Novel Therapeutic Enzyme Fusion Protein and Its Use

Abstract

The present invention relates to therapeutic enzyme / immunoglobulin Fc region fusion proteins, methods for their production, and compositions containing them.

Description

Technical Field

[0001] The present invention relates to a therapeutic enzyme fusion protein in which an immunoglobulin Fc region is fused to an enzyme for the purpose of extending the in vivo half-life of biotherapeutic enzymes, a method for producing the same, and a composition containing the same.

Background Art

[0002] Lysosomes are cytoplasmic organelles that have the function of degrading macromolecules such as proteins, polynucleotides, polysaccharides, and lipids. The interior of lysosomes is in an acidic atmosphere and contains hydrolyase enzymes that promote the hydrolysis of biological macromolecules. Lysosomes are also known to play some role in the absorption of molecules by endocytosis.

[0003] Lysosomal Storage Disorders (LSDs) are a type of genetic metabolic disorder that manifests due to the loss of lysosomal function. Lysosomal Storage Disorders occur at a rate of approximately 1 in 100,000 people, usually due to a deficiency of enzymes that break down substances such as lipids, proteins, and polysaccharides, and are inherited as recessive diseases. Lysosomal Storage Disorders occur when a specific enzyme responsible for such breakdown is lacking or present in very small amounts. When such a digestive enzyme is lacking, an excessive amount of substances accumulates without being broken down, ultimately causing problems in cell function. Similar to various other genetic diseases, Lysosomal Storage Disorders are inherited from parents. Additionally, each disease is caused by mutations in various genes that translate various enzymes. The enzymes causing such diseases generally have similar biochemical properties, and all Lysosomal Storage Disorders occur due to abnormal substance accumulation within lysosomes. Representative Lysosomal Storage Disorders currently known include approximately 50 or more diseases such as Niemann-Pick disease, Fabry’s disease, Gaucher disease, Hunter syndrome, Maroteaux-Lamy syndrome, etc. A typical method for treating these Lysosomal Storage Disorders is enzyme-replacement therapy (ERT), and many studies have been conducted on it (Non-Patent Document 1).

[0004] Hunter syndrome, a representative lysosomal storage disorder, is a disease that appears due to the accumulation of glycosaminoglycan (GAG) in lysosomes without being degraded due to the deficiency of iduronate-2-sulfatase (IDS). Specific facial features, a large head, abdominal distension due to enlargement of the liver and spleen, etc. are observed, and it is also accompanied by hearing loss, heart valve disease, obstructive respiratory disease, sleep apnea, etc. Hunter syndrome is known to occur at a rate of 1 in 162,000 people and is inherited in an X-linked recessive pattern. Currently, Elaprase (recombinant IDS, Shire) is used as a drug for enzyme replacement therapy for Hunter syndrome.

[0005] Proteins such as these therapeutic enzymes are generally low in stability, prone to denaturation, degraded by proteases in the blood, and need to be frequently administered to patients to maintain blood concentration and activity. However, in protein pharmaceuticals mainly administered to patients in the form of injections, frequent injections to maintain the blood concentration of the active polypeptide cause great pain to the patients. To solve such problems, many efforts have been made to improve the blood stability of therapeutic enzymes and maximize the drug efficacy by maintaining a high blood drug concentration for a long time. Such sustained formulations of therapeutic enzymes must improve the stability of therapeutic enzymes, maintain the activity of the drug itself at a sufficiently high level, and not induce an immune response in patients.

[0006] In particular, lysosomal storage disorders are fatal diseases caused by genetic defects in specific enzymes, and replacement therapy for the defective enzymes is essential. Enzyme replacement therapy is the standard treatment for lysosomal storage disorders and exerts the effect of alleviating existing symptoms and delaying the progression of the disease by supplementing the deficient enzyme. However, the drug must be continuously administered intravenously every 1 to 2 weeks over 2 to 6 hours, restricting the daily lives of patients and their families.

