Development of a novel DDS targeting lysosomes

Abstract

To provide a hydrolytic enzyme for use in enzyme replacement therapy for lysosomal disease.SOLUTION: The hydrolytic enzyme is one present in lysosomes that cause lysosomal storage diseases due to deficiency and comprises at least one sugar chain having a bifurcated sugar chain with sialic acid at the end.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a lysosome-targeted drug delivery system (DDS), specifically, a technology for delivering an enzyme containing a sugar chain with sialic acid at its terminal to the lysosome via binding to a sialic acid receptor on the cell surface.

[0002] The present invention also relates to a hydrolase whose deficiency causes a lysosomal disease, the hydrolase comprising at least one sugar chain having a biantennary sugar chain with sialic acid attached to its terminal, and a therapeutic agent for a lysosomal disease comprising the hydrolase. [Background technology]

[0003] Lysosomes are intracellular organelles that break down biopolymers such as proteins, sugars, and lipids. Approximately 60 types of hydrolytic enzymes are involved in lysosomes, breaking down biopolymers that have been taken up into the lysosomes by endocytosis or autophagy. Congenital metabolic disorders in which the lysosomal degradative function is lost due to a deficiency or abnormality of these enzymes, causing substances that should be broken down to accumulate in the body as waste products, are called lysosomal storage diseases. There are approximately 50 types of diseases depending on the enzyme that is deficient. Many of these are "incurable neurological diseases" that cause neurological symptoms.

[0004] Enzyme replacement therapy, which involves taking up a dysfunctional enzyme into cells, is used to treat lysosomal diseases (see Non-Patent Document 1). Many lysosomal hydrolases undergo post-translational glycosylation, resulting in the addition of mannose (Man) and the addition of a phosphate group to mannose to form mannose 6-phosphate (M6P), and have a sugar chain structure with terminal mannose (Man) or mannose 6-phosphate (M6P). In enzyme replacement therapy, the enzyme is taken up into cells by utilizing the binding of the terminal mannose or mannose 6-phosphate-containing sugar chain of the enzyme to the mannose receptor (MR) or mannose 6-phosphate receptor (M6PR) on the cell surface.

[0005] Currently, recombinant enzyme preparations are being used clinically for 10 types of lysosomal storage diseases. Enzyme preparations are expensive to manufacture, and large amounts of enzyme preparations must be administered to allow them to be taken up into cells. For example, recombinant enzyme preparations are administered intravenously at doses of 1-20 mg / kg body weight every 1-2 weeks.

[0006] It is known that glycoproteins with desired properties can be produced by replacing the sugar chains of proteins. To replace the sugar chains that can produce such glycoproteins (see Patent Document 1), it is necessary to cleave the original sugar chains and attach sugar chains from other proteins. However, an endoglycosidase has been developed that can replace sugar chains in just one step (one-pot reaction) (see Patent Document 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-80453 [Patent Document 2] Japanese Patent Publication No. 2020-10662 [Non-patent literature]

[0008] [Non-Patent Document 1] Wraith JM et al., PEDIATRICS, Volume 120, Number 1, July 2007, e37-e46 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a hydrolase for use in enzyme replacement therapy for lysosomal diseases. [Means for solving the problem]

[0010] The present inventors have conducted extensive research into a method for producing, at low cost, an enzyme that can be used in enzyme replacement therapy for lysosomal diseases and that is taken up into lysosomes more efficiently than conventional enzymes.

[0011] The present inventors have discovered that a hydrolase present in lysosomes, the deficiency of which causes lysosomal storage diseases, and which contains at least one sugar chain having a biantennary sugar chain with sialic acid attached to its terminal, is efficiently taken up into lysosomes in cells via sialic acid receptors on the cell surface, and that the hydrolase taken up into lysosomes can degrade high molecular weight compounds in lysosomes, preventing the accumulation of waste products and allowing it to be used as a therapeutic agent for lysosomal storage diseases, thereby completing the present invention.

[0012] That is, the present invention is as follows. [1] A hydrolase present in lysosomes whose deficiency causes lysosomal storage diseases, and which contains at least one sugar chain with a biantennary sugar chain attached with sialic acid at the end. [2] A hydrolase present in lysosomes whose deficiency causes lysosomal storage diseases, in which at least one of the original sugar chains with terminal mannose has been replaced with a sugar chain with terminal sialic acid, i.e., a hydrolase containing at least one sugar chain with a biantennary sugar chain with terminal sialic acid as in [1]. [3] The hydrolase of [1] or [2], which is α-L-iduronidase (IDUA) or cathepsin A (CTSA). [4] A drug for treating lysosomal diseases containing the hydrolase of [1] or [2] as an active ingredient. [5] A lysosomal disease treatment drug [4] containing the hydrolase of [3] as an active ingredient, in which the lysosomal disease is mucopolysaccharidosis type I. [6] A therapeutic agent for lysosomal diseases according to [4] or [5], which is taken up into lysosomes via binding between sialic acid contained in the terminal sugar chain and sialic acid receptors on the cell surface. [7] A method for producing a hydrolase present in lysosomes, the deficiency of which causes a lysosomal disease, comprising at least one sugar chain having a biantennary sugar chain with a terminal sialic acid, selected from the following (i) to (vi): (i) A gene encoding a hydrolase whose deficiency causes a lysosomal disease is introduced into mammalian cells and expressed in the mammalian cells; (ii) A gene encoding a hydrolase whose deficiency causes a lysosomal disease is expressed in insect cells or in silkworms themselves, into which a sialyltransferase gene has been introduced; (iii) Expression in yeast that has been genetically engineered to have human-type glycans attached to the expressed protein; (iv) glycosylation of peptides expressed in prokaryotic microorganisms via disulfide bonds, followed by peptide ligation; (v) adding a sugar having a biantennary sugar chain with a terminal sialic acid to the primary amine of a lysine residue of a protein expressed in a prokaryotic microorganism to the primary amine of the lysine residue of the protein by using a sugar oxazoline having a biantennary sugar chain with a terminal sialic acid as a sugar donor and the protein as a sugar acceptor; or (vi) Proteins are prepared by peptide synthesis using asparagine having a biantennary glycan with terminal sialic acid as a starting material. [8] A method for producing a hydrolase containing at least one glycan having a biantennary glycan with a terminal sialic acid by mixing and reacting a hydrolase whose deficiency causes a lysosomal disease, a glycoprotein or glycopeptide that serves as a donor of a glycan having a terminal sialic acid, and an endoglycosidase. [9] The method of [8], wherein the endoglycosidase is selected from the group consisting of Endo-M, Endo-M D175Q, Endo-A, Endo-S, Endo-S D233Q, Endo-CC, Endo-SB, Endo-CoM, Endo-CE, Endo-HS, Endo-Tsp1006, Endo-Tsp1263, and Endo-Tsp1457, as well as Endo-CC N180H, Endo-CC N180A, Endo-CC N180D, and Endo-CC N180Q, in which the asparagine at position 180 of the endoglycosidase has been replaced with another amino acid.

[10] The method of [8] or [9], wherein the hydrolase is α-L-iduronidase (IDUA) or cathepsin A (CTSA).

[11] A method for delivering a hydrolase present in lysosomes, the deficiency of which causes a lysosomal disease, and which contains at least one glycan having a biantennary glycan with sialic acid attached at its end, to a lysosome, comprising taking up the hydrolase into the lysosome via binding between the sialic acid at the end of the glycan and a sialic acid receptor on the cell surface.

[12] The method of

[11] , wherein the hydrolase is α-L-iduronidase (IDUA) or cathepsin A (CTSA). [Effects of the Invention]

[0013] A biantennary terminal sialic acid-containing hydrolase obtained in a one-pot reaction system containing an enzyme that hydrolyzes chitobiose bonds and simultaneously catalyzes the transfer reaction of sugar chains, a glycoprotein or glycopeptide that serves as a donor of sugar chains containing terminal sialic acid, and an acceptor glycoprotein whose deficiency causes lysosomal storage diseases is taken up by cells of patients with lysosomal storage diseases in a sialic acid residue-dependent manner, transported to lysosomes, and can treat lysosomal storage diseases by restoring the deficient hydrolase activity. This hydrolase is taken up into lysosomes more efficiently than conventional enzymes. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows an outline of a one-pot method for replacing at least one of the sugar chains of IDUA with a sugar chain having a terminal sialic acid. [Figure 2] FIG. 1 shows the effect of enzyme supplementation on MSPSI-jp. [Figure 3] FIG. 1 shows the competitive inhibitory effect of SG-IDUA on the intracellular uptake of MPSI-jp. [Figure 4] FIG. 1 shows the effect of SG-IDUA on the intracellular uptake of F17. [Figure 5] FIG. 5 shows the results of CBB staining (FIG. 5A), SSA lectin blotting (FIG. 5B), and Con A lectin blotting (FIG. 5C) in the case of cathepsin A (CTSA) sugar chain replacement by Endo-CC (N180H). [Figure 6] FIG. 1 shows the results of SSA lectin blotting when the N-glycan of either the 32 kDa or 20 kDa domain of SG-CTSA was replaced. [Figure 7] FIG. 1 shows the results of CBB staining when the N-glycan of either the 32 kDa or 20 kDa domain of SG-CTSA was replaced. [Figure 8] FIG. 1 shows the administration site of cocoon-derived CTSA precursor (proCTSA) and α2,6-SG-CTSA in GS model mice when administered intracerebroventricularly. [Figure 9] FIG. 9A shows the Ctsa specific activity (FIG. 9A) and Neu specific activity (FIG. 9B) after intracerebroventricular administration of cocoon-derived CTSA precursor (proCTSA) and α2,6-SG-CTSA to GS model mice. [Figure 10] 10A, 10B, and 10C show the reduction of accumulated sialyl substrates in the liver (FIG. 10A), spleen (FIG. 10B), and heart (FIG. 10C) of mice administered cocoon-derived CTSA precursor (proCTSA). [Figure 11] FIG. 11A shows a decrease in accumulated sialyl substrates in the kidneys (FIG. 11A) and lungs (FIG. 11B) of mice administered cocoon-derived CTSA precursor (proCTSA). [Figure 12]FIG. 1 shows the inhibitory effect of cocoon-derived CTSA precursor (proCTSA) on urinary excretion of sialyl substrates in mice administered with the precursor. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below. The present invention relates to a lysosome-targeted DDS (drug delivery system). The DDS of the present invention enables enzyme replacement therapy by incorporating the enzyme that causes lysosomal storage disease due to its deficiency into the lysosomes in the cells of patients with lysosomal storage disease.

[0016] The enzymes whose deficiency causes lysosomal storage diseases are hydrolases (lysosomal enzymes) present in lysosomes, which hydrolyze biopolymers such as proteins, sugars, and lipids that have been taken into lysosomes by endocytosis or autophagy. Deficiency or abnormality of these enzymes results in the biopolymers in lysosomes not being broken down and accumulating as waste products, leading to the onset of lysosomal storage diseases.

[0017] Examples of enzymes whose deficiency causes lysosomal storage diseases include the following enzymes. The lysosomal storage disease caused by the deficiency of that enzyme is indicated in parentheses. These enzymes are only examples, and the enzymes that cause lysosomal storage diseases in the present invention are not limited to these enzymes.

[0018] α-L-iduronidase (mucopolysaccharidosis type I), iduronate-2-sulfidase (mucopolysaccharidosis type II, Hunter syndrome), heparan-N-sulfatase (mucopolysaccharidosis type III(A)), α-N-acetylglucosaminidase (mucopolysaccharidosis type III(B)), acetyl-CoA α-glucosaminide-N-acetyltransferase (mucopolysaccharidosis type III(C)), N-acetylglucosamine-6-sulfatase (mucopolysaccharidosis type III(D)), N-acetylgalactosamine-6-sulfate sulfatase (mucopolysaccharidosis type IV(A)), β-galactosidase (mucopolysaccharidosis type IV(B), GM1-gangliosidosis), N-acetylgalactosamine-4-sulfatase (mucopolysaccharidosis type VI), β-glucuronidase (mucopolysaccharidosis type VII), hyaluronidase (mucopolysaccharidosis type IX), β-glucocerebrosidase (Gaucher disease), α-galactosidase (Fabry disease), acid sphingomyelinase (Niemann-Pick disease types A and B), acid ceramidase (Farber disease), β-hexosaminidase A, B (GM2-gangliosidosis), arylsulfatase A, B, C (metachromatic leukodystrophy, multiple sulfatase deficiency), β-galactocerebrosidase (Krabbe disease), acid α-glucosidase (Pompe disease), cathepsin A (CTSA) (galactosialidosis), neuraminidase 1 (sialidosis), α-mannosidase (α-mannosidosis), β-mannosidase (β-mannosidosis), fucosidase (fucosidosis), aspartylglucosaminidase (aspathylglucosaminuria), prosaposin (prosaposin deficiency), saposin A (saposin A deficiency), saposin Saposin B (saposin B deficiency), saposin C (saposin C deficiency), α-N-acetylglucosaminidase (Schindler-Kanzaki disease), acid lipase (Wolman disease), cathepsin K (cathepsin K deficiency), palmitoyl protein thioesterase (neuronal ceroid lipofuscinosis type 1), tripeptidyl peptidase 1 (neuronal ceroid lipofuscinosis type 2), CLN5 protein (neuronal ceroid lipofuscinosis type 5), cathepsin D (neuronal ceroid lipofuscinosis type 10), acid phosphatase (acid phosphatase deficiency).The substances that accumulate in lysosomes in each lysosomal storage disease are mucopolysaccharides in mucopolysaccharidoses; for example, dermatan sulfate and heparan sulfate accumulate in mucopolysaccharidoses types I and II, heparan sulfate accumulates in mucopolysaccharidoses type III, keratan sulfate accumulates in mucopolysaccharidoses type IV, and dermatan sulfate accumulates in mucopolysaccharidoses type VI.

[0019] In the present invention, a hydrolase whose deficiency causes a lysosomal disease and which contains at least one sugar chain having a biantennary sugar chain with sialic acid attached to its terminal is used.

[0020] These enzymes are originally post-translationally glycosylated, with mannose (Man) and phosphate groups added to mannose to form mannose 6-phosphate (M6P), resulting in a sugar chain structure with terminal mannose (Man) or mannose 6-phosphate (M6P). These enzymes are taken up into intracellular endosomes via the mannose receptor (MR) or mannose 6-phosphate receptor (M6PR), and then fuse with lysosomes, where they are taken up into lysosomes and exert their hydrolytic activity.

[0021] A hydrolase whose deficiency causes a lysosomal disease and which contains at least one sugar chain having a biantennary sugar chain with sialic acid attached to its terminal can be produced, for example, by the following method.

[0022] (i) Expression is performed using mammalian cells capable of producing recombinant proteins bearing biantennary glycans with terminal sialic acid. A gene encoding a hydrolase whose deficiency causes a lysosomal disease is cloned, incorporated into an expression vector, and then introduced into host mammalian cells. The cells are then cultured to obtain a hydrolase bearing a biantennary glycan with terminal sialic acid. Known vectors can be used to construct the expression vector. Examples include Flexi (registered trademark) vector (Promega), pUC19, pTV118N (Takara Shuzo), pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), and pMAM-neo (Clontech). The expression vector can be introduced into host cells by known methods, which can then transform the host cells. Examples include electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. Such mammalian cells include human embryonic kidney cell lines HEK293 cells or HEK293T cells, Chinese hamster ovary (CHO) cells, and monkey COS cells.

[0023] (ii) A sialyltransferase gene is introduced into insect cells such as silkworm cells or into silkworms themselves by genetic recombination, and then expressed in an expression system using the sialyltransferase-introduced insect cells or silkworms. Lepidopteran insect cells such as silkworms, such as Sf21 cells, Sf9 cells, and TN5 cells, can be used as insect cells. For example, a gene encoding a hydrolase whose deficiency causes lysosomal storage disease can be incorporated into a baculovirus vector, and the vector can be introduced into insect cells. The sialyltransferase gene can be introduced into cells and silkworm bodies by known methods. Production using silkworm bodies can be carried out by known methods. These methods can be carried out according to the description of Japanese Patent No. 3,598,374.

[0024] (iii) A microorganism belonging to the fungus family, such as yeast, capable of expressing sugar chains is expressed in an expression system in which a human-type sugar chain is attached to the expressed protein by genetic engineering. That is, a cytidine monophosphate-sialic acid (CMP-Sia) synthesis pathway is created in a fungus, such as yeast, lacking CMP-Sia by genetic engineering techniques, allowing CMP-Sia to be synthesized. The CMP-Sia synthesis pathway can be achieved, for example, by introducing at least one enzyme selected from mammalian-derived UDP-GlcNAc epimerase, sialic acid synthase, CMP-sialic acid synthase, UDP-N-acetylglucosamine-2-epimerase, N-acetylmannosamine kinase, N-acetylneuraminate-9-phosphate synthase, N-acetylneuraminate-9-phosphatase, and CMP-sialic acid synthase into a fungus, such as yeast.

[0025] Examples of yeast include yeasts of the genus Pichia, Candida, Hansenula, Saccharomyces, Kluyveromyces, etc. Other eukaryotic organisms include koji molds of the genus Aspergillus, ascomycetes of the genus Trichoderma, fungi of the genus Chrysosporium, fungi of the genus Fusarium, and ascomycetes of the genus Neurospora.

[0026] Yeasts of the genus Pichia include Pichia pastoris, Pichia methanolica, and Pichia burtonii; yeasts of the genus Candida include Candida ulitis, Candida boidinii, and Candida mycoderma; yeasts of the genus Hansenula include Hansenula polymorpha, Hansenula anomala, and Hansenula capsulata; and yeasts of the genus Saccharomyces include Saccharomyces cerevisiae. cerevisiae, and examples of yeasts of the genus Kluyveromyces include Kluyveromyces lactis. Other examples of Aspergillus koji molds include Aspergillus niger and Aspergillus oryzae, Trichoderma ascomycetes include Trichoderma reesei, Chrysosporium fungi include Chrysosporium lucknowense, Fusarium fungi include Fusarium sp., and Neurospora ascomycetes include Neurospora crassa. These methods can be carried out according to the description in Japanese Patent No. 4932699.

[0027] (iv) Proteins are produced by adding sugar chains to peptides expressed in prokaryotic microorganisms such as Escherichia coli, which do not produce glycosylated proteins, using disulfide bonds, and then linking the peptides. Examples of prokaryotic microorganisms include Escherichia coli, Bacillus subtilis, filamentous fungi, Aspergillus oryzae, and actinomycetes. These methods can be carried out as described in N. Yamamoto et al., Tetrahedron Lett, (2004), 45(16), 3287-3290.

[0028] (v) A sugar oxazoline having a biantennary sugar chain with a terminal sialic acid is used as a sugar donor for a protein expressed in a prokaryotic microorganism, and the protein is used as a sugar acceptor, and a sugar having a biantennary sugar chain with a terminal sialic acid is added to the primary amine of a lysine residue of the protein. Examples of prokaryotic microorganisms include Escherichia coli, Bacillus subtilis, filamentous fungi, Aspergillus oryzae, and actinomycetes. These methods can be carried out according to the description of Japanese Patent No. 6342968.

[0029] (vi) Proteins are prepared by peptide synthesis using asparagine having a biantennary glycan with a terminal sialic acid as a starting material. These methods can be carried out according to the method described in N. Yamamoto et al., J. Am. Chem. Soc., (2008), 130(2), 501-510.

[0030] Furthermore, by replacing at least one N-linked glycan having mannose or mannose 6-phosphate at the end bound to the asparagine of the hydrolase with a glycan having sialic acid (Neu5Ac) at the end, a hydrolase containing at least one glycan having a biantennary glycan with sialic acid at the end can be prepared.

[0031] The remodeling of glycans involves first cleaving N-linked glycans originally bound to the hydrolase, which have terminal mannose or mannose 6-phosphate. Then, a biantennary glycan with a terminal sialic acid is transferred from the donor. Specifically, the N,N'-diacetylchitobiose residue at the reducing end of the N-linked glycan of the hydrolase is hydrolyzed to release the glycan into an endo-type. The glycan is then cleaved, leaving a single N-acetylglucosamine (GlcNAc) residue at the reducing end of the N-linked complex glycan bound to the asparagine (Asn) of the hydrolase. N-linked glycans with terminal mannose or mannose 6-phosphate include high-mannose glycans, designated Man3, Man5, Man6, Man8, and Man9, depending on the number of mannose residues. High-mannose glycans with five or fewer mannose residues are cleaved. Next, a glycan with terminal sialic acid is transferred from the donor. As the donor, a glycoprotein or glycopeptide having a sugar chain with a biantennary sugar chain (sialyl sugar chain) with a terminal sialic acid can be used. For example, a sialyl glycopeptide (α2,6-SGP) having a biantennary terminal sialic acid can be used. Sialyl glycopeptide can be prepared from egg yolk. CAS No. 189035-43-6 (C 112 H 189 N 15 O 70 The sugar chain replacement enzyme may be produced by expression in prokaryotic microorganisms, fungi, insect cells, silkworm cells, or mammalian cells.

[0032] The sugar chain replacement can be achieved using enzymes that hydrolyze chitobiose bonds and simultaneously catalyze the sugar chain transfer reaction, such as endo-β-N-acetylglucosaminidases (ENGases) belonging to the GH85 (glycoside hydrolase) family and the GH18 family. These endo-β-N-acetylglucosaminidases include Endo-M (derived from Mucor hiemalis: M. Umekawa et al., J. Biol. Chem., 2008, Feb. 22; 283(8):4469-79), Endo-A (derived from Arthrobacter protophormiae), Endo-S (derived from Streptococcus pyogenes; Collin, M. et al., (2001) The EMBO Journal, 20, 3046-3055), Endo-CC (derived from Coprinus cinreus), Endo-SB (Japanese Patent Application Laid-Open No. 2019-17259), Endo-CoM (Japanese Patent Application Laid-Open No. 2019-17259), and Endo-CE (T. Kato, Glycobiology, Volume 12, Issue 10, October 1, 2002). Examples of enzymes that can be used include Endo-HS (Japanese Patent No. 6341571), Endo-Tsp1006, Endo-Tsp1263, and Endo-Tsp1457 (S. Takashima et al., Glycobiology, Volume 30, Issue 11, November 2020, pp. 923-934). Using these enzymes, glycan replacement can be achieved in a single step (one-pot reaction) by cleaving the original mannose-containing glycan and transferring a sialic acid-containing glycan from a donor. In particular, Endo-CC, an endo-β-N-acetylglucosaminidase derived from Coprinopsis cinerea (Coprinus cinereus), a member of the family Coprinoceae in the order Agaricales of the Basidiomycetes, is suitable. Endo-CC is described in Japanese Patent Publication No. 2015-080453.The nucleotide sequence of the Endo-CC enzyme is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. By using Endo-CC N180H, a mutant of the enzyme in which the 180th asparagine has been converted to histidine, glycan replacement can be performed in a single step (one-pot reaction) by cleaving the original mannose-containing glycan and transferring a sialic acid-containing glycan from a donor. Endo-CC N180H and methods for glycan replacement using it are described in JP 2020-10662 A and Manabe S. et al., R Soc Open Sci, 5(5), 171521 2018 16 eCollection May (2018). Alternatively, a commercially available enzyme (Fushimi Pharmaceutical Co., Ltd., product number: 171832) can be used as Endo-CC N180H. Enzymes such as Endo-CC N180A, a mutant in which asparagine at position 180 is converted to alanine; N180Q, a mutant in which asparagine at position 180 is converted to glutamine; and N180D, a mutant in which asparagine at position 180 is converted to aspartic acid, also possess hydrolytic and transglycosylation activities and can be used in the same manner as Endo-CC N180H and are believed to exhibit effects similar to those of the present invention. A method for producing Endo-CC N180A, N180Q, and N180D is described in "Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline" at https: / / www.ncbi.nlm.nih.gov / pubmed / 27714557. Furthermore, instead of a one-pot reaction, the degradation and transglycosylation reactions may be carried out using multiple enzymes.

[0033] This method can be carried out by mixing and reacting an enzyme whose deficiency causes a lysosomal disease, a glycoprotein or glycopeptide that serves as a donor of a glycan having a biantennary glycan with sialic acid at its terminal, and Endo-CC N180H or an endo-β-N-acetylglucosaminidase with equivalent activity.

[0034] In the production method of the present invention, endo-β-N-acetylglucosaminidase is preferably immobilized on magnetic resin beads and mixed with an enzyme whose deficiency causes lysosomal storage disease and a glycoprotein or glycopeptide that serves as a donor sugar chain having a biantennary sugar chain with a terminal sialic acid. The enzyme whose deficiency causes lysosomal storage disease is called an acceptor protein because it can replace the sugar chain.

[0035] By immobilizing endo-β-N-acetylglucosaminidase on magnetic resin beads, the enzyme is uniformly dispersed in the reaction system, allowing the reaction to be carried out efficiently. Furthermore, after the reaction, the magnetic resin beads can be collected in one part of the reaction system by the action of a magnet, making it easy to separate from the rest of the reaction system, which is preferable as it makes it easy to separate and remove the reaction system.

[0036] Endo-β-N-acetylglucosaminidase immobilized on magnetic resin beads can be produced by adding magnetic resin beads and endo-β-N-acetylglucosaminidase to a buffer solution and bringing them into contact. Commercially available magnetic resin beads available from Cytiva (Global Life Technologies Japan, Inc.) and Tamagawa Seiki Co., Ltd. can be used.

[0037] The enzyme whose deficiency causes lysosomal disease, the glycoprotein or glycopeptide (SGP donor) that serves as a donor of a sugar chain having terminal sialic acid, and endo-β-N-acetylglucosaminidase can be mixed and reacted, for example, by placing them in a container and shaking the container.

[0038] The molar reaction ratio of the enzyme whose deficiency causes lysosomal disease to the glycoprotein or glycopeptide that will become the donor sugar chain with terminal sialic acid may be selected from the range of approximately 1:1 to 10,000:1. The amount of endo-β-N-acetylglucosaminidase used may be selected from the range of 1 to 300 munits per nanomole of the glycoprotein or glycopeptide that will become the donor sugar chain with terminal sialic acid. The reaction temperature may be selected from the range of approximately 10 to 60°C, and the reaction time may be selected from the range of several minutes to several tens of hours, specifically from the range of approximately 5 minutes to 30 hours. The pH during the reaction is preferably 5.0 to 8.0. The preferred range of the reaction conditions varies depending on the type and molar ratio of the compound and the glycoprotein or glycopeptide, the amount of endo-β-N-acetylglucosaminidase used, etc., and therefore it is preferable to select optimal conditions in advance through simple preliminary experiments, etc.

[0039] When endo-β-N-acetylglucosaminidase is used after immobilization on magnetic resin beads, the magnetic resin beads can be collected in one part of a reaction vessel using a magnet, thereby separating the enzyme from other reaction systems. The separated reaction system can then be purified by chromatography or the like to obtain the reaction product, a hydrolase having a sugar chain containing terminal sialic acid.

[0040] The enzyme causing a lysosomal storage disease due to a defect produced by any of the above methods (i) to (vi) contains a sugar chain having a biantennary sugar chain with sialic acid attached to at least one terminus. Furthermore, the enzyme causing a lysosomal storage disease due to a defect, whose sugar chain has been replaced by the above method using endo-β-N-acetylglucosaminidase, contains a sugar chain having a biantennary sugar chain with sialic acid attached to at least one terminus and a sugar chain having mannose or mannose 6-phosphate at its terminus. The sugar chain containing the sugar chain having a biantennary sugar chain with sialic acid attached to at least one terminus is taken up into lysosomes in cells via binding to α2,6 sialic acid receptors on the cell surface. Furthermore, the enzyme with the replaced sugar chain is taken up into lysosomes in cells via binding to mannose receptors or mannose 6-phosphate receptors on the cell surface with the original sugar chain having mannose or mannose 6-phosphate at its terminus, or via binding to α2,6 sialic acid receptors on the cell surface. That is, an enzyme whose deficiency causes a lysosomal storage disease, produced by any of the above methods (i) to (vi), which contains a sugar chain with a biantennary sugar chain with sialic acid attached to at least one terminus, or an enzyme with a replaced sugar chain, can be incorporated into cellular lysosomes using the cellular transport mechanism for proteins or peptides with sialic acid-containing sugar chains. Therefore, it is efficiently incorporated into lysosomes. Because it is efficiently incorporated, a small dosage is required. The hydrolase incorporated into lysosomes restores the deficient hydrolase activity, thereby decomposing waste products in the lysosomes. As a result, it is possible to achieve treatment of lysosomal storage diseases or alleviate their symptoms.

[0041] The following describes in detail the case where α-L-iduronidase (IDUA) is used as the hydrolase and Endo-CC N180H is used as the endo-β-N-acetylglucosaminidase.

[0042] IDUA can be produced, for example, by producing transgenic silkworms (TG silkworms) into which the human alpha-iduronidase (IDUA) gene has been introduced by the method described in paragraph

[0108] of JP 2017-184736 A, and then from these TG silkworms by the method described in paragraphs

[0112] to

[0114] of the same publication (IDUA derived from TG silkworms).

[0043] Figure 1 shows a one-pot method for replacing at least one of the IDUA glycans with a sialic acid-terminated glycan. As shown in Figure 1, IDUA has six glycans, one or two of which are high-mannose glycans with more than five mannoses. Because these high-mannose glycans are essential for enzymatic activity, at least one high-mannose glycan must be maintained without replacement. Therefore, it is sufficient to replace one to five of the six glycans. As described above, glycan replacement can be achieved by reacting IDUA with the donor glycan (sialylglycopeptide) and Endo-CC 180H. The number of glycans replaced can be controlled by adjusting the amount of enzyme, the amount of donor glycan, and the reaction time. However, high-mannose glycans with more than five mannoses are not cleaved and therefore are not replaced. In SG-IDUA, the product of the enzymatic reaction shown in Figure 1, one high-mannose glycan is maintained without replacement, and the other five glycans have been replaced with glycans containing terminal sialic acids.

[0044] SG-IDUA contains a sugar chain with at least one terminal sialic acid and a sugar chain with a terminal mannose or mannose 6-phosphate. The SG-IDUA with the replaced sugar chain is taken up into lysosomes in cells via binding between the original sugar chain with terminal mannose or mannose 6-phosphate and a cell surface mannose receptor or mannose 6-phosphate receptor, or via binding to a cell surface α2,6 sialic acid receptor. SG-IDUA taken up into lysosomes replaces the defective IDUA and degrades heparan sulfate and dermatan sulfate in lysosomes that accumulate in the cells of mucopolysaccharidosis type I patients. As a result, treatment or alleviation of symptoms of mucopolysaccharidosis type I, which is caused by a deficiency of IUDA, can be achieved.

[0045] The present invention encompasses hydrolases produced by any of the above methods (i) to (vi), which are enzymes whose deficiency causes lysosomal disease and which contain a sugar chain with a biantennary sugar chain attached to at least one terminus of sialic acid; hydrolases in which at least one sugar chain of a hydrolase having a sugar chain with mannose or mannose 6-phosphate at its terminus has been replaced by a sugar chain with sialic acid at its terminus by the above method; and therapeutic agents for lysosomal disease which contain these enzymes as active ingredients.

[0046] An example of such an enzyme is IDUA (SG-IDUA) or CTSA (SG-CTSA) in which at least one of the six terminal mannose-containing sugar chains, excluding at least one high-mannose type sugar chain necessary for enzymatic activity, i.e., one, two, three, four, or five sugar chains, has been replaced with a sugar chain having a biantennary terminal sialic acid at the terminal. This enzyme can be used to treat or alleviate the symptoms of mucopolysaccharidosis type I.

[0047] The administration route of the therapeutic agent for lysosomal storage disease of the present invention is not limited, and it can be administered orally, parenterally, or the like. Parenteral administration includes intravenous, subcutaneous, intramuscular, and intraperitoneal injections. The administration route of the formulation includes oral liquids, tablets, capsules, pills, powders, and the like. Capsules, tablets, powders, granules, and the like can be produced using additives such as excipients such as lactose, glucose, sucrose, and mannitol; disintegrants such as starch and sodium alginate; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin.

[0048] Liquid preparations such as emulsions and syrups can be produced using additives such as water, sugars such as sucrose, sorbitol, and fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil, and soybean oil; preservatives such as p-hydroxybenzoic acid esters; and flavors such as strawberry flavor and peppermint.

[0049] The injection preparation may contain additives such as water, sugars such as sucrose, sorbitol, xylose, trehalose, and fructose; sugar alcohols such as mannitol, xylitol, and sorbitol; buffers such as phosphate buffer, citrate buffer, and glutamate buffer; and surfactants such as fatty acid esters.

[0050] The dosage of a therapeutic drug for lysosomal storage disease required for treatment can vary depending on the age, sex, severity, etc. of the patient, but can ultimately be decided by the attending physician. For example, the therapeutic drug for lysosomal storage disease may be administered at a dose of 0.05 to 10 mg / kg body weight, preferably 0.1 to 2 mg / kg body weight per dose. The prescribed dosage may be administered in a single dose, or in divided doses two, three, four, or more times per day at appropriate intervals over a period ranging from one day to several years.

[0051] The present invention encompasses a method for producing any of the above-mentioned (i) to (vi) hydrolases, the deficiency of which causes a lysosomal disease, and which contain a sugar chain having a biantennary sugar chain with sialic acid attached to at least one terminus.

[0052] The present invention also encompasses a method for producing a hydrolase in which at least one of the sugar chains of a hydrolase having a sugar chain containing mannose or mannose 6-phosphate at its terminal is replaced with a sugar chain containing sialic acid at its terminal. This method produces a hydrolase containing at least one sugar chain containing sialic acid at its terminal by mixing and reacting a hydrolase whose deficiency causes a lysosomal storage disease, a glycoprotein or glycopeptide that serves as a donor of the sugar chain containing terminal sialic acid, and Endo-CC N180H, in which the asparagine at position 180 of an endoglycosidase has been converted to histidine. This method enables the production of a therapeutic drug for lysosomal storage disease at low cost. Furthermore, by standardizing the production conditions using Endo-CC N180H, in which the asparagine at position 180 of an endoglycosidase has been converted to histidine, a hydrolase with a uniform sugar chain structure can be produced.

[0053] A hydrolase containing a sugar chain having a biantennary sugar chain with terminal sialic acid can be delivered into cells by a new drug delivery system that binds to the α2,6 sialic acid receptor on the cell surface. Furthermore, by replacing at least one sugar chain of a hydrolase having a sugar chain with terminal mannose or mannose 6-phosphate with a sugar chain having terminal sialic acid, the enzyme can be delivered into cells by a new drug delivery system that binds to the α2,6 sialic acid receptor on the cell surface. [Example]

[0054] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0055] [Example 1] One-pot glycosylation using Endo-CC (N180H) SG-IDUA was prepared by replacing the sugar chain structure of IDUA expressed in silkworm cocoons with SG using the methods described in Production Examples 1 to 3 and Examples 1 and 2 of JP 2020-10662 A.

[0056] [Example 2] Effect of enzyme supplementation on MPSI-jp As a model cell line for spontaneous mucopolysaccharidosis type I, ear fibroblasts (MPSI-jp) isolated from Japanese macaques were used, and as a control, Japanese macaque skin fibroblasts (jm1481) were used. 5 cell, jm1481 is 2.0 × 10 5Cells were seeded onto a 35 mm dish and confirmed to have adhered to the dish and proliferated. After confirming that the cells had grown, 1 μg each of buffer alone, IDUA expressed in silkworm cocoons (silkworm-IDUA), and SG-IDUA, which had been expressed in silkworm cocoons and had its glycan replaced as described in Example 1, was added to MPSI-jp. Buffer alone was added to JM1481 Japanese macaque skin fibroblasts, which served as a control, and each was incubated overnight. The medium was discarded, and the cells were washed with 1 mL of PBS. Then, 1 mL of PBS was added and the cells were detached with a scraper. After collecting the cells in a 1.5 mL tube, 500 μL of PBS was added to the dish, and the remaining cells were also collected. The cells were centrifuged at 500 × g for 5 minutes, and the supernatant was removed. One hundred microliters of cell extract buffer (50 mM NaOAc (pH 4.5) / 150 mM NaCl / 1 (w / v)% Triton X-100) containing protease inhibitors (EDTA, pepstatin A, and leupeptin) was added and mixed thoroughly with a pipette. The mixture was sonicated on ice for 10 minutes and centrifuged at 18,000 × g for 5 minutes to collect the supernatant. 15 μL of this cell extract supernatant was mixed with 15 μL of 0.1 M sodium acetate buffer (pH 4.5) containing 2 mM 4-MU-Idopyranoside and 500 mM NaCl and incubated at 37°C for 30 minutes. The reaction was stopped by adding 380 μL of 0.2 M glycine-NaOH (pH 10.7). 300 μL of the mixture was dispensed into a 96-well plate and the fluorescence intensity was measured at Ex 360 nm and Em 448 nm.

[0057] The amount of 4-MU-Idopyranoside enzymatically digested into 4-MU per hour per mg of cell extract supernatant protein, and the amount of fluorescence emitted was evaluated (Figure 2).

[0058] When the amount of 4-MU-Idopyranside decomposed into 4-MU in the control jm1481 was taken as 100%, the enzyme activity of MPSI-jp decreased to 10%, but that of Silkworm-IDUA increased to 100.7%, and that of SG-IDUA increased to 153.5%.

[0059] Since silkworm-IDUA was expressed in silkworm cocoons, its sugar chain structure is known to be either paucimannose or high mannose. It has already been shown that these sugar chains are not taken up into cells, and although the enzyme activity increased to 111%, we believe that this increase in enzyme activity is due to nonspecific adsorption to the cell membrane. SG-IDUA also showed a similar increase in enzyme activity. Since this case is also thought to be nonspecifically adsorbed to the cell membrane, as with silkworm-IDUA, we believe that the enzyme activity actually taken up into cells represents the difference between SG-IDUA and silkworm-IDUA, or 43% of the enzyme activity shown within the cells.

[0060] [Example 3] Competitive inhibitory effect of SG-IDUA on the intracellular uptake of MPSI-jp MPSI-jp 1.0×10 5Cells were seeded onto a 35 mm dish. After confirming that the cells had adhered to the dish and multiplied, the MPSI-jp medium was supplemented with (1) buffer alone, (2) 1 μg of SG-IDUA, (3) 1 μg of SG-IDUA and 5 mM sialic acid (SA), (4) 1 μg of SG-IDUA and 5 mM SG, or (5) 1 μg of SG-IDUA and 5 mM mannose 6-phosphate (M6P). After overnight incubation, the medium was discarded, the cells were washed with 1 mL of PBS, and then 1 mL of PBS was added and the cells were detached with a scraper. After collecting the cells in a 1.5 mL tube, 500 μL of PBS was added to the dish to collect the remaining cells. The cells were centrifuged at 500 × g for 5 minutes and the supernatant was removed. 100 μL of cell extraction buffer (50 mM NaOAc (pH 4.5) / 150 mM NaCl / 1 (w / v)% Triton X-100) containing protease inhibitors (EDTA, pepstatin A, leupeptin) was added and mixed thoroughly by pipetting. The mixture was sonicated on ice for 10 minutes and centrifuged at 18,000 × g for 5 minutes to collect the supernatant. 15 μL of this cell extract supernatant was added to 15 μL of 0.1 M sodium acetate buffer (pH 4.5) containing 2 mM 4-MU-Idopyranoside and 500 mM NaCl and incubated at 37°C for 30 minutes. The reaction was stopped by adding 380 μL of 0.2 M glycine-NaOH (pH 10.7). 300 μL aliquots were dispensed into a 96-well plate and the fluorescence intensity was measured at Ex 360 nm and Em 448 nm (Figure 3).

[0061] When SG-IDUA alone was added, the enzyme activity was taken as 100%, and when 5 mM SA, 5 mM SG, and 5 mM M6P were added, the enzyme activity decreased, suggesting that SG-IDUA competes with SA, SG, and M6P.

[0062] [Example 4] Intracellular uptake of F17 (MPSI patient skin fibroblasts) To assess uptake into human MPS1 patient skin fibroblasts (F17), IDUA was pre-labeled using AcidiFluor (Goryo Chemical), a reagent that fluoresces in the acidic range. 10 μg of Silkworm-IDUA and 10 μg of SG-IDUA were each diluted to 300 μL with 0.1 M sodium bicarbonate buffer (pH 8.3). 2.1 μL of AcidiFluor ORANGE was added to each solution, and the mixture was stirred at room temperature for 2 hours in the dark. SG-IDUA-AFO was ultrafiltered using an Amicon Ultra-0.5 mL (10 kDa; Merck Millipore) at 5,000 × g for 5 minutes. This was repeated five times to obtain SG-IDUA-APO.

[0063] An 8-well chamber (Thermo Scientific) was coated with collagen and left to stand in a clean bench for 1 hour. After removing the collagen from the surface and washing with 1 mL of PBS, 1 × 10 cells were added. 4 100 cells were seeded into each well. After confirming cell adhesion, the medium was removed and the cells were washed twice with PBS. 1 μg of SG-IDUA-APO was diluted to 200 μL with medium and added to the cells. To examine the inhibitory effect, a sample containing 5 mM sialic acid was also prepared and added to the cells. The cells were incubated at 37°C and 5% CO2 for 24 hours. Images were observed using a fluorescence microscope (BZ9000, BIO-REVO) at an excitation wavelength of 532 nm and an emission wavelength of 568 nm. SG-IDUA-APO exhibits red fluorescence in an acidic environment. The results are shown in Figure 4. As shown in Figure 4, SG-IDUA-APO was taken up by F17 cells, resulting in red fluorescence. However, 5 mM sialic acid inhibited cellular uptake, resulting in a decrease in red fluorescence. These results demonstrate that SG-IDUA-APO is internalized into cells in an SG-dependent manner and transported to lysosomes under an acidic pH environment.

[0064] [Example 5] Sugar chain replacement of cathepsin A (CTSA) by Endo-CC (N180H) We prepared SG-CTSA by replacing the sugar chain structure of CTSA expressed in silkworm cocoons with SG using Endo-CC (N180H).

[0065] method The sample used was CTSA derived from U-32-2a×mari1-2 cocoons (butyl 0M, 8.3 mg / mL, purified on October 8, 2019, stored at -30°C). Glycosylation was performed using Endo-CC (N180H) 1 mU / μL, 352 μg / mL (Fushimi Pharmaceutical, J2717A) and SGP 250 nmol / μL (Fushimi Pharmaceutical, Product code: 171801, #N1617vB) as follows.

[0066] 1. Glycosylation reaction The reaction mixture was prepared as shown in Table 1. Donor:acceptor=1000:1 (molar ratio).

[0067] [Table 1]

[0068] The reaction was incubated at 30°C in an air incubator, and 1 μL of each was collected at 0, 4, 8, and 24 hours, boiled for 3 minutes with 6x dye(+), and then subjected to SDS-PAGE, CBB staining, SSA rectiblotting, and ConA lectin blotting.

[0069] 2.CBB staining The sections were stained overnight with staining solution and destained with 10% acetic acid.

[0070] 3. Lectin Blotting (1) The PVDF membrane was hydrophilized with MeOH. (2) After electrophoresis, the gel, filter paper, and PVDF membrane were shaken in blotting buffer. (3) Blotting was performed at 15 V for 1 hour. (4) The membrane was blotted with 50 (w / v)% Blocking One / TBS at room temperature for 1 hour. (5) 1 st Biotin-labeled SSA (500-fold dilution) was used as a probe, and the reaction was carried out at 4°C overnight, followed by washing three times with PBS-0.1 (w / v)% Tween 20 for 5 minutes each. (6) 2 nd Anti-biotin HRP linked Ab #32 (CST) (1000-fold dilution) was used as a probe, and the reaction was carried out at room temperature for 1 hour, followed by washing three times with PBS-0.1 (w / v)% Tween 20 for 5 minutes each. (7) Wash with PBS for 5 minutes. (8) Detected by Western lightning ultra-ECL. (9) The sample was reprobed with stripping buffer (10% SDS 5 mL, 4x Upper buffer 3.125 mL, 2-mercaptoethanol 176 μL, MQ up to 25 mL) at 50°C for 1 hour. (10) Blocking was performed with 50 (w / v)% Blocking One / TBS at RT for 1 hour. (11) 1 st Biotin-labeled ConA (250-fold diluted) was used as a probe, and the reaction was carried out at 4°C overnight. (12) The sections were washed three times with PBS-0.1 (w / v)% Tween 20 for 5 minutes each. (13) 2 nd Anti-biotin HRP linked Ab #32 (CST) (1000-fold diluted) was used as a probe, and the reaction was carried out at RT for 1 hour. (14) The sections were washed three times with PBS-0.1 (w / v)% Tween 20 for 5 minutes each. (15) Washed with PBS for 5 minutes. (16) Detection was performed using Western lightning plus-ECL.

[0071] result FIG. 5 shows the results of CBB staining (FIG. 5A), SSA lectin blotting (FIG. 5B), and Con A lectin blotting (FIG. 5C). The results of SSA lectin blotting indicate that replacement with SGP has occurred in the 4, 8, and 24-hour samples. Furthermore, the results of CBB staining indicate that the bands considered to have been replaced with SGP in the 24-hour sample are darker than those in the 4 and 8-hour samples. This indicates that the longer the reaction time, the more likely the sugar chains are to be replaced. The replacement efficiency was approximately 50%.

[0072] [Example 6] SG-CTSA trypsin treatment Example 5 confirmed that the cocoon-derived CTSA precursor (proCTSA) can be replaced with an SG-type glycan by Endo-CC (N180H). Therefore, we confirmed which N-glycan of the 32 kDa and 20 kDa domains of SG-CTSA, in which the glycan prepared in Example 5 was replaced with an SG-type glycan, had been replaced.

[0073] method SG-CTSA (1 mg / mL, stored at -30°C) was used as a sample. The reaction conditions were SG-CTSA:trypsin = 1:3 (molar ratio), and the reaction was carried out at pH 6.0 for 2 hours at 37° C. The composition of the reaction solution is shown in Table 2.

[0074] [Table 2]

[0075] The sugar chain replacement was confirmed by the following steps. (1) The reaction was initiated under the above conditions, and after 2 hours, 3 μg of the solution was taken, 6× dye was added, and the mixture was boiled. (2) SDS-PAGE was performed (12.5 (w / v)% SDS-PAGE gel, stacking: 15 mA, running: 20 mA). (3) After electrophoresis, the gel was shaken in blotting buffer for 30 minutes. (4) Blotting was performed at 15 V for 1 hour. (5) Blotting was performed with Blocking ONE / TBS for 1 hour. (6) 1 st As a probe, SSA-biotin (500-fold diluted, Blocking ONE 500 μL: lectin 1 μL) was used, and the reaction was carried out overnight at 4°C. (7) The plate was washed three times with PBS-0.1 (w / v)% Tween 20 for 5 minutes each. (8) 2 nd Anti-biotin HRP-linked Ab (CST) #33 (1000-fold diluted, Blocking ONE 500 μL: Ab 0.5 μL) was used as a probe, and the reaction was carried out at rt for 1 hour. (9) The plate was washed three times with PBS-0.1 (w / v)% Tween 20 for 5 minutes each. (10) Wash with PBS for 5 minutes. (11) Detection was performed using Western lightning ultra-ECL (Chemi Hi Resolution, 23.588 sec). (12) Because the 20 kDa domain was difficult to transfer to the PVDF membrane, SDS-PAGE was performed again (1 μg) and CBB staining was performed.

[0076] result The results are shown in Figures 6 and 7. Figure 6 shows the results of SSA lectin blotting, and Figure 7 shows the results of CBB staining. Lane a in Figure 7 shows the results of staining with CTSA + trypsin, and lane b shows the results of staining with SG-CTSA + trypsin. The results of SSA lectin blotting suggest that the N-glycans of both the 32kDa and 20kDa domains of SG-CTSA have been replaced. Furthermore, CBB staining confirmed a shift toward higher molecular weights in the 32kDa domain compared to the 20kDa domain, indicating that the high-mannose glycan on the 32kDa side has been replaced by a sialyl glycan. Therefore, it is thought that the N-glycan on the 32kDa domain is more likely to be replaced.

[0077] [Example 7] Intracerebroventricular administration of cocoon-derived CTSA precursor (proCTSA) and α2,6-SG-CTSA to GS model mice (enzyme replacement therapy for the central nervous system) Cocoon-derived CTSA precursor and SG-CTSA were administered at 3 mg / kg body weight to GS (galactosialidosis) model mice (6-7 week-old adult mice), and then dissected 24 hours later to determine the extent to which Ctsa and Neu activity were restored. GS model mice were produced by Unitech Co., Ltd.

[0078] Neu activity refers to the catalytic activity of the hydrolase neuraminidase (sialidase), which cleaves sialic acid residues in terminal α2,3, α2,6, or α2,8-linked sialic acid-containing glycans. In this example, the decomposition activity of the artificial fluorescent substrate 4-MU-N-acetyl-neuraminic acid was measured. Pro-CTSA or the mature form taken up into cells associates with endogenous Neu1 and becomes activated, exhibiting Neu activity. This demonstrates the therapeutic effect (efficacy) of restoring both the activities of Ctsa and Neu1, which are deficient in GS mice.

[0079] method The samples administered to mice were the cocoon-derived CTSA precursor, α2,6-SG-CTSA prepared in Example 5, and PBS. Table 3 shows the mouse numbers, genotypes, sexes, ages in weeks, and administered samples.

[0080] [Table 3]

[0081] The experiment was carried out in the following steps. 1. Intracerebroventricular administration to mice (1) Pre-anesthesia was administered with isoflurane. (2) 150 μL of Somnopentyl was administered intraperitoneally. (3) The scalp was incised, and each sample was administered intracerebroventricularly to the right brain at 3 mg / kg body weight. The administration site is shown in Figure 8. (4) The scalp was replaced and placed in a cage.

[0082] 2.Dissection (1) The animals were anesthetized with isoflurane. (2) The heart was perfused with approximately 20 mL of PBS. (3) The brain and liver were removed, placed in a 1.5 mL tube, and cooled on ice. (4) Each organ was stored at -80°C.

[0083] 3. Extract Preparation (1) An extraction buffer was prepared with the composition shown in Table 4. At this time, the amount prepared was the number of samples plus one. [Table 4] (2) Five volumes of extraction buffer were added to the sample, and the mixture was homogenized with a pestle. (3) For protein quantification and NEU1 activity measurement, half of the extract was dispensed into a 1.5 mL tube (pre-centrifugation sample). (4) The remaining extract was centrifuged at 12,000 rpm, 4°C, for 15 minutes. (5) The supernatant was collected and centrifuged to prepare a sample.

[0084] 4.NEU1 activity measurement (1) A substrate was prepared with the composition shown in Table 5. At this time, two samples were prepared in amounts equal to the number of samples x 2 + 2. [Table 5] (2) The prepared substrate was dispensed into 1.5 mL tubes in 20 μL portions. At this time, the enzyme (-) was also prepared. (3) 20 μL of the extract was added to each of the dispensed substrates, and the mixture was incubated at 37°C for 30 minutes. (4) 370 μL of 0.2 M Gly-NaOH (pH 10.7) was added to stop the reaction. (5) A calibration curve was prepared as shown in Table 6. [Table 6] (6) 300 μL of each sample and calibration curve was applied to a 96-well plate, and fluorescence was measured at 460 nm.

[0085] 5. Cathepsin A Enzyme Activity Measurement 5-1. Primary reaction: Reaction that liberates Leu (1) A reaction solution was prepared with the composition shown in Table 7. At this time, two samples were prepared in amounts equal to the number of samples x 2 + 2. [Table 7] (2) 25 μL of each sample was dispensed into duplicate 1.5 mL tubes. One substrate (-) was prepared for each sample (a total of three tubes per sample). (3) All 1.5 mL tubes were opened, and 25 μL of the reaction mixture was quickly poured into them. However, 25 μL of 0.2 M NaOAc buffer (pH 5.6) was added to the substrate (-) tube instead of the reaction mixture. (4) The reaction was carried out for 30 minutes at 25°C. (5) The reaction was stopped by placing the mixture in boiling water at 100°C for 2 minutes. The resulting reaction mixture was used as the primary reaction mixture. (6) A calibration curve was prepared as shown in Table 8. [Table 8]

[0086] 5-2. Secondary reaction: Color reaction (1) The secondary reaction mixture was prepared as shown in Table 9. At this time, a calibration curve was also prepared. [Table 9] (2) 500 μL of the secondary reaction mixture was added to each sample and the standard curve, and incubated at 37°C for 40 minutes. (3) 500 μL of 6N HCl was added to each well to stop the reaction. (4) 300 μL of each solution was applied to a 96-well plate, and the absorbance was measured at OD540 nm. (5) CathA activity (Ctsa activity) was calculated from the results of (4) and protein quantification.

[0087] result Figure 9 shows the Ctsa specific activity (Figure 9A) and Neu specific activity (Figure 9B). 1.Ctsa activity The mean for WT was 108 nmol / h / mg protein. With PBS administration, the value was 2.2 nmol / h / mg protein. The mean for the CTSA-treated group was 32 nmol / h / mg protein. The mean for the SG-CTSA group was 54 nmol / h / mg protein.

[0088] 2.Neu activity With PBS administration, the value was 5.56 nmol / h / mg protein. The mean for the CTSA-treated group was 0.34 nmol / h / mg protein. The mean for the SG-CTSA group was 1.35 nmol / h / mg protein. The mean for WT was 0.84 nmol / h / mg protein.

[0089] Twenty-four hours after intracerebroventricular administration of Man-proCTSA (a cocoon-derived CTSA precursor) or SG-proCTSA, Ctsa and Neu1 activity were restored in the cerebrum of GS mice, with the degree of recovery being greater with SG-CTSA. It is thought that cocoon-derived Man-proCTSA was only taken up by microglia expressing the Man receptor. On the other hand, because SG-proCTSA contains both terminal mannose and sialic acid-containing glycans, it was taken up by cells other than microglia, suggesting that it showed a greater recovery of activity than Man-proCTSA. The recovery of activity suggests that it was taken up into lysosomes.

[0090] [Example 8] Administration of cocoon-derived CTSA precursor (proCTSA) and α2,6-SG-CTSA to the tail vein of GS model mice SG-proCTSA (SGW group in Figure 10), an SG-glycosylated precursor of CTSA derived from TG silkworm cocoons, was administered to GS (galactosialidosis) model mice (6- to 7-week-old adult mice) via the tail vein at 3 mg / kg body weight weekly (4 times in total). The experiment was carried out three times. Seven days after the final administration, each mouse was dissected, and the liver, spleen, heart, kidneys, and lungs were removed. Neu activity in the extracts of each organ and the decrease in its accumulated sialyl substrate (sialic acid amount) were used as indicators for comparison among the non-administration group (PBS-administered control, CT group in Figure 10), CHO cell-derived CTSA precursor (CHO-proCTSA, CHO group in Figure 10), and TG silkworm cocoon-derived CTSA precursor (Man-type proCTSA, SW group in Figure 10). The WT group in Figure 10 represents wild-type mice. In addition, the amount of terminal sialic acid-containing glycans (sialyl glycans) in urine collected for each CTSA precursor before the first administration (Pre), before the second administration (Day 7), and before the fourth administration (Day 22) was quantified using the resorcinol method, and the effectiveness of administration was evaluated using the decrease in urinary excretion after administration as an indicator.

[0091] method Preparation of extract (1) An extraction buffer was prepared with the composition shown in Table 10. The amount of the buffer prepared was the number of samples plus one. [Table 10] (2) Five volumes of extraction buffer were added to the sample, and the mixture was homogenized with a pestle. (3) For protein quantification and NEU1 activity measurement, half of the extract was dispensed into a 1.5 mL tube (pre-centrifugation sample). (4) The remaining extract was centrifuged at 12,000 rpm, 4°C, for 15 minutes. (5) The supernatant was collected (post-centrifugation sample).

[0092] NEU1 activity measurement (1) A substrate was prepared with the composition shown in Table 11. Two tubes were prepared (number of samples x 2 + 2 tubes). [Table 11] (2) The pre-centrifugation sample was dispensed into 1.5 mL tubes in 20 μL portions. Enzyme (-) was also prepared. (3) 20 μL of the prepared substrate was added to each well and incubated at 37°C for 30 minutes. (4) 370 μL of 0.2 M Gly-NaOH (pH 10.7) was added to stop the reaction. (5) A calibration curve was created as shown in Table 12. [Table 12] (6) 200 μL of each sample and calibration curve was applied to a 96-well plate, and fluorescence was measured at 460 nm.

[0093] Cathepsin A enzyme activity measurement Primary reaction: Reaction that liberates Leu (1) A reaction solution was prepared with the composition shown in Table 13. Two bottles were prepared (number of samples x 2 + 2). [Table 13] (2) After centrifugation, 25 μL of each sample was dispensed into duplicate 1.5 mL tubes, and one substrate (-) sample was prepared for each. A total of three tubes per sample were prepared. (3) All 1.5 mL tubes were opened, and 25 μL of the reaction mixture was quickly poured into them. 25 μL of 0.2 M NaOAc buffer (pH 5.6) was added to the substrate (-) tube instead of the reaction mixture. (4) Incubated for 30 minutes at 25°C. (5) The reaction was stopped by placing the mixture in boiling water at 100°C for 2 minutes (primary reaction solution). (6) A calibration curve was prepared as shown in Table 14. [Table 14]

[0094] Secondary reaction: color reaction (1) The secondary reaction mixture was prepared as shown in Table 15. A calibration curve was also prepared. [Table 15] (2) 500 μL of the secondary reaction mixture was added to each sample and the standard curve, and incubated at 37°C for 40 minutes. (3) 500 μL of 6N HCl was added to each well to stop the reaction. (4) 300 μL of each solution was applied to a 96-well plate, and the absorbance was measured at OD540 nm. (5) CathA activity was calculated from the results of (4) and protein quantification.

[0095] β-Hex enzyme activity measurement (1) After centrifugation, 15 μL of the sample was dispensed (duplicate). (2) 15 μL of MQ-HO was dispensed into a 1.5 mL tube as the enzyme (-). (3) 15 μL of MUG was added to each well, and the mixture was incubated at 37°C for 15 minutes. (4) 380 μL of 0.2 M Gly-NaOH (pH 10.7) was added to stop the reaction. (5) A calibration curve was prepared as shown in Table 16. [Table 16] (6) 200 μL of each sample and calibration curve was applied to a 96-well plate, and fluorescence was measured at 460 nm.

[0096] β-Gal enzyme activity measurement (1) After centrifugation, 15 μL of the sample was dispensed (duplicate). (2) 15 μL of MQ-HO was dispensed into a 1.5 mL tube as the enzyme (-). (3) 15 μL of 4MU-Gal was added to each well, and the mixture was incubated at 37°C for 30 minutes. (4) 380 μL of 0.2 M Gly-NaOH (pH 10.7) was added to stop the reaction. (5) A calibration curve was prepared as shown in Table 17. [Table 17] (6) 200 μL of each sample and calibration curve was applied to a 96-well plate, and fluorescence was measured at 460 nm.

[0097] Sialic acid quantification (1) For the calibration curve, sialic acid was prepared as shown in Table 18. 0.2 mL tubes were used. [Table 18] (2) The pre-centrifugation sample was dispensed into three 0.2 mL tubes (50 μL each). (3) Add 10 μL of 40 mM NaIO4 to each sample and mix. (4) Resorcinol (-) solution and resorcinol solution were prepared as follows. Resorcinol solution composition (per 1 mL) 6 (w / v)% resorcinol / 18% HCl solution 100 μL 300 μL of 6N HCl 0.185(w / v)% CuSO4 16μL MilliQ 584μL Resorcinol (-) solution composition (per 1 mL) 334 μL 6N HCl 0.185(w / v)% CuSO4 16μL MilliQ 650μL (5) 125 μL of resorcinol (-) solution was added to one tube, and 125 μL of resorcinol solution was added to two tubes, and the samples were mixed. (6) The following treatment was carried out using a thermal cycler. 4.0℃ 5min 99.9℃ 15min 37.0℃ (7) 125 μL of tert-butyl alcohol was dispensed into a 96-well plate. (8) The reaction solution and 125 μL of tert-butyl alcohol were mixed in the well. (9) The absorbance at OD630nm was measured (plate reader in the central equipment room).

[0098] Figure 10 shows the accumulation of sialyl substrates, which are Neu substrates, in the liver (Figure 10A), spleen (Figure 10B), and heart (Figure 10C) of the SG-proCTSA-administered (SGW) group, relative to the PBS-administered (CT) group, which is defined as 100%. As shown in Figure 10, the sialic acid content in the liver, spleen, and heart was reduced to the same level as in WT. Figure 11 similarly shows the accumulation of sialyl substrates, which are Neu substrates, in the kidney (Figure 11A) and lung (Figure 11B). As shown in Figure 11, the amount of sialic acid in the kidney was reduced in the CHO-CTSA-administered group compared to the control. In the lung, the amount of sialic acid was significantly reduced in the CHO-CTSA- and SG-CTSA-administered groups compared to the control, and there was also a tendency for it to decrease in the SW-CTSA-administered group.

[0099] Figure 12 shows the accumulation of sialic acid, a Neu substrate, in urine excretion. The amount of sialic acid in urine Pre (before administration) for each individual is set at 100, and the amounts of sialic acid in urine on D7 (the day after the first administration) and D22 (the day after the third administration) are shown. The amount of sialic acid in urine was significantly reduced on D22 in the CHO-CTSA and SG-CTSA administration groups compared to CT. The amount of sialic acid in urine also tended to decrease in the SW-CTSA administration group, although this was not significantly different.

[0100] In the liver, spleen, and heart, CHO-derived M6P-type proCTSA, TG silkworm cocoon-derived Man-type proCTSA, or SG-proCTSA was taken up into cells via M6PR, ManR, or SiaR present in macrophages, monocytes, etc., resulting in a significant reduction in accumulated sialyl substrates, which is thought to be the therapeutic effect (efficacy).

[0101] In the kidney and lung, CHO-derived M6P-type proCTSA is taken up into cells via M6PR in tissue-constituting cells, resulting in a significant reduction in accumulated sialyl substrate (effectiveness). SG-type proCTSA is taken up into lung-constituting cells and shows a significant reduction in substrate. However, TG silkworm cocoon-derived Man-type proCTSA is not taken up into cells, and therefore is not effective.

[0102] Multiple intravenous administration of CHO-derived M6P-type normal proCTSA or SG-type proCTSA inhibited the urinary excretion of accumulated sialyl substrates in this GS model mouse.

[0103] The results for the liver, spleen, heart (shown in slide 8) and lungs (no significant difference with Man-type proCTSA derived from TG silkworm cocoons) and the significant decrease in urinary excretion of sialyl substrates (glycans) demonstrate the specific function and efficacy of SG-proCTSA. [Industrial Applicability]

[0104] The hydrolase of the present invention in which a sugar chain having a sialic acid at its terminal is substituted can be used in the treatment of lysosomal diseases.

Claims

1. A hydrolase that is α-L-iduronidase (IDUA) or cathepsin A (CTSA) present in lysosomes, the deficiency of which causes a lysosomal disease, and that contains at least one sugar chain having a biantennary sugar chain with α2,6 sialic acid attached to its terminal, and at least one high-mannose sugar chain that is essential for enzymatic activity, and that is delivered to the lysosome by being taken up into the lysosome via binding between the α2,6 sialic acid contained in the terminal sugar chain and a sialic acid receptor on the cell surface.

2. The hydrolase is α-L-iduronidase (IDUA) or cathepsin A (CTSA) present in lysosomes, the deficiency of which causes a lysosomal disease, and at least one of the originally present sugar chains having a mannose terminal is replaced with a sugar chain having an α2,6 sialic acid terminal, while at least one of the high-mannose sugar chains essential for enzymatic activity is maintained without being replaced, said hydrolase being α-L-iduronidase (IDUA) or cathepsin A (CTSA) according to claim 1.

3. A therapeutic agent for lysosomal diseases, comprising as an active ingredient the hydrolase α-L-iduronidase (IDUA) or cathepsin A (CTSA) according to claim 1 or 2, wherein the hydrolase is taken up into lysosomes via binding between α2,6 sialic acid contained at the end of the sugar chain and a sialic acid receptor on the cell surface.

4. A therapeutic agent for lysosomal disease as described in claim 3, wherein the lysosomal disease is mucopolysaccharidosis type I.

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