terminal -OCH), 3.71 (PEG OCHCH). A calibration curve was prepared at the uricase absorption wavelength of 280 nm to determine the uricase concentration in the PEG-modified uricase solution. From these data, the number of PEGs (n) modified per uricase tetramer was calculated to be 7.5.

[0051] [Example 3] The following PEG-modified uricase was obtained in the same manner as in Example 1, except that spacer molecule 3 was used instead of spacer molecule 1. [ka] [wherein a is the number of repeating units in the polyethylene glycol residue, corresponding to a polyethylene glycol residue with a molecular weight of 12,000, and P is a uricase residue.] [ka]

[0052] PEG-NH2 (Yuka Sangyo Co., Ltd., trade name: SUNBRIGHT MEPA-12T) (470 mg, 0.0392 mmol) (corresponding to the compound represented by formula (2)) was mixed with 33 equivalents of spacer molecule 3 (Tokyo Chemical Industry Co., Ltd.) (361.2 mg, 1.29 mmol) in dehydrated DMF (7.8 mL) and stirred at room temperature for 2.5 hours. After completion of the reaction, purification was carried out in the same manner as in Example 1 (yield: 411.4 mg). The spacer introduction rate for PEG was 1 The product was identified by H NMR (400 MHz), and the introduction rate was determined from the peak ratio of the terminal OCH3 peak of PEG to the 2CH peak of the maleimide group, confirming that it was 90% or more. δ / ppm: 1.78 (CH2), 2.15 (CH2), 3.35 (CH2), 3.38 (terminal -OCH3), 3.65 (OCH2CH2 of PEG), 6.71 (2H of CH). The reaction with thiolated uricase was carried out in the same manner as in Example 1. A portion of the obtained PEG-modified uricase solution was freeze-dried, and the solution concentration was calculated from the weight of the resulting white solid. The obtained white solid was dissolved in D2O and 1H NMR (400 MHz) was measured. A calibration curve in DO was prepared using PEG of the same molecular weight as used as the initiator in Reference Example 2 as a standard substance, and the PEG concentration in the PEG-modified uricase solution was determined. δ / ppm: 3.39 (terminal -OCH), 3.71 (PEG OCHCH). A calibration curve was prepared at the uricase absorption wavelength of 280 nm, and the uricase concentration in the PEG-modified uricase solution was determined. From these data, the number of PEGs (n) modified per uricase tetramer was calculated to be 7.5.

[0053] [Example 4] PEG-P(Asp) prepared in Reference Example 3 instead of PEG-NH 50 -NH2 to form PEG-P(Asp) before reacting with spacer molecule 1. 50 PEG-modified uricase was obtained in the same manner as in Example 1, except that NEt3 was added to -NH2 in dehydrated DMF. [ka] [wherein a is the number of repeating units in the polyethylene glycol residue, corresponding to a polyethylene glycol residue with a molecular weight of 12,000, b is 50, and P is a uricase residue.]

[0054] PEG-P(Asp) represented by Reference Example 3 50 To a solution of -NH2 (709 mg, 0.040 mmol) in anhydrous DMF (9.2 mL), 3.5 mL (2.0 mmol) of a separately prepared triethylamine solution in anhydrous DMF (0.586 M) was added, and spacer molecule 1 (Tokyo Chemical Industry Co., Ltd.) (911.5 mg, 4.03 mmol) was added and mixed, followed by stirring at room temperature for 3 hours. After completion of the reaction, purification was carried out in the same manner as in Example 1 (yield: 687.9 mg). The spacer introduction rate for PEG was 1.0% in DMSO-d6. 1The product was identified by H NMR (400 MHz), and the introduction rate was determined from the peak ratio of the terminal OCH3 peak of PEG to the 2CH peak of the maleimide group. δ / ppm: 3.19 (terminal -OCH3), 3.48 (OCH2CH2 of PEG), 6.85 (2H of CH). The reaction with thiolated uricase was carried out in the same manner as in Example 1. A portion of the obtained PEG-modified uricase solution was freeze-dried, and the solution concentration was calculated from the weight of the obtained white solid. The obtained white solid was dissolved in D2O and 1 H NMR (400 MHz) was measured. A calibration curve in DO was prepared using PEG of the same molecular weight as used as the initiator in Reference Example 2 as a standard substance, and the PEG concentration in the PEG-modified uricase solution was determined. δ / ppm: 3.39 (terminal -OCH), 3.71 (PEG OCHCH). A calibration curve was prepared at the uricase absorption wavelength of 280 nm to determine the uricase concentration in the PEG-modified uricase solution. From these data, the number of PEGs (n) modified per uricase tetramer was calculated to be 6.2.

[0055] [Comparative Example 1] The following PEG-modified uricase was obtained by the conventional PEGylation method, the NHS method. [ka] where a is the number of repeating units in the polyethylene glycol residue and P is the uricase residue. Uricase (Merck, trade name: Uricase from Candida sp. recombinant, expressed in E. coli) was dissolved in HEPES buffer (pH = 8.5) and reacted with PEG-NHS (Yuka Sangyo Co., Ltd., trade name: SUNBRIGHT ME-100AS) at 0°C for 3 hours. After the reaction was completed, the reaction solution was centrifuged using an AmiconUltra 4 (molecular weight cutoff = 100kJ). The solution was then washed with D-PBS using HPLC (Toso G4000PWXL, eluent = D-PBS, flow rate = 1.0 mL / min, detection = RI / UV @ 280 nm) until the excess PEG-NHS disappeared. The PEG-modified uricase was then sterilized through a 0.45 μm PVDF filter. A portion of the resulting PEG-NHS-modified uricase solution was lyophilized, and the solution concentration was determined from the weight of the resulting white solid. The resulting white solid was dissolved in DO and washed with D-PBS until the excess PEG-NHS disappeared. 1 H NMR (400 MHz) was measured. The PEG concentration in the PEG-modified uricase solution was determined using the same method as in Example 1(b). δ / ppm: 3.36 (terminal -OCH3), 3.72 (PEG OCH2CH2). The GPC elution curve showed a single peak, and the number-average molecular weight of the PEG-modified uricase solution was 205,000 and the weight-average molecular weight was 253,000. A calibration curve was prepared at the uricase absorption wavelength of 280 nm to determine the uricase concentration in the PEG-modified uricase solution. The number of PEGs (n) modified onto the uricase was calculated from the PEG and uricase concentrations in the solution. Next, the molecular weight of the PEG-modified uricase solution was determined by GPC in aqueous solution using PEG as a standard.

[0056] 2. Identification of PEG-modified uricase The results of identifying the PEG-modified uricases prepared in Examples 1 to 4 and Comparative Example 1 are shown below. [Table 1]

[0057] [3. Evaluation of anti-PEG IgM antibody production using PEG-modified uricase] 3.1 Experimental Procedure The production of anti-PEG IgM antibodies was evaluated in animal experiments for the PEG-modified uricases prepared in Examples 1 to 4 and Comparative Example 1. The animal experiments and binding evaluation by ELISA were carried out according to the following procedures.

[0058] (1) Animal experiments Each uricase solution was administered at 2 units / kg (1.0 mg uricase / kg) via the tail vein of C57BL / 6 mice (6 weeks old, male) (n=3). One week later, the tail vein of each mouse was slightly incised, and 35-45 μL of blood was collected using a blood collection tube. The collected blood was centrifuged to recover serum. PEG-PBLA (Reference Example 2) was used as a positive control and administered at 0.04 μmol PEG / kg.

[0059] (2) Binding evaluation by ELISA (2-1) For the PEG-coated plate, 100 μL of a 1 / 1 solution of PEG (molecular weight 12 kJ)-poly(benzyl L-aspartate) (PEG-PBLA) in EtOH / HO was added to the Nunc Maxisorp plate, and the plate was allowed to bond overnight at 4°C. (2-2) The PEG-coated plate was washed three times with a washing solution (50 mM Tris-buffered saline, pH 8.0, 0.05% Tween 20), and 100 L of a blocking solution (50 mM Tris-buffered saline, pH 8.0, 1% BSA) was added to the plate for blocking for 1 hour. The plate was then washed with the washing solution. (2-3) A solution diluted with 100 μL of saline was added to 1 μL of serum, allowed to bind to the PEG-coated plate for 1 hour, and then washed with a washing solution. (2-4) 0.010 μg / mL anti-mouse IgM (HRP conjugate) was used as a detection antibody, and the antibody was allowed to bind for 1 hour and then washed with a washing solution. (2-5) 100 μL of TMB solution was added, and the reaction was stopped after 15 minutes with 0.36 M H2SO4 solution, and the absorbance at 450 nm was detected using a plate reader.

[0060] [3.2 Results] The results for Examples 1 to 3 and Comparative Example 1 are shown in Figure 1. Physiological saline was used as a control in Figure 1. Figure 1 shows that the PEG-modified uricases of Examples 1 to 3 can reduce anti-PEG IgM production and have reduced immunogenicity compared to the PEG-modified uricase of Comparative Example 1 prepared by the conventional NHS method.

[0061] The results for Examples 1 and 4 and Comparative Example 1 are shown in Figure 2. PEG-PBLA in Figure 2 was used as a positive control. Figure 2 shows that the PEG-modified uricases of Examples 1 and 4 were able to reduce anti-PEG IgM production and thus had reduced immunogenicity compared to the PEG-modified uricase of Comparative Example 1, which was prepared by the conventional NHS method. [Industrial Applicability]

[0062] The PEG-modified protein or PEG-modified hydrophobic substance of the present invention can provide a PEGylated pharmaceutical product with reduced immunogenicity compared to conventional products.

Claims

1. The following formula (1): 【Chemical 1】 [In the formula, P is a protein residue, A is a group containing a polyethylene glycol residue having a molecular weight of 1,000 to 50,000, L is an alkylene group having 1 to 10 carbon atoms which may contain one or more bonds selected from an ether bond and an amide bond in the molecular chain, X is a single bond, a divalent amino acid residue consisting of an amino acid selected from aspartic acid, glutamic acid, glycine, serine, and threonine, or a divalent peptide residue consisting of 2 to 100 amino acids selected from aspartic acid, glutamic acid, glycine, serine, and threonine; Y is a single bond or -CH 2 -CO-NH-* or -CH 2 CH 2 -CO-NH-*, * denotes the point of attachment to the protein residue, n is 1 to 100. A PEG-modified protein represented by the formula:

2. The PEG-modified protein according to claim 1, wherein L is an alkylene group having 1 to 10 carbon atoms.

3. The PEG-modified protein according to claim 1 or 2, wherein X is a single bond.

4. The PEG-modified protein according to claim 1 or 2, wherein X is a divalent peptide residue, and the divalent peptide residue consists of 20 to 100 amino acids.

5. The divalent peptide residue contains an amino acid selected from aspartic acid and glutamic acid. The PEG-modified protein according to claim 4.

6. The following formula (1): 【Chemistry 2】 [In the formula, P is a protein residue, A is a group containing a polyethylene glycol residue having a molecular weight of 1,000 to 50,000, L is an alkylene group having 1 to 10 carbon atoms which may contain one or more bonds selected from an ether bond and an amide bond in the molecular chain, X is a single bond, a divalent amino acid residue consisting of an amino acid selected from aspartic acid, glutamic acid, glycine, serine, and threonine, or a divalent peptide residue consisting of 2 to 100 amino acids selected from aspartic acid, glutamic acid, glycine, serine, and threonine; Y is a single bond or -CH 2 -CO-NH-* or -CH 2 CH 2 -CO-NH-*, * denotes the point of attachment to the protein residue, n is 1 to 100. A method for producing a PEG-modified protein represented by the formula: (a) The following formula (2) A-X-NH 2 (2) [In the formula, A and X have the same meanings as above.] The compound represented by the following formula (3): 【Chemistry 3】 [In the formula, L has the same meaning as above.] in a solvent to obtain a compound represented by the following formula (4): 【Chemistry 4】 [In the formula, A, L, and X have the same meanings as above.] forming a PEG maleimide derivative represented by (b) Adding an excess amount of a PEG maleimide derivative represented by formula (4) to a compound represented by formula (5): 【Chemistry 5】 [In the formula, P and n have the same meanings as above.] or a protein having a thiol group represented by the following formula (6): 【Chemistry 6】 wherein P and n are as defined above, m is 1 or 2. In a solvent, the thiolated protein represented by the formula (1) is reacted with the thiolated protein represented by the formula (1) to form a PEG-modified protein represented by the formula (1). The above method, further comprising the step of obtaining the protein.

7. A method for PEG-modifying a protein, comprising steps (a) and (b) according to claim 6.

8. A pharmaceutical composition comprising the PEG-modified protein according to any one of claims 1 to 5.

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