[0007] The half-life of recombinant enzymes used in the treatment of lysosomal storage diseases in humans is as short as 10 minutes or at most less than 3 hours. Since its duration is extremely short, it causes inconvenience to patients who must receive lifelong enzyme administration, and there is a strong demand for an extension of its half-life.

[0008] Recently, in order to stabilize proteins and prevent their removal from the kidneys, extensive research has been conducted on fusion proteins using the immunoglobulin Fc region. Immunoglobulins are major components of blood and there are five types: IgG, IgM, IgA, IgD, and IgE. The type mainly used in the research of fusion proteins is IgG, which is classified into four subtypes: IgG1 - 4. Fusion proteins using the immunoglobulin Fc region increase the size of the protein to prevent its removal from the kidneys and play a role in extending the blood half-life by binding to the FcRn receptor and undergoing endocytosis and recycling into cells.

[0009] However, the immunoglobulin Fc region has the drawback of causing unintended immune reactions. It has effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Such functions are caused by the binding of the Fc receptor of the immunoglobulin Fc region, complement binding, or glycosylation of the Fc region. Also, the in vivo instability of Fc itself is likely to occur.

[0010] Therefore, there are drawbacks that the target fusion protein is not stable in vivo, its persistence is not improved, and the activity of the fusion protein is not maintained.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

[0012] [Non-Patent Document 1] Frances M. Platt et al., J Cell Biol. 2012 Nov 26;199(5):723-34 [Non-Patent Document 2] van der Neut Kolfschoten, et at., Science, 317: 1554-1557.2007 [Non-Patent Document 3] H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979 [Non-Patent Document 4] Urlaub et al., Somat. Cell. Mol. Genet., 12, 555-566, 1986 [Summary of the Invention] [Problems to be Solved by the Invention]

[0013] An object of the present invention is to provide an enzyme fusion protein in which an immunoglobulin Fc region is fused to a therapeutic enzyme and which has improved in vivo persistence as compared to a therapeutic enzyme not fused with the Fc region.

[0014] Another object of the present invention is to provide a pharmaceutical composition containing the therapeutic enzyme fusion protein. Furthermore, an object of the present invention is to provide a polynucleotide encoding the therapeutic enzyme fusion protein, an expression vector containing the polynucleotide, and a transformant into which the expression vector has been introduced. Furthermore, an object of the present invention is to provide a method for producing an enzyme fusion protein, which includes the step of culturing the transformant. [Means for Solving the Problems]

[0015] One aspect of the present invention is an enzyme fusion protein in which a therapeutic enzyme is fused with an immunoglobulin Fc region.

[0016] In one specific example, the present invention relates to an enzyme fusion protein in which an immunoglobulin Fc region is fused to a therapeutic enzyme and which has improved in vivo persistence compared to a therapeutic enzyme without the fused Fc region.

[0017] Hereinafter, further specific examples of the present invention will be described.

[0018] Specifically, it is the enzyme fusion protein according to the specific example, and the enzyme is beta-glucosidase, alpha-galactosidase, beta-galactosidase, iduronidase, iduronate-2-sulfatase, Galactose-6-sulfatase, acid alpha-glucosidase, acid ceramidase, acid sphingomyelinsase, galactocerebrosidsase, arylsulfatase A, arylsulfatase B, beta-hexosaminidase A, beta-hexosaminidase B, heparin N-sulfatase, alpha-D-mannosidase, beta-glucuronidase, N-acetylgalactosamine-6 sulfatase, lysosomal acid lipase, alpha-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholinesterase, Chitinase, glutamate decarboxylase, imiglucerase, lipase, Uricase, Platelet-Activating Factor Acetylhydrolase, neutral endopeptidase (neutralcharacterized by being selected from the group consisting of endopeptidase and myeloperoxidase.

[0019] An enzyme fusion protein according to any of the above specific examples, wherein the enzyme fusion protein is characterized in that a therapeutic enzyme and an immunoglobulin Fc region are fused with a peptide linker.

[0020] An enzyme fusion protein according to any of the above specific examples, wherein the enzyme fusion protein is characterized in that one immunoglobulin Fc region molