WO2015009052A1 - Fusion protein of immunoglobulin hybrid fc and enzyme - Google Patents

Abstract

The present invention relates to a fusion protein of hybrid immunoglobulin Fc and enzyme and, more particularly, to a fusion protein in which an enzyme is linked to human immunoglobulin hybrid Fc represented by an amino acid sequence of SEQ ID NO: 4, and to a method for producing the same. The fusion protein in which an enzyme is linked to human immunoglobulin hybrid Fc represented by an amino acid sequence of SEQ ID NO: 4 according to the present invention maintains activity of the enzyme, has extended half-life and bioavailability when compared with enzymatic treatment agents on the market regardless of the administration method, and exhibits excellent distribution efficiency in specific organs in association with causes of diseases, and thus is effective in preventing diseases caused by the lack of various enzymes in the body, including lysosomal accumulation disease, or producing agents for treating the diseases.

Description

 【Specification】

 [Name of invention]

 Hybrid immunoglobulin Fc and enzyme fusion protein

 This application claims the priority of Korean Patent Application No. 10-2013-0083860 filed on July 16, 2013, the entirety of which is a reference of the present application.

 The present invention relates to a hybrid immunoglobulin Fc and a fusion protein of an enzyme, and more particularly, to a fusion protein conjugated with an enzyme and a human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4, and a method for preparing the same.

Background technology

One of the various methods used to increase the in vivo persistence of proteins and peptides is the fusion of the Fc portion of the immunoglobulin with the target protein. Fc and raised hapdoen protein are recycled to the child as lysosomes via FcRn therefore increase the tissue permeability, because the transmission is possible through the placenta FIG FcRn as well as to increase the in vivo half-life and to cross the placenta in _yae matrix it can be delivered to the fetus The fetal state can be treated, and since the protein delivered in the fetal state is recognized as a self protein, there is a merit such that a neutralizing antibody is unlikely to occur even after administration after birth. Conventional Fc fusion proteins are used for the production of immunoglobulin IgGl-derived Fc. In the case of IgGl Fc, antibody-dependent cel l-mediaated cytotoxicity (ADCC) and complement-mediated cytotoxicity (complement- dependent cytotoxicity (CDC), causing the target cells to die. In other words, even if the function of the target protein to be fused does not require apoptosis, the target cells to be bound by the fusion of IgGl Fc are killed, which may result in fatal side effects that result in no therapeutic effect. In order to overcome this problem, a method of eliminating cytotoxicity of a fusion protein made by mutating a gene of a part related to cell-mediated cytotoxicity and complement-mediated cytotoxicity of IgGl Fc, but in this case, a natural immunoglobulin portion In this case, antibody formation and a decrease in efficacy occur due to Fc-immune reaction when administered in vivo. In addition, the conventional antibody fusion technology is specifically two antibody The therapeutic protein binds to one Fc to form a dimer, which has a problem in that the activity of the bound protein and peptide therapeutic agent is reduced. Particularly in the case of enzymes with high molecular weights, there is a possibility that the two molecules' enzymes can be approached closely through fusion with Fc, thereby suppressing the mutual screening effect of the active site that binds the substrate and the sudden decrease in efficacy due to the restriction of the short or coenzyme binding sites. exist. Lysosomes are a major site of intracellular digestion, see Dan, RT et al. , 1976, consists of membrane-encapsulated vesicles containing hundreds of enzymes, including more than 40 acid hydrolases capable of breaking down most biologically important polymers. Each enzyme has a specialized job of breaking down a specific class of molecules. Gene mutations affecting any one of these degrading enzymes result in the accumulation of lysosomes and the accumulation of large amounts of substances that they cannot degrade, which is referred to as "lysosomal storage disease (LSD)". . For example, Gaucher's disease is caused by a genetic defect in the production of an enzyme called glucocerebrosidase, which breaks down a carbohydrate called glucocerebroside. Reduction or loss of cerebrosidase activity results in lysosomal accumulation of glucocerebroside.

There are more than 30 lysosomal diseases, each of which is usually the result of a gene mutation, and the lack of certain lysosomal proteins usually results in a harmful accumulation of metabolites. For example, in Heller syndrome, Hunter syndrome, Morquio syndrome, and San Filippo syndrome, there is an accumulation of mucopolysaccharides; In Ty-Sachs syndrome, Gaucher syndrome, Crabbe syndrome, Niemann-Pick syndrome, and Fabry syndrome, accumulation of sphingolipids is present; In fucoside accumulation and mannose accumulation, there are accumulations of fucose-containing sphingolipids and glycoprotein fragments, and mannose-containing loligosaccharides, respectively (Cotran et al., Robbins Pathologi c Basi s of Di sease (4th ed. 1989)). There are no known therapies for lysosomal accumulation, but a number of different therapies have been studied for various LSDs, including bone marrow and umbilical cord blood transplantation, enzyme replacement therapy (ERT), substrate reduction therapy (SRT), and pharmacological heparon therapy. Gene therapy is also under development but not clinically tested. Among these treatments, ERT is best established, and ERT is the most versatile in the treatment of Gaucher's disease, Fabry's disease, Pumpe disease, MPS I, MPS II, One SRT drug is approved for the treatment of Gaucher disease, while the other is approved for the treatment of various LSDs, including MPS VI. As above, enzyme replacement therapy (ERT) is an established strategy for the treatment of several lysosomal accumulation diseases (LSD). Genetically deficient lysosomal enzyme precursors in LSD patients are provided by systemic injection. Enzymes internalized from external media by somatic cells reach the dysfunctional lysosomes and are activated there, fulfilling their normal potential. With regard to existing enzyme replacement therapies, systemically delivered enzymes do not cross the blood brain barrier and are therefore very limited for the treatment of CNS symptoms (Kaye, EM, 2001). The blood-brain barrier (BBB) resists the transport of therapeutic enzymes from the blood and therefore does not allow access to dysfunctional cells in the central nervous system. BBB is a capillary barrier comprising a continuous layer of tightly bound endothelial cells. BBB is described by Neuwelt, EA et al. 1980; Rappaport, S. I. , 1976, excludes molecules in the blood from entering the brain based on both molecular weight and lipid solubility. For example, BBB normally excludes molecules with molecular weights greater than 180 Daltons. It also occurs on the basis of similar exclusion lipid solubility.

[Detailed Description of the Invention]

[SUMMARY] ^"

Accordingly, the present inventors have completed the present invention by finding a hybrid Fc that increases the half-life and bioavailability, and the long-term distribution effect related to the etiology, regardless of the administration method without significantly reducing the activity of the enzyme and causing cytotoxicity. . It is an object of the present invention to provide a fusion protein conjugated with an enzyme and an immunoglobulin (Ig) hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4. Another object of the present invention is to provide a polynucleotide encoding the fusion protein. Another object of the present invention is to provide a recombinant vector containing the polynucleotide. Another object of the present invention is to provide a recombinant cell line transformed with the recombinant vector. 목적 Another object of the present invention is to cultivate the recombinant cell line; And (b) separating the fusion protein of the present invention from the cell culture. Another object of the present invention is a lysosomal accumulation. A pharmaceutical composition for preventing or treating a lysosomal accumulation disease comprising a fusion protein conjugated with a disease enzyme and a human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 ᅳ as an active ingredient. To provide. Another object of the present invention is to provide a lysosomal accumulation disease enzyme and a human: immunoglobulin hybrid Fc conjugated with an amino acid sequence of SEQ ID NO: 4 to a fusion protein conjugated with a lysosomal accumulation disease. Another object of the present invention is to prevent the lysosomal accumulation disease, characterized in that the lysosomal accumulation disease enzyme and the human immunoglobulin hybrid Fc conjugated with the amino acid sequence of SEQ ID NO: 4 conjugated to a subject in need thereof Or to provide a treatment method.

Technical Solution

In order to achieve the above object of the present invention, the present invention provides a fusion protein conjugated with an enzyme and human immunoglobulin (Ig) hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4. In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the fusion protein. In order to achieve another object of the present invention, the present invention provides a recombinant vector containing the polynucleotide. In order to achieve another object of the present invention, the present invention provides a recombinant cell line transformed with the recombinant vector. In order to achieve another object of the present invention, the present invention comprises the steps of (a) culturing the recombinant cell line; And (b) isolating the fusion protein of the present invention from the cell line culture medium. In order to achieve another object of the present invention, the present invention provides a prophylactic or therapeutic treatment for a lysosomal accumulation disease comprising a fusion protein conjugated with a lysosomal accumulation disease enzyme and a human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 as an active ingredient. It provides a pharmaceutical composition. In order to achieve another object of the present invention, the present invention provides a use for the preparation of a lysosomal accumulation disease of the lysosomal accumulation disease enzyme and the human immunoglobulin hybrid Fc conjugated with the amino acid sequence of SEQ ID NO: 4 for the prevention or treatment of lysosomal accumulation disease to provide. In order to achieve another object of the present invention, the present invention is characterized by administering an fusion protein conjugated with a lysosomal accumulation disease enzyme and a human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 to an individual in need thereof. Provides a method for preventing or treating lysosomal accumulation disease. Hereinafter, the present invention will be described in detail. The present invention provides a fusion protein conjugated with an enzyme and a human immunoglobulin (Ig) hybrid Fc followed by an amino acid sequence of SEQ ID NO. The hybrid Fc is represented by the amino acid sequence of SEQ ID NO: 4, comprising a hinge region, a CH2 domain and a CH3 domain from the N-terminal direction, wherein the hinge The region comprises at least a human IgD hinge region, wherein the CH2 domain comprises at least part of amino acid residues of the human IgD and IgG4 domains, wherein the CH3 domain comprises at least part of amino acid residues of the human gG4 CH3 domain. Due to the hybrid Fc of the present invention, the hinge region flexibility having an IgD than the enzyme Fc a unfused ^"does reduced efficacy size do not have a region binding to the binding receptors of the Fc with their neutrophil cells FcyR is this side effect Is reduced. Because IgG4 does not have an effect function such as complement mediated cytotoxicity, it can reduce unintended immune response and has the best ability to bind FcRn associated with intracellular recycling, resulting in increased stability. And increased serum half-life. ^"That is, while a hybrid of the Fc balttang maintains the activity of the enzyme is bonded, to prevent the side effects caused by the common Fc, also increase its half-life. The enzyme and hybrid Fc can be linked by a linker. The linker may be linked to the N-terminus of the hybrid Fc or the C-terminus of the enzyme. When the enzyme and hybrid Fc are separately expressed and then conjugated to each other, the coupling can be accomplished using any of several crosslinkers known in the art. Examples of crosslinking agents include 1,1-bis (diazoacetyl) -2-phenylethane (1,1-bis (diazoacetyl) -2-pheny 1 et hane), glutaraldehyde, 4- N-hydroxysuGcinimide esters, such as 4-azidosalicylic acid, 3,3'-dithiobis (succinimidylpropionate) (3,3 ' imidoesters, including disuccinimidyl esters such as -dithiobis (succinimidyipropionate), and bi functional raaleimides such as bis-N-maleimido-1,8-octane ), But is not limited thereto. In addition, the hybrid Fc of the present invention may bind the C-terminus of the biologically active enzyme at its N-terminus, or the C-terminus of the hybrid Fc and the N-terminus of the enzyme. Enzymes that can be conjugated with the hybrid Fc include, but are not limited to, glucocerebrosidase, alpha-L-iduronidase, and eduronate-2-sulfate. Izu (iduronat e-2-su lfatase), alglucosidase alfa, alpha-galactosidase-A, agalsidase alpha and beta, beauty Lil Colline Raise (butyrylcholinesterase), chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrase platelet-activating factor acetylhydrolase), neutral endopeptidase and myeloperoxidase, preferably glucocerebrosidase, alpha -L-edue Ronidayes (alpha-L-iduronidase), iduronate-2-sulfatase

(iduronate-2-sulfatase), alglucosidase alfa, alpha-galactosidase-A, agalsidase alpha, agalsidase beta Lysosomal activating enzyme, most preferably alpha-galactosidase-A, agalsidase alpha, agalsidase beta, glucocerebrosi Days, Eduro May be the 2-Sulfate. The enzyme of the present invention may preferably be a lysosomal storage disease enzyme. In the present invention, the term 'lysosomal storage disease (LSD)' refers to metabolic diseases caused by a deficiency in specific lysosomal proteins that result in the accumulation of various substances in the endosome / lysosomal compartment when activity is reduced or lost. It also refers to lysosomal accumulation, lysosomal accumulation, and the like. The 'deficiency' is

Means a significant decrease in the activity of the enzyme (to the extent that a malfunction is induced) or a state substantially free of enzyme activity, may be due to the cause of the non-expression, mutation or inactivation of the enzyme. In the present invention, the lysosomal accumulation disease is not limited as long as it is a metabolic disease caused by intracellular water accumulation related to deficiency of the lysosomal protein, but for example, aspartylglucosaminuria, cholesterol esters axis enemy ^'increased (cholesterol ester storage disease), May bottles (Wolman disease), ^City, Destin increase (cystinosis), the nonbyeong (Danon disease), Fabry disease (Fabry disease), Farber local granulomas increase (Farber 1 i ogranulomatosis ), Farber disease, fucosidosis, galactosial idosis types I / II, Gaucher disease types 1/11 / III, Gaucher disease, globoid cell leucodystrophy, Krabbe disease, II glycogen accumulation disease II ), Pompe disease, Ι / Ιί / ΠΙ GMl-gangHosidosis types 1/11/111, Type I GM2-gangliosidosis ¾12- 31 1 0 ^ 00 ^ 3 type I), Tay Sachs disease, type II & 1 \ 12-gangliosidosis ½¾12 ^ 31 110 ^ (10 ^ 3 type 11), ^ Shoffhoff disease, GM2-gangliosido GM2-gangliosidosis (AB variant), type I / II alpha-mannosidosis types 1/11, mannosidosis, met achromatic leucodystrophy, type I Mucol ipidosis type I, sialidosis types 1/11, Π / ΙΠ mucolipidosis, mucol ipidosis types ll / III 1-cell disease ), Type IIIC mucolipidosis-Herrel Mucolipidosis type IIIC pseudo-Hurl er polydys trophy, mucopolysaccharidosis type I, mucopolysaccharidosis type II, Hunter syndrome, Mucopolysaccharidosis type IIIA, Sanf i Πρρο syndrome, mucopolysaccharidosis type IIIB, type IIIC mucopolysaccharidosis type IIIA Mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA Morquio syndrome, mucopolysaccharidosis type IVB Morquio syndrome, VI Mucopolysaccharidosis type VI, Mucopolysaccharidosis type VII, Sliosis Group (Sly syndrome), mucopolysaccharidosis type IX, multiple sulphatase deficiency, neuronal ceroid lipofuscinosis, CLNl Batten ^' disease ), Type A / B Niemanxi-Pick disease types A / B, Niiemann-Pick disease, Type CI Niiemann-Pick disease type CI, Type C2 Nii Niemann Pick disease type C2, pycnodysostosis, Schindler disease types VII, Schindler disease, CM2 gangliosidosis, beta I Beta—mannosidosis, infanti le sialic acid storage disease and sal la (sialuria) disease _. Mucopl ipidosis (ML) IV, I-cell disease (MLII), and pseudo- Hurler polydystrophy, MLIII), including but not limited to lysosomal accumulation diseases selected from the group comprising. In the present invention, the term 'lysosomal storage disease enzyme' refers to a specific lysosomal protein (enzyme) that causes metabolic diseases by causing decomposition of various substances in the endosomal / lysosomal compartment when deficient. Means. In the present invention, the lysosomal accumulation disease enzyme is not particularly limited as long as it is a known lysosomal enzyme, for example, glucocerebrosidase, eduronate— 2-sulfate (iduronate-2-sulfatase) , Alpha-galactosidase A, agalsidase alpha, agalsidase beta, alpha-L-i duronidase, hepa Heparan—N-su If atase, alpha-N-acetylglucosaminidase, arylsul f atase A, galactosyl ceramids Galactosylceramidase, acid-alpha-glucosidase, thioesterase, hexosaminidase A, acid sphingomyelinase, alpha -Galactosidase and α-galactosidase Butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydride Platelet-activating factor acetylhydrolase, neutral endopeptidase, mucolipin 1, UPD-N-Ac-glucosaminyl phosphotransferase), beta-mannosidase and β-mannosidase and myeloperoxidase, but may be one or more enzymes selected from the group. Preferably, the lysosomal accumulation disease enzyme of the present invention is glucocerebrosidase, iduronate-2-sulfatase, alpha-galactosidase A (alpha-galactosidase A), agalsidase alpha and agalsidase beta may be one or more enzymes selected from the group consisting of. Although the proteins of the alpha galactosidase A have the same sequence, the agalsidase beta (repolalgal) produced in the agalsidase beta (fabrazyme) and human f ibroblast (HT-1080, etc.) cells produced in CH0 cells It is commercialized in two kinds. They have identical protein sequences but different patterns of sugar chains, resulting in differences in usage and dosage, and pharmacological effects are the same. The fusion protein in which alpha-galactosidase A and the hybrid Fc are conjugated is not limited thereto, but may be represented by the amino acid sequence of SEQ ID NO.

The fusion protein conjugated with the glucocerebrosidase and the hybrid Fc is not limited thereto, but may be represented by the amino acid sequence of SEQ ID NO. The fusion protein to which the duronate-2-sulfatase and the hybrid Fc are conjugated is not limited thereto, but may be represented by the amino acid sequence of SEQ ID NO: 8. In an embodiment of the present invention, a protein in which alpha ᅳ galactosidase A, glucocerebrosidase and eduronate-2-sulfate was fused with a hybrid Fc was gradated. As a result of sequencing analysis, alpha-galactosidase A-hybrid Fc shows the nucleotide sequence of SEQ ID NO: 2 and amino acid sequence of SEQ ID NO: 3, and glucocerebrosides-hybrid Fc shows the nucleotide sequence of SEQ ID NO: 5 The amino acid sequence of 6, it was confirmed that the eduronate-2-sulfatase-hybrid Fc has the nucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8. As a result of measuring the enzymatic activity of these fusion proteins, alpha-galactoside azu A-hybrid Fc maintained about 6 activities compared to the control (alpha-galactosidase A protein), and its half-life in the body was commercially available alpha. It was significantly longer than Galactosides A fillers (Fabrazyme and Replagal) (see Examples 5 and 7). Glucocerebrosides-Hybrid Fc had 26% of the activator activity compared to the control (glucocerebrosides protein), and eduronate-2-sulfate-hybrid Fc had the control substance (eduuronate-2). -Sulfatase protein) was about 12-fold higher activity (see Examples 15 and 16). Enzyme activity was confirmed to be different according to the type of enzyme binding to the hybrid Fc, the activity is different depending on the type of control used in the production method changes and testing, such as culture and purification conditions, but ultimately provided by the present invention There is no problem that the enzyme conjugate is not produced properly by the fusion of a relatively high molecular weight enzyme with the hybrid Fc, or the activity of the enzyme is canceled by a change in the tertiary structure. It was. Therefore, it can be seen that the hybrid Fc represented by SEQ ID NO: 4 of the present invention is suitable as an enzyme transporter.

^The present invention provides a polynucleotide (nucleic acid molecule) encoding the fusion protein conjugated with the enzyme and the hybrid Fc, a recombinant vector containing the polynucleotide, and a recombinant cell line transformed with the recombinant vector. The polynucleotides encoding the fusion proteins of the invention can be isolated from nature or prepared using chemical synthesis, can be single or double-stranded, and can be DNA molecules (genome, cDNA) or RNA molecules. When chemically synthesizing polynucleotides encoding the fusion proteins of the present invention, synthesis methods well known in the art, for example, Engels and Uhlmann, Angew Chem Int Ed Engl. 37: 73- 127, 1988), such as tryster phosphate, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, and ligonucleotide synthesis on solid supports. Vectors of the present invention include, but are not limited to, polsmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. The vector of the present invention may be a conventional cloning vector or expression vector, the expression vector being membrane targeting or secreted in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers (promoter). It includes a signal sequence or leader sequence for and can be prepared in various ways depending on the purpose. The polynucleotide sequence according to the present invention may be operably linked to an expression control sequence, wherein the operably linked gene sequence and the expression control sequence include a selection marker and a rep icat ion origin. It can be included in the expression vector of. “Operably l inked” may be genes and expression control sequences that are linked in such a way as to enable gene expression when the appropriate molecule is bound to the expression control sequences. "Expression control sequence" refers to a DNA sequence that regulates the expression of a polynucleotide sequence operably linked in a particular host cell. Such control sequence regulates a promoter, transcription, for transcription. And any operator sequence to encode a suitable _m RNA ribosomal binding site and a sequence to control the termination of transcription and translation. A selection marker for selecting the artillery, and, in the case of a replicable vector, a replication origin. Specifically, the alpha-galactosidase A-hybrid Fc fusion protein expression vector of the present invention may be a Gal-hyFc / pAD15 vector having a cleavage map disclosed in FIG. 1. In addition, the vector for expressing the glucocerebrosidase-hybrid Fc fusion protein may be a GCB-hyFc / pAD15 vector having a cleavage map disclosed in FIG. In addition, the vector for expressing the eduron-2-sulfatase-hybrid Fc fusion protein may be an IDS-hyFc / pAD15 vector having a cleavage map disclosed in FIG. 11B. Transformation with the vector can be carried out by transformation techniques known to those skilled in the art. Preferably, microprojectile bombardment, electroporat ion, calcium phosphate (CaP0 ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation,

PEG-mediated fusion, microinject ion, and liposome-mediated methods can be used, and the transformed recombinant cell can be a prokaryotic or eukaryotic cell line (animal, Plant, microbial cell line), preferably animal cell line. Examples of animal cell lines include CHO Chinese Hamster Ovary cells, Hepatocytes, Human Embryonic Kidney cells, Fibrosarcoma cells, VER0 cells, HeLa cells, W138, BHK, COS-7, 293, HepG2, 3T3 'NSO, NSl, RIN, MDCK cell line and HLFCHuman Lung Fibroblast) cells, CH0 cells are preferred among the animal cell lines. The present invention (a) culturing the recombinant cell line; And (b) isolating the fusion protein of the present invention from cell line culture medium. ^"The production method is carried out by culturing under suitable culture medium and conditions such that the polynucleotide is expressed that encodes the fusion protein of the invention in the recombinant vector introduced into the transformed cell of the present invention. Methods for culturing the transformed cells to express the recombinant protein are known in the art, for example, inoculating them in a suitable medium in which the transformed cells can be grown and cultured, and then contacting them with the culture medium. The expression of the protein can be induced by culturing in suitable conditions. Since then Isolation and purification of the fusion protein of the present invention induced expression in the previously transformed cells can be carried out through various separation and purification methods known in the art, for example, after lysing the cells, centrifuged the lysate Separation, salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange chromatography, reverse phase column chromatography and affinity chromatography Techniques may be applied alone or in combination to produce fusion proteins of the invention (Maniat is et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Gold Spring Harbor, NY (1982); Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Puricat ion Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990)). The present invention provides a pharmaceutical composition for preventing and treating a lysosomal accumulation disease comprising a fusion protein conjugated with the lysosomal accumulation disease enzyme and the human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 as an active ingredient. . The lysosomal accumulation disease enzyme and lysosomal accumulation disease are as described above. Specifically, the present invention provides a pharmaceutical composition for preventing or treating Fabry disease comprising a fusion protein conjugated with alpha-galactosidase A and a human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 as an active ingredient. to provide.

 Alpha-galactosidase A is a lysosomal enzyme that breaks down globotriaosy leer amide (Gtr3) into lactosy leer amide, which is composed of three sugars and one lipid called cer amide. Lack or lack of alpha-galactosidase A activity results in the accumulation of glycolipids, such as Gb-3, in the vessel wall, which impairs the function of tissues and organs, leading to Fabry disease, which presents a wide variety of symptoms. In another aspect, the present invention provides a pharmaceutical composition for preventing or treating Gaucher's disease, comprising a fusion protein conjugated with glucocerebrosidase and a human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 as an active ingredient.

Glucocerebrosides is a lysosomal enzyme that breaks down glucocerebroside. Lack of or lack of glucocerebrosidase activity causes glucocerebrosides to accumulate in macrophages, resulting in hypertrophy and result in Gaucher's disease. This onset. Gaucher cells accumulate in the spleen, liver, and bone marrow, and accumulate in the spleen when they accumulate in the spleen, and the spleen's activity is excessive, leading to anemia, fatigue and poor blood function, and sometimes neurological symptoms. It is divided into three types according to the time of onset and progression rate, and the appearance of neurological symptoms. Type 1 is chronic, adult, and no neurological symptoms. The most common type is acute and infant type. do. Type 3 is a subaqueous, juvenile type with neurological symptoms, but progression is slower than the type 2 and various clinical features. The present invention provides a pharmaceutical composition for the prevention or treatment of Hunter syndrome, comprising a fusion protein conjugated with a duronate-2-sulfatase and a human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 as an active ingredient. .

 Irononate-2-sulfatase is a lysosomal enzyme that breaks down glycosatninoglycan. Lack of and lack of eduron-2-sulfatase activity leads to coarse facial morphology, hypertrophy of the liver and spleen, and involvement of the central nervous system induces Hunter syndrome, which causes neurological symptoms and delayed development. Ultimately, premature death is associated with upper respiratory tract obstruction, cardiopulmonary disease, and malformations aftereffects. The pharmaceutical composition according to the present invention may include the fusion protein of the present invention alone or further include one or more pharmaceutically acceptable carriers, excipients or diluents. As used herein, 'pharmaceutically acceptable' refers to a composition which is physiologically acceptable and does not normally cause an allergic or similar reaction when administered to humans. Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like.

In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose, glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following references (Remington's Pharmaceutical Sciences, 19th ed. , Mack Publishing Company, East on, PA, 1995). The pharmaceutical composition for preventing or treating the lysosomal accumulation disease of the present invention can be administered to any mammal, including humans. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal Could be administration. In the above, transdermal administration 'means that the active ingredient contained in the pharmaceutical composition for preventing or treating lysosomal accumulation disease is delivered to the skin by administering to the cell or skin of the pharmaceutical composition of the present invention. For example, the pharmaceutical composition of the present invention may be prepared by injectable formulations, and may be administered by lightly pricking the skin with a 30 gauge thin injection needle, or directly applying to the skin. In addition, the pharmaceutical composition may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.

 In the case of preparations for oral administration, the compositions of the present invention are formulated using powders, granules, tablets, pills, sugar tablets, capsulants, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. Can be. For example, oral formulations can be obtained by tablets or dragees by combining the active ingredients with solid excipients and then grinding them, adding suitable auxiliaries and processing them into granular mixtures. Examples of suitable excipients include sugars and corn starch, wheat starch, rice starch and potato starch, including lactose, dextrose, sucrose, solbi, manny, xili, erythritol and malty. Layering agents such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including starch, cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropylmethyl- cellulose, and the like. In some cases, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical compositions of the present invention may further comprise anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives.

Formulations for parenteral administration may be formulated by methods known in the art in the form of injections, creams, lotions, external ointments, oils, humectants, gels, aerosols and nasal inhalants. These formulations are prepared by Remington ¹ s Pharmaceutical Science, 15th Edit ion, 1975. Mack Publishing, a prescription commonly known in all pharmaceutical chemistries. Company, East on, Pennsylvania 18042, Chapter 87: Blaug, Seymour.

^"The pharmaceutical compositions of the present invention is characterized in that the enzyme treatment was extended than that of the lysosomal storage diseases and the biological half-life, regardless of the method of administration that is non-commercial utilization is excellent associated specific organ distribution efficiency in the pathogenesis.

 Commercially available enzyme therapies for lysosomal accumulation (ex. Frabazyme) used in enzyme replacement therapy have been limited in their method of administration by intravenous administration. Because of the lack of crossover, the treatment of CNS symptoms was very limited.

 However, the fusion proteins of the enzyme of the present invention (particularly, lysosomal accumulation disease enzyme) and the hybrid immunoglobulin Fc of SEQ ID NO: 4 have a half-life and bioavailability even when injected into the body through other administration routes such as subcutaneous administration. Excellent (Examples 7, 9, and 10). In addition, the fusion protein of the present invention is not limited to the route of administration, but also distributed to the brain tissues in which the existing enzyme therapeutic agent was not distributed (acted), which has excellent distribution efficiency for various organs. (Examples 8 and 11).

As described above, the fusion proteins of the enzyme (particularly, the lysosomal accumulation disease enzyme) of the present invention and the hybrid immunoglobulin Fc of SEQ ID NO: 4 are not particularly limited in the route of administration, and preferably, intravenous (intravenous) or subcutaneous. Administration (subcutaneous injection). The total effective amount of the fusion protein of the present invention may be administered to a patient in a single dose, and may be administered by a fraction ionated treatment protocol administered for a long time in a multiple iple dose. Can be. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease. Preferably the preferred total dose of the fusion protein of the present invention may be from about 0.01 β to 1,000 mg, most preferably 0.1 to 100 mg per kg of patient body weight per day. However, the dose of the fusion protein may be determined based on various factors such as the age, weight, health condition, sex, severity of the disease, diet, and excretion rate, as well as the route and frequency of treatment of the pharmaceutical composition. Given this, one of ordinary skill in the art can determine the appropriate effective dosage for the particular use of the fusion protein as a prophylactic or therapeutic agent for lysosomal accumulation diseases. will be. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, route of administration and method of administration as long as the effect of the present invention is shown. Furthermore, the pharmaceutical composition may further include an anticoagulant lubricant, a humectant, a perfume, an emulsifier, and a preservative.

 Formulations for parenteral administration may be formulated in the form of injections by methods known in the art. These formulations are statements that are commonly known prescriptions for all pharmaceutical chemistries.

¾ (Remington's Pharmaceut i cal Science, 15th Edison, 1975. Mack Publ i shing Company, East on, Pennsylvani a 18042, Chapter 87: Blaug, Seymour). Furthermore, the pharmaceutical composition of the present invention can be administered in combination with a known compound having the effect of preventing or treating lysosomal accumulation disease. The present invention provides a use for the prevention or treatment of a lysosomal accumulation disease of the lysosomal accumulation disease enzyme and the fusion protein conjugated with human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4.

 The present invention provides a use for the preparation of a prophylactic or therapeutic agent for Fabry disease of a fusion protein conjugated with alpha-galactosidase A and a human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4.

 In another aspect, the present invention provides a use for the prevention or treatment of Gaucher disease of the fusion protein conjugated to glucocerebrosidase and the human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4.

 The present invention provides a use for the prevention or treatment of Hunter syndrome of the fusion protein conjugated with the duronate-2-sulfatase and the human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4. The present invention provides a method for the prevention of lysosomal accumulation disease, characterized in that the lysosomal accumulation disease enzyme and the human immunoglobulin hybrid Fc conjugated with the amino acid sequence of SEQ ID NO: 4 are conjugated to an individual in need thereof. Provide a method of treatment.

The present invention is represented by the alpha-galactosides A and the amino acid sequence of SEQ ID NO: 4 Provided is a method for preventing or treating Fabry disease, wherein the human immunoglobulin hybrid Fc conjugated fusion protein is administered to an individual in need thereof.

 In another aspect, the present invention is to prevent or treat Gaucher disease, characterized in that the fusion protein conjugated to glucocerebrosidase and the human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 is administered to an individual in need thereof. Provide a method.

The present invention provides a fusion protein conjugated with iduroneart-2-sulfatase and a human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 to an individual in need thereof in a ≤ amount. Provide methods for the prevention or treatment of medical treatment. The term 'subj ect' in the present invention may be an animal, preferably a mammal, particularly an animal including a human, or may be a cell, tissue, organ or the like derived from the animal. The subject may be a patient (pat i ent) in need of treatment. In the present invention, the term 'subject in need thereof' may be a subject in need of prevention or treatment of lysosomal accumulation disease. ^In addition, the term 'effective amount' in the present invention means an amount that indicates the effective effect, that is, the prevention or treatment of lysosomal accumulation disease in the subject to which the fusion protein of the present invention is administered. ^'

 The fusion protein of the present invention may be administered as it is or may be prepared and administered in various formulations as described above, preferably until a desired effect is obtained, i.e., until a prophylactic or therapeutic effect of lysosome accumulation disease is obtained Can be. The fusion protein of the present invention can be administered by various routes according to methods known in the art. Oral or parenteral, such as oral, intramuscular, intravenous, intradermal, intraarterial, intramedullary, intradural, intraperitoneal, intranasal, intravaginal, intrarectal, sublingual or subcutaneous, gastrointestinal tract, mucosa or hop It can be administered by the group.

Advantageous Effects

Fusion proteins conjugated with the enzyme of the present invention and the human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 maintain the enzyme activity while Regardless of the law, half-life and bioavailability have been prolonged over commercially available enzyme therapeutics, and the specific long-term distribution efficiency associated with the etiology is excellent, preventing or treating diseases caused by various types of enzyme deficiencies in the body, including lysosomal accumulation diseases. It is effective to prepare.

[Drawing and brief description]

Figure 1 shows a recombinant plasmid finally constructed by inserting DNA encoding a fusion protein (a -gal A-hyFc) conjugated with alpha-galactosidase A and hybrid Fc into the vector. Figure 2 shows the results confirmed by Western blotting the expression of the alpha-galactosidase A-hybrid Fc fusion protein of the present invention in CHO DG44 cells. Figure 3 is confirmed by Western blotting in non-reducing conditions and reducing conditions of the alpha-galactosidase A-hybrid Fc fusion protein of the present invention expressed in each cell line (Lane 1 ~ 12: Gal-hyFc Transfectant 1-12). 4 shows the results of qualitatively and quantitatively confirming the fractions of which UV values are measured after antibody affinity column chromatography with a culture medium recovered from an alpha-galactosidase A-hybrid Fc-expressing cell line. non—recognized in a reducing condition and a reducing condition. 5 is an analysis of the alpha-galactosidase A-hybrid Fc fusion protein of the present invention using RP-HPLC. Figure 6 shows the pharmacokinetic values of the alpha-galactosidase A-hybrid Fc fusion protein of the present invention as protein quantitative values (A) and enzyme activity values (B). FIG. 7 shows brain, spleen, kidney, heart, and liver tissues after 1 hour of administration of alpha ᅳ galactosidase A-Hybrid Fc fusion protein (A) and Fabrazyme (B) of the present invention to 5 mg / kg of SD rats. It was extracted and observed by Western blotting alpha-galactosidase A protein in each tissue (V: excipient group, Gal-hyFc: alpha-galactosidase A-hybrid) De Fc fusion protein administration group, Fabra: Fabrazyme administration group). FIG. 8 shows the brain, spleen, kidney, heart and liver tissues extracted 72 hours after the administration of the alpha-galactosidase A ᅳ hybrid Fc fusion protein and Fabrazyme to Fabry model mice. (Wi ld: normal rat). Figure 9 shows the pharmacokinetic values as protein quantitative values (A) and enzyme activity values (B) when the alpha-galactosidase A-hybrid Fc fusion protein of the present invention is administered by subcutaneous injection. FIG. 10 shows that the brain, spleen, kidney, heart, and liver tissues were extracted 48 hours after administration of the alpha-galactosidase A-hybrid Fc fusion protein of the present invention to 5 mg / kg of subcutaneous injection (SC) in SD rats. Proteins in each tissue were observed by Western blotting. 11 shows a fusion protein (GCB-hyFc) conjugated to glucocerebrosidase and hybrid Fc (FIG. 11 a) and a fusion protein conjugated to eduron-2-sulfatase and hybrid Fc (IDS-hyFc) ( Figure 11 b) shows the recombinant plasmid finally constructed by inserting the DNA encoding the vector into the vector. Figure 12 Days when a celebrity glucoside of the present invention in CHO DG44 three ^"for-shows the results confirm that the hybrid Fc fusion protein expression by Western blotting - a hybrid Fc fusion protein and carbonate-2-sulfamic te rise to yidyu Lane 1: GCB-hyFc, Trans feet ant 1; Lane 2: GCB-hyFc, Trans feet ant 2; Lane 3: IDS-hyFc, Trans feet ant 1; Lane 4: IDS-hyFc, Transfectant 2; Lane 5: GCB -hyFc, Trans feet ant 3; Lane 6: GCB-hyFc, Transfectant 4; Lane 7: IDS-hyFc, Transfectant 3; Lane 8: IDS-hyFc, Transfectant 4). Figure 13 shows the UV values after antibody affinity column chromatography with the culture of each cell line producing the glucocerebrosidase-hybrid Fc fusion protein and the duronate-2-sulfatase-hybrid Fc fusion protein. This highly measured fraction shows the results of qualitative and quantitative confirmation, which was confirmed under non-reducing conditions (A) and reducing conditions (B) (lane A: GCB-hyFC, Lane 2: IDS- in FIG. 13A). hyFc 13, Lane 1: GCB-hyFc production cell line culture medium, Lane 2: GCB-hyFc protein A ^■ Tablet f low through, Lane 3: GCB-hyFc protein A eluate, Lane 4: IDS-hyFc Cell line culture medium, Lane 5: IDS-hyFc protein A purified f low through, Lane 6: IDS-hyFc protein A eluate).

[Form for implementation of invention]

 Hereinafter, the present invention will be described in detail by way of examples.

 However, the following examples are merely to illustrate the present invention, the content of the present invention is not limited to the following embodiments.

<Example 1>

 Construction of Synthetic Synthetic Plasmids Encoding Fusion Proteins of α-galactosidase A with Hybrid Fc Fragments

 For gene cloning, the DNA of human alpha-galactosidase A (Genbank Accession No. BT007835) and hybrid Fc (SEQ ID NO: 1) were synthesized and fused, and finally, the entire gene sequence DNA (SEQ ID NO: 2) was obtained.

 Specifically, pAD15 vector was used for gene construction, and the insert DNA (human alpha-galactosidase A DNA) and vector (pAD15 vector containing hybr id Fc) were cut with EcoRI and BstEI I restriction enzymes and purified. Clean DNA was extracted. The final product inserted into the pAD15 vector was obtained by using l igat ion. The final product was inserted into efficient DH5 a competent cel l, plated on an agar plate containing ampici l l in antibiotics for antibiotic resistance genes in the vector, colonies were selected, and sequencing was performed to confirm the nucleotide sequence. The base sequence encoding the fusion protein is SEQ ID NO: 2, the amino acid sequence of the fusion protein is SEQ ID NO: 3, and the amino acid sequence of the hybrid Fc fragment is SEQ ID NO: 4.

 The recombinant plasmid finally constructed in Figure 1 is shown graphically. A 7,345 bp vector was constructed by inserting the DNA of alpha-galactosides A ᅳ Hybrid Fc obtained through synthesis into the cloning site of the pAD15 vector.

<Example 2>

Establishment of Alpha-galactosidase A-Hybrid Fc Fusion Protein Expressing Cell Line <2-l> quality introduction

 The final vector obtained through the cloning of Example 1 was introduced into CH0-DG44 mammalian cells to express the fusion protein.

Specifically, 24-wel l piate containing DNA in ⁵ × 10 ⁶ cells and introducing DNA into cells with a Neon ™ transfect ion system (Invi trogen) and containing 0.5 ml of medium (Hyclone SFM4CH0 with Hypoxanthine). Was added.

<2-2> expression cell line classification

 Hypoxanthine (HT) was used to fractionate cell lines expressing the alpha-galactosidase A-hybrid Fc fusion protein of the invention. The transfected CH0-DG44 cells transfected with the vector containing the dihydrofolate reductase (DHFR) gene and the fusion protein gene of the present invention obtained in Example 1 were replaced with a medium without HT after 72 hours. Expressing cell lines were selected by changing the medium every 3-4 days until colonies were formed.

Figure 2 is a result confirmed by Western blotting (westernbloUing, the same method as in Example 3) that the alpha-galactosidase A-Hybrid Fc fusion protein of the present invention is expressed in the cell. As shown in Figure 2 it can be seen that the fusion protein is normally expressed in cells and secreted into the medium. Selected cells were treated with various concentrations of methotrexate (MTX), and every 3-4 days ^' cells were selected while changing medium and reached 1-cel l / wel l in 96-wel l piate after reaching normal cell growth rate. Dispense and incubate until colony is formed. After about two weeks, when colony was formed, the medium was recovered, and cell lines with high expression level were selected using a dot-blot system (Bio-rad).

<2-3> Cellular productivity evaluation

 The protein-expressing medium was collected and the number of cells was counted 24 hours after replacing the medium of the cell transfected with the vector encoding the alpha-galactosidase A-hybrid Fc fusion protein of the present invention the day before. . Cellular productivity was measured using Human IgG quantitative ion ELISA kit (Betyl lab., Inc., E8 ()-104). In this experiment, 6 cell strains were used and numbered from 1 to 6 arbitrarily.

100 ^ each of the coating antibodies dilute in the coating solution was added to Nunc 96-wel l plates. And incubated at room temperature for 1 hour. The plate was washed 5 times with TBST (Tr is-buf fered saline Tween-20) solution and then added with a blocking buffer (200% / well) with 1% BSA (Bovine serum albumin) for 30 minutes. In fact, it was left at. The plate was washed five times with TBST washing solution, and then 100% of the standard solution diluted in 1/2 to 0ng / ml ~ 500ng / ml or diluted media in an appropriate proportion. After 1 hour incubation at room temperature, washed 5 times with TBST washing solution and diluted to 10 / 100,000 HRP (Horse radish peroxidase) detection antibody (Detection antibody) was added 100 / well ^'and incubated at room temperature for 1 hour. Wash 5 times with TBST washing solution, add 100 / we 11 TMB substrate solution, and react for 15 minutes at dark room temperature. Then, stop the reaction by adding 100 fd / mll reaction termination solution and measure the absorbance with a plate reader at 450 nm. The results are shown in Table 1. One unit productivity was obtained by dividing the mass calculated by ELISA by the total cell number to obtain pg / cell / day (pcd).

 [Table 1] Unit productivity evaluation

 As shown in Table 1, it can be seen that several cell lines selected 1 to 6 express the fusion protein with various pcd values.

<Example 3>

 Expressed Protein through Western Blotting of Alpha—Galactosides A-Hybrid Fc ¾

The protein expressed in each cell line (transfectant 1-12 transduced with Gal-hyFc) was verified by western blotting. A medium sample of the protein expressed in the Eppendorf tube (E-tube) was added, a loading dye was added to IX, and a 6¾ SDS-PAGE gel was mounted in a running ^' tank and electrophoresed between 80 and 100 V. Proteins were transferred to PVDF membrane using Trans-Blot SD Semi Dry Transfer Cell (Bio-Rad). After transfer, the membrane was blocked for 10 minutes at room temperature using TBST containing 5% skim milk powder. IX TBST to goat human IgG-HRP antibody (Santacruz biotechnology) was diluted to 1/2000, adhered to the membrane for 1 hour to 2 hours at room temperature, and washed three times with IX TBST for 5 minutes. ECL soul t ion (iNtRON) was evenly soaked in membrane, and the signal was confirmed in chemidoc (Bio-rad). The results are shown in FIG. 3. As shown in Figure 3, the fusion protein expressed in each cell line has a molecular weight of about 80 kDa when the disul f ide-bond is removed, Fc forms a dimer when the disul f ide-bond is not removed It can be confirmed that the molecular weight of about 170 kDa, two sizes.

<Example 4>

Purification and Concentration of Alpha-Galactosidase A-Hybrid Fc Synthesized Protein Cells having a pore size of 0.2 μπι were obtained by culturing the medium obtained as adherent cell production using the transfectant cell line prepared in Example 2. The impurities were removed by filtration on the membrane and the filtered medium was prepared by storing in 4 ^° C or ice until loading to the column. The column was stratified with Mabselect Sure (GE healthcare), one of a class of antibody-affinity column resins labeled with Protein A protein, and mobile phases were prepared. First, buffer A (20 mM sodium phosphate, 150 mM NaCl, PH7.2) was prepared to equilibrate the column, and low pH buffer B (lOOmM sodium ci trate, pH3.0) was prepared to elute the bound protein under acidic conditions. . The antibody affinity column was connected to a line of chromatography (AKTA puri ier, GE healthcare) system with buffer A and the resin was equilibrated to 10 CV (column volume). After confirming that the UV values were equilibrated, pre-prepared alpha-galactosidase A 준비 hybrid Fc fusion proteins were loaded onto the column by pump loading of FPLC to carry out antibody affinity chromatography. After binding by lowering the pH of the complete layer B, the eluted protein was recovered by fractionation of 200 ^. The recovered fraction was ^"Chemistry of the UV to determine the value of 0.015 or more fractions collected only into the neutralization solution (1M Tris-Cl, pH8.0) . The fractionated protein was identified as non-reducing phase in 8¾ SDS-PAGE and reducing phase protein size in 10% SDS-PAGE (FIG. 4).

 The amino acid sequence analysis of the fusion protein, it was confirmed by SEQ ID NO: 3.

Example 5 Determination and Comparison of Enzyme Activity between ALPHA-Galactosides A-Hybrid Fc of the Present Invention

4-methyhimbel liferyl-α-El-gal act opyranoside (Sigma) was used as a substrate to compare the substrate resolution of Fabrazyme (Genzyme) and alpha-galactosidase A-Hybrid Fc fusion protein expressed in the present invention. It was. Dilute the Fabrazyme and Alpha 2-galactosides A ᅳ hybrid Fc fusion protein (Gal-hyFc) with ^'' assay buffer (50 mM sodium citrate, 50 mM NaCl, pH4), respectively, and use the 4-methylumbelliferyl-aD-galactopyranoside assay buffer. Dilute the protein and substrate 50 ^ by diluting it into 800μΜ concentration in black 96-well piate. 6) «^ 3 ^ 011 is 365nm wavelength and 611 ^ 5 ^ 011 is kinetic for 5 minutes at 445 ^ wavelength. Measured in mode. Specific activity was calculated by calibrating the immediate Vmax by standard 4-methyl umbel 1 iferone ≦. Calculated by multiplying the conversion factor (N = 3, specific activity SEM). As a result, as shown in Table 2, the alpha-galactosidase A-hybrid Fc fusion protein of the present invention had activity of 0.9X10 ⁶ pmol / minA «g, and the control group was 1.4XK) ⁶ pmol / min / // shows g-activity, the alpha-galactosidase A-hybrid Fc fusion protein of the present invention maintains 60% of its weight relative to Fabrazyme, which is conventionally used for Fabry disease treatment, and converted to molarity. It was confirmed that the level was almost the same.

[Table ² ]

In vitro Activity Comparison of Alpha-galactosidase A-Hybrid Fc (Gal-hyFc) and Fabrazyme

<Example 6>

Alpha-galactosidase A-Hybrid Fusion Protein Analysis by Reverse Phase HPLC (RP-HPLC) RP-HPLC analysis was performed on alpha-galactosidase A-hybrid Fc fusion protein purified using Mabselect Sure (GE healthcare), one of the types of antibody-affinity column resin labeled Protein A protein. . RP-HPLC is acetonite reel containing 0.095% TFA in tertiary distilled water containing 0.1% trifluoroacetic acid (TFA) at a flow rate of l.Oml / min using an Xbridge BEH300 C4 (4.6 x 150 mm) column. Protein was eluted using a linear gradient of (35-55% in 40min). Elution peak was observed at 280nm. 5 shows that the protein of the present invention was analyzed using RP-HPLC. A single peak was confirmed at 280 nm at 12.5 minutes. Whereas both Fabrazyme and Replagal products have one or both of the two leucine amino acid sequences present at the C-terminus, three peaks are identified by RP-HPLC analysis (Lee et al. Glycobiology, 13: 305-313 , 2003), the alpha-galactosidase A-hybrid Fc fusion protein was found to be more favorable for quality control than the previous two products because the C terminus is protected by Fc and observed as a single peak in RP-HPLC analysis.

<Example 7>

Pharmacokinetics of Alpha-galactosidase A-Hybrid Fc SD rats weighing about 250 g were intravenously administered Gal-hyFc protein expressed in the present invention at 5 mg / kg and concentration, respectively. Gal-hyFc protein expressed in the present invention which remains in plasma by taking a blood sample before administration, 5, 10, 20, 30, 60, 120, 240, 360, 480, 1440, 2880, 4320, 8640 minutes after administration The concentration and activity of the was measured. Protein concentration was measured using ELISA. Affinity purified Human IG capture antibody (Bethyl Laboratories, Inc.) diluted in the coating solution was added 100 N each to Nunc 96 well plates and allowed to stand at room temperature for 1 hour. Antibody-coated plates were washed five times with TBST (Tr is—Buffered Saline Tween-20) wash solution, followed by adding 200% of blocking buffer with 1% BSA (Bovine serum albumin) per well for 30 minutes at room temperature. Replied at. The plate was washed five times with TBST washing solution, and then 100 parts of plasma samples diluted in half or 0 to 500 pg / ml or appropriate proportions were added and allowed to stand at room temperature for 1 hour. Wash 5 times with TBST washing solution and dilute HRP Conjugated Human IgG Detection Antibody to 100 ^ for each well. The shoots were allowed to react for 1 hour at room temperature, and the plates were washed 5 times with TBST washing solution, and then mixed with TMB substrate solution per well for 15 minutes at dark room temperature. And the absorbance was measured with a late reader at 450nm. The protein concentration was calculated by multiplying the amount calculated from the ELISA by the dilution factor to calculate the protein concentration in plasma.

 Activity was recovered by diluting the recovered plasma with assay buffer (50mM sodium citrate, 50mM NaCl, pH4.0). 4-methyhimbelliferyl—α-D-galactopyranoside, which is diluted to 800 μΜ in assay buffer, is then mixed with 50 / ^ of diluted plasma and substrate in a black 96-well plate, followed by excitation at 365 nm wavelength and emission at 445 nm. It was measured in kinetic mode for 5 minutes at the wavelength. Specific activity was determined by Vmax of cal ibrat ion standard 4-me t hy 1 umbe 11 i f e r one ≦. Calculated by multiplying the conversion factor (N = 3, specific activity SEM). FIG. 6 shows the pharmacokinetic values of alpha-galactosidase A-hybrid Fc of the present invention as protein quantitative values (A) and enzyme activity values (B). Has a terminal half-life of more than 20 hours, which is much longer than the two-hour half-life of Fabrazyme and Replagal on the market for Fabry disease.

<Example 8>

Tissue Distribution Measurement of Alpha-Galactosidase A-Hybrid Fc SD rats weighing about 250 g were administered intravenously with the protein expressed in the present invention and Fabrazyme (Genzyme) at a concentration of 5 mg / kg, respectively. In the non-drug group, the same volume excipient (20 mM sodium phosphate, 165 mM mannitol, pH7.0) was administered in the same manner as the drug-treated group. After 1 hour of administration, liver, heart, kidney, spleen and brain were removed, lysis buffer (27 mM citric acid, 46 mM sodium phosphate dibasic, 0.15% Triton X— 100, pH4.6) was added, crushed on ice and centrifuged. Only the supernatant was recovered. The recovered supernatant was confirmed by Western blotting using anti-GLA antibody (Abeam) on SDS-PAGE. 7 shows Alpha-galactosides A—Hybrid Fc (A) and Fabrazyme (B), respectively. Fabrazyme and alpha-galactosidase A-hybrid Fc were identified by West turn blotting using ant i-α gal actos idase A ant ibody (Abeam) in the protein of each tissue extract after administration to SD rats. Alpha-galactosidase A is rarely or very weakly expressed in the brain and heart, whereas alpha-galactosidase A-hybrid Fc has been observed in all tissues including the brain. This means that the distribution of alpha-galactosidase A-hybrid Fc may be different from that of Fabrazyme, suggesting the possibility of distributing it to tissues in which Fabrazyme is not well distributed. As shown in FIG. 7A, not only alpha-galactosidase A-hybrid Fc fusion protein bodies but also proteins of alpha-galactosidase A molecular weight size were observed, which resulted in some alpha ᅳ galactosidase in each organ. It is necessary to confirm whether A and Fc are cleaved or are the result of protein extraction process.

Example 9

Measurement of the effect of increasing alpha-galactosidase A activity in Fabry model rats 3 days (72 hours) by intravenous administration of alpha-galactosidase A-hybrid Fc and Fabrazyme to Fabry model rats without alpha-galactosidase A activity ), Liver, spleen, kidney, kidney and liver were extracted for enzyme activity. Two normal rats, two Fabry-model rats with an excipient (20 mM sodium phosphate, 165 mM manni tol, pH7.0), alpha-galactosidase A-hybrid Fc at lmg / kg and 2 mg / kg The activity was compared with three rats each and two rats receiving Fabrazyme lmg / kg. The activity of the enzyme was performed by the same method as described in <Example 7>. FIG. 8 shows the activity of Fabrazyme and Alpha-galactosidase A-Hybrid Fc after 72 hours in Fabry model rats. The enzyme activity was not measured in all tissues in the excipient-only group. In the livers of the Fabrazyme and alpha-galactosides A-Hybrid Fc groups, activity was measured above normal rats, and cardiac activity was measured at normal rat levels. However, the spleen, kidney and brain were below normal rat levels and Fabrazyme activity was high in the spleen, but the activity of alpha-galactosidase A-hybrid Fc group was high in the kidney, especially in the brain. In the alpha-galactosidase A-hybrid Fc-administered group, both lmg / kg and 2mg / kg showed approximately 4¾ of activity in normal rats. It became. These results suggest that alpha-galactosidase A-Hybrid Fc shows concentration-dependent distribution and activity of each organ and may improve the distributional weakness in kidney and heart, which is actually a clinical problem of Fabrazyme. In particular, as shown in FIG. 7A, as well as protein expression results in the brain not observed in Fabrazyme, enzymatic activity confirmation in brain tissues represents the specificity of the alpha-galactosidase A-hybrid Fc. By not crossing the Blood brain barrier, I can improve the fatal weakness that does not improve brain lesions ^; It shows the possibility of Fc fusion protein as a new concept treatment.

<Example 10>

 Bioavailability test of alpha-galactosides A-hybrid Fc fusion protein Subcutaneous expression of the protein alpha-galactosides A-hybrid Fc fusion protein expressed in the present invention to male rats weighing about 250 g was 5 mg / kg. Blood samples were taken by injection and intravenous injection, and blood samples were taken before administration, 5, 10, 20, 30, 60, 120, 240, 360, 480, 1440, 2880, 4320, 8640 minutes before administration. The concentration and activity of the protein expressed in the invention was measured. Protein concentration and activity were measured in the same manner as in <Example 7>. FIG. 9 shows that the pharmacokinetic values were confirmed as protein quantitative values (A) and enzyme activity values (B) when the alpha-galactosidase A-Hybrid Fc fusion protein of the present invention was administered by subcutaneous injection. It was confirmed that the terminal half-life (terminal hal f ᅳ li fe) in which the lactosidase A-hybrid Fc fusion protein persists in the blood is more than 50 hours. Alpha-galactosidase A—hybrid Fc fusion protein has a BA of 30-70%, while bioavailability of bioplai labi li ty, BA 10-25% (see US2010 / 0291060 Al) when administered by subcutaneous injection This is due to the low bioavailability of conventional enzyme treatments, which is typically possible only by slowly injecting a high-volume protein composition into the vein for 1-4 hours, whereas the half-life of the drug through fusion between the enzyme and Fc of the present invention and Increased bioavailability confirms the possibility of administration through various routes such as dose reduction and subcutaneous injection.

<Example 11>

Tissue upon administration with alpha-galactosidase A-hybrid Fc fusion protein subcutaneous injection The weight distribution measuring 250g degree of SD rat the expressed protein in the present invention the male alpha-galactosyl side rise A- hybrid Fc administration of the fusion protein by subcutaneous injection with 5mg / kg and between the back 48 ^hours', kidney, heart, The spleen and brain were extracted and the enzyme was crushed on ice using lysi s buffer (27 mM citric acid, 46 mM sodium phosphate dibasic, 0.15% Tr i ton-X 100, pH4.6) and centrifuged to recover only the supernatant. . Proteins were quantified by BCA quantification, proteins were isolated using SDS-PAGE, and alpha ᅳ galactosidase A-hybrid Fc fusion proteins were identified by Western blotting using ant i-GLA ant ibody. 10 shows that the alpha-galactosidase A-hybrid Fc fusion protein was confirmed by Western blotting, and it was confirmed that the protein was also observed in all tissues including the brain through subcutaneous injection. These results suggest that Fabrazyme and Replagal, as well as target organs such as heart and kidney, are rarely distributed even when alpha-galactosidase A-hybrid Fc fusion protein is administered by various routes such as subcutaneous injection rather than conventional intravenous injection. It is distributed in the poor brain, suggesting the possibility of developing therapeutics of various usages that overcome the limitations of existing treatments and increase patient convenience.

<Example 12>

 Construction of Recombinant Plasmid Encoding Fusion Protein of Glucocerebrosidase and Hybrid Fc Fragment and Fusion Protein of Eduronate-2-Sulfate and Hybrid Fc Fragment

For gene cloning, human glucocerebrosidase (Genbank Accession No. 丽 _000157) and human eduronate-2-sulfatase (Genbank Accession. 醒 _000202), respectively, were synthesized with DNA of hybrid Fc (SEQ ID NO: 1). After synthesis and fusion, the entire gene sequence DNA of SEQ ID NO: 5 and SEQ ID NO: 7 encoding the respective fusion proteins was finally obtained.

Specifically, the pAD15 vector was used for gene construction, and EcoRI was used as the insert DNA of human glucocerebrosidase and human eduronate-2-sulfatase DNA and vector (pAD15 vector including hybr id Fc). And cut with BstEI I restriction enzyme After purification, the purified DNA was extracted. The resulting vector and each insert DNA were inserted into the pAD15 vector via l igat ion. The final product was inserted into an efficient DH5 a competent cel l and plated on an agar plate containing an ampici ll in antibiotic that matches the antibiotic resistance gene in the vector, colonies were selected, and sequenced. The base sequence encoding the fusion protein is SEQ ID NO: 5 in which the glucocerebrosidase and the hybrid Fc are fused, and DNA in which the eduronate-2-sulfatase and the hybrid Fc is fused is SEQ ID NO: 7. The amino acid sequence of the Glucocerebrosidase-hybrid Fc fusion protein is SEQ ID NO: 6, and the amino acid sequence of the eduron-2-sulfatase-hybrid Fc fusion protein is SEQ ID NO: 8. The amino acid sequence of the hybrid Fc fragment is SEQ ID NO: 4.

 The recombinant pollamide finally constructed in Figure 11 is shown as a picture. A glucoserbrosidase-hybrid Fc DNA obtained by synthesis was inserted into the cloning site of the pAD15 vector to prepare a vector having a size of 7,672 bp (FIG. 11 a), and the duronate-2-sulfatase obtained by the same method. DNA of hybrid Fc was inserted to prepare a vector having a size of 7,714 bp (FIG. 11 b).

Example 13

 Establishment of Glucoprotein Expression Cell Line of Glucocerebrosidase-Hybrid FcWireuronate-2-Sulfate-Hybrid Fc

<13-1> Introduction of quality

 The final vector obtained through the cloning of <Example 12> was introduced into CH0-DG44 mammalian cells, respectively, to express the fusion protein.

Specifically, DNA was incorporated into ⁵ × 10 ⁶ cells and the Neon ™ transfect ion system

(Invi trogen) each DNA was introduced into cells and added to a 24-wel l plate containing 0.5 ml of medium (Hyclone SFM4CH0 with HT).

<13-2> Expression cell line classification

Using HT, cell lines expressing the glucocerebrosidase-hybrid Fc of the present invention and the eduronate-2-sulfate-hybrid Fc fusion protein were fractionated, respectively. After 72 hours, HT was not contained in CH0-DG44 cells transduced with the vector containing the DHFR gene and each of the fusion protein genes of the present invention obtained in Example 12. Was replaced with medium. Expression cell lines were selected with medium replacement every 3-4 days until colonies were formed.

 FIG. 12 shows Western blotting using Human IgG ant ibody (Santa Cruz Biotechnology) to express glucocerebrosidase-hybrid Fc and eduronate 2-sulfate-hybrid Fc fusion protein of the present invention in cells. The result is confirmed. As shown in Figure 12, it can be seen that the fusion protein is normally expressed in cells and secreted into the medium. In addition, as shown in FIG. 12, each of the fusion proteins expressed in each cell line had a molecular weight of about 95 kDa when Glucocerebrosidase-Hybrid Fc was removed when disul f ide-bond was removed. -2- Sulfatase-Hybrid Fc has a molecular weight of about 106 kDa, and when the disul fide-bond is not removed, Fc forms a dimer, and each of the fusion proteins has a molecular weight of 190 kDa and 212 kDa. Can be.

<Example 14>

Purification and Concentration of Glucocerebrosidase-Hybrid FcWyduronate-2-Sulfate-Hybrid Fc Fusion Protein The glucocerebrosidase-Hybrid Fc or Edu of the present invention obtained in Example 13 above was obtained. The cultures recovered from each of the Ronate-2-Sulfate-Hybrid Fc expressing cell lines were filtered through a 0.2 um pore size cell filter to remove impurities and the filtered medium was loaded until the column was loaded. Prepared by storage at ° C or ice bath. A column was filled with Mabselect Sure (GE healthcare), one of a kind of antibody-affinity column resin labeled with Protein A protein, and a mobile phase was prepared. First, prepare buffer A (20 mM sodium phosphate, 150 mM NaCl, pH 7.2) to equilibrate the column, and low pH supernatant B (lOOmM sodium ci trate, pH 3.0) to elute the bound protein under acidic conditions. Ready. A complete cheungaek by chromatography (AKTA puri f ier, _GE. Healthcare) connecting the antibody affinity columns of the line of the system to equilibrium and gave a resin with 10 CV (column volumes). After confirming that the UV values are equilibrated, the previously prepared glucocerebrosidase-hybrid Fc and eduronate-2-sulfate-hybrid Fc fusion proteins were loaded onto the column by pump loading of FPLC, respectively, to carry out antibody affinity chromatography. Photography was performed. The pH was lowered with the complete solution B, and then the eluted protein was recovered by fractionation. Recovered fractions are checked for UV values to at least 0.05 Only the fractions were collected and neutralized with neutralizing solution (1M Tris-Cl, ρΗ8 · 0). The protein obtained by fractionation was confirmed protein size by SDS-PAGE on non-reducing and reducing (Fig. 13). As a result of amino acid sequence analysis of the fusion protein, glucocerebrosidase—Hybrid Fc was identified by SEQ ID NO: 6, and eduronate-2-sulfate-hybrid Fc was identified by SEQ ID NO: 8.

<Example 15>

 Measurement and Comparison of Enzyme Activity of Glucocerebrosidase-Hybrid Fc

Comparing the substrate resolution of recombinant human glucocerebrosidase (rhGBA, R & D Systems) and the glucocerebrosidase-hybrid Fc fusion protein expressed in the present invention. 4 methyl umbel 1 i feryl-β -D-glucopyranoside (Sigma) was used as a substrate. GBA protein and glucocerebrosidase-hybrid Fc fusion protein were diluted in assay buffer (50 mM Sodium Citrate, 25 mM Sodium Choiate, 5 mM DTT, pH 6.0), respectively, and 4_methylumbelliferyl ᅳ β ᅳ D_glucopyranoside was also used as assay buffer. Diluted by concentration, the protein and substrate were mixed 25 / ^ in black 96 well plate and reacted at 37 ^° C for 20 minutes. Excitation was measured at 365nm wavelength and emission was measured at 445nm wavelength. Specific activity is measured after the type gwanggap banung divided by time and the enzyme amount was calculated by multiplying the calibration standard 4- methylumbel 1 iferone≤- determined ^{conversion factor '(Ν = 3,} specific activity土SEM) ·

 As a result, as shown in Table 3, the glucocerebrosidase-hybrid Fc fusion protein of the present invention exhibited an activity of 809 pmol / min /, and the control group exhibited an activity of 3062 pmol / min / lig, which is about 26.4 at the same weight. It was confirmed to maintain% activity. Since the glucocerebrosidase-hybrid Fc fusion protein has a molecular weight of about 30% higher than that of the control substance, the enzyme activity corresponds to about 40% of the control substance in terms of molarity. Since glucocerebrosidase has a large change in enzyme activity and productivity depending on culture and purification conditions (Humphreys JD & Ihler GJ, Lab. Clin. Med., 96: 682, 1980), Because it is not a state, the enzyme activity was measured lower than that of the control material, and it is expected that further activity can be increased through the change of the culture method and the method later.

Table 3 In vitro activity comparison with Glucocerebrosidase-Hybrid Fc (GCB-hyFc) and recombinant human glucocerebrosides (rhGBA)

<Example 16>

 Comparison and Comparison of Iduronate-2-Sulfate-Hybrid Fc Enzyme Activity

In order to compare the substrate resolution of the recombinant human eduronate 2-sulfatase (rhlDS, R & D Systems) and the eduron-2-sulfatase-hybrid Fc fusion protein expressed in the present invention, 4-nitrocatechol sulfate ( Sigma) was used as substrate. rhlDS protein and eduronate-2-sulfate-hybrid Fc fusion protein were ligated into assay buffer (50 mM sodium acetate, 100 mM NaCl, pH 5.0), respectively, and 4-nitrocatechol sulfate was also added to the assay buffer at 2 mM concentration. Diluted, the protein and substrate were mixed by 50 ^ and reacted at 37 ^° C for 24 hours. In order to stop the reaction, 50 μί of 0.2M NaOH was added and transferred to a transparent 96 weir plate, and the absorbance was measured at 510 nm. Specific activity was calculated by multiplying the measured absorbance by the conversion standard obtained from calibration standard P-nitrocatechol, then dividing by reaction time and amount of enzyme (N = 3, specific activity 士 SEM).

 As a result, as shown in Table 4, the invention and the duronate-2-sulfate-hybrid Fc fusion protein showed the activity of 14.2 pmol / min / ^ g, the control group showed the same 1.2 pmol / min / / zg activity It was confirmed that the activity was about 12 times higher in weight than the control group.

Table 4

Comparison of in vitro activity of eduron-2-sulfate-hybrid Fc (IDS-hyFc) with recombinant human eduronate -2-sulfate (rhlDS)

Industrial Applicability

 As described above, the present invention relates to a hybrid immunoglobulin Fc and a fusion protein of an enzyme, and more particularly, a fusion protein conjugated with a human immunoglobulin hybrid Fc represented by an amino acid sequence of SEQ ID NO. It relates to a manufacturing method.

 . The fusion proteins conjugated to the enzyme of the present invention and human immunoglobulin hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4 maintain the activity of the enzyme, and are commercially available enzyme therapeutic agents having a half-life and bioavailability regardless of the administration method. It is more prolonged and has an excellent efficiency of specific organ distribution related to the etiology, and thus is highly industrially useful as it is effective in preparing a prophylactic or therapeutic agent for diseases caused by various kinds of enzyme deficiency including lysosomal accumulation disease.

Claims

[Claim]

 [Claim 1]

 A fusion protein conjugated with an enzyme and a human immunoglobulin (Ig) hybrid Fc represented by the amino acid sequence of SEQ ID NO: 4.

[Claim 2]

 The fusion protein of claim 1, wherein the enzyme is a lysosomal storage disease enzyme.

[Claim 3]

 The method of claim 2, wherein the lysosomal animal disease enzyme is glucocerebrosidase, eduronate-2-sulfatase, alpha-galactosidase A (alpha-galactosidase A) ), Agalsidase alpha, agalsidase beta, alpha-L- iduronidase, heparan-N-suIfatase , Α-Ν-acetylglucosa minidase, arylsulfatase A, galactosylceramidase, acid-alpha-glucosidase ), Thioesterase, hexosaminidase A, acid sphingomyelinase, alpha-galactosidase and butyrylcholinesterase , Chitinase, glutamate Glutamate decarboxylase, imiglucerase, lipase, uri case, platelet-activating factor acetylhydrolase, neutral endopeptides ( nuetral endopept i dase, Mucolipin 1, UPD-N-Ac-glucosaminyl phosphotransferase, β-mannosidase And one or more enzymes selected from the group consisting of myeloperoxidase.

[Claim 4]

4. The method of claim 3, wherein the lysosomal accumulation disease enzyme is glucocerebrosidase, eduronate—iduronate-2-sul fatase, alpha- A fusion protein, characterized in that it is at least one enzyme selected from the group consisting of galactosidase A, agalsidase alpha and agalsidase—a beta.

[Claim 5]

 A polynucleotide encoding the fusion protein of claim 1.

[Claim 6]

 The polynucleotide of claim 5, wherein the polynucleotide is represented by any one nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 7. 7.

[Claim 7

 A recombinant vector containing the polynucleotide of claim 5.

[Claim 8]

 A recombinant cell line transformed with the recombinant vector of claim 7.

[Claim 9]

 (a) culturing the recombinant cell line of paragraph 8; And

 (b) A method for producing the fusion protein of claim 1, comprising the step of separating the fusion protein of claim 1 from the cell line culture.

[Claim 10]

 Claim 3 composition for the prevention and treatment of lysosomal storage disease (lysosomal storage disease) comprising the fusion protein.

[Claim 11]

The method of claim 10, wherein the lysosomal accumulation disease is aspartylglucosaminuria, cholesterol ester storage disease, Wolman disease, cyst inosis, Danone disease ( Danon di sease), Fabry disease, Farber liposarcoma 1 ipogranulomatosis, Farber disease, fucosidosis, galactosialidosis types I / II, Gaucher disease types I / II / III), Gaucher disease, globoid cell leucodystrophy, Krabbe disease, II glycogen storage disease II, Pompe disease, Ι / Π / ΙΠ GM1—ganglisidosis types 1/11/111, GM I gangliosidosis type 1, Tay Sachs disease, type II GM2- Gangliosidosis cis 2 ᅳ & 110 (10 ^ 3 type II), Sandhof f disease, GM2—Ganglio sidosis (AB variant), type I / II alpha-mannose accumulation ( alpha- mannosidosis types 1/11), mannosidosis '' metachromatic leucodystrophy, type mucol ipidosis type I, type I / II Sialidosis types 1/11, ΙΙ / ΙΠ mucolipidosis, mucolipidosis types ll / III 1-cell disease, type IIIC mucolipidosis-like-Heller polydistrophy ( mucolipidosis type IIIC pseudo-Hurl er polydys trophy, mucopolysaccharidosis type I, mucopolysaccharidosis type II, Hunter syndrome, and ΙΠΑ ^'' mucopolysaccharidosis type I Mucopolysaccharidosis type IIIA, Sanfilippo syndrome, mucopolysaccharidosis type 11 IB, mucopolysaccharidosis type IIIC, type IIID mucopolysaccharides (Mucopolysaccharidosis type IIID), mucopolysaccharidosis type IVA Morquio syndrome, mucopolysaccharidosis type IVB Morchio syndrome (mucopolysaccharidosis type IVB Morqui) o syndrome, mucopolysaccharidosis type VI, type VII mucopolysaccharidosis type VII, Sly syndrome, type IX mucopolysaccharidosis ( _{腿 copo} l _ysacc haridosis type IX), multiple sulphatase deficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease, A / B type Niimann-Pick disease types a / B), call it eu pikbyeong (Niemann-Pick disease), CI-type kneader only - pikbyeong (一Niemann Pick disease type CI), only the C2-type kneader-pikbyeong ^{(Niemann- Pick 'disease type C2)} , concentrated Pycnodysostosis, Schindler disease types VII, Schindler disease, CM2 gangliosidosis, beta- mannosidosis, Sala disease (Infanti) le sialic, acid storage disease and sal la (sialuria) disease, Mucopl ipidosis (ML) IV, I-cell disease (MLII) and pseudo-Hurler polydystrophy , MLIII) is a lysosomal accumulation disease selected from the group comprising.

[Claim 12]

 4. The fusion protein of claim 3, wherein the enzyme is glucocerebr osidase.

[Claim 13]

 The fusion protein according to claim 12, wherein the fusion protein is represented by the amino acid sequence of SEQ ID NO.

[Claim 14]

 A pharmaceutical composition for preventing or treating Gaucher's disease comprising the fusion protein of claim 12 as an active ingredient.

[Claim 15]

 4. The fusion protein of claim 3, wherein the enzyme is iduronate-sulfatase.

[Claim 16]

 The fusion protein according to claim 15, wherein the fusion protein is represented by the amino acid sequence of SEQ ID NO: 8.

[Claim 17]

 Claim 15 composition for the prevention and treatment of Hunter syndrome (hunter syndrom) comprising the fusion protein as an active ingredient.

[Claim 18]

The method of claim 3, wherein the enzyme is alpha-galactosidase A, agalsidase alpha and agalsidase beta fusion protein, characterized in that the enzyme selected from the group consisting of.

[Claim 19]

 19. The fusion protein of claim 18, wherein the fusion protein is represented by the amino acid sequence of SEQ ID NO.

[Claim 20]

 A composition for preventing and treating Fabry di sease, comprising the fusion protein of claim 18 as an active ingredient.

[Claim 21]

 Use of the fusion protein of claim 18 for the manufacture of a prophylactic or therapeutic agent for Fabry disease.

[Claim 22]

 A method for preventing or treating lysosomal accumulation disease, comprising administering the fusion protein of claim 18 to an individual in need thereof.

[Claim 23]

 For the preparation of a prophylactic or therapeutic agent for lysosomal accumulation of the fusion protein of claim 3

[Claim 24]

^"The method of treating or preventing lysosomal storage disease characterized by administering an effective amount to a subject in need thereof a fusion protein 3 above.

[Claim 25]

 Use of the fusion protein of claim 12 for the manufacture of a prophylactic or therapeutic agent of Gaucher disease. [Claim 26]

13. The method for preventing or treating Gaucher's disease, wherein the fusion protein of claim 12 is administered to an individual in need thereof. [Claim 27]

 Use of the fusion protein of claim 15 for the prophylaxis or treatment of Hunter syndrome. [Claim 28]

 A method for preventing or treating hunter syndrome, comprising administering an effective amount of the fusion protein of claim 15 to an individual in need thereof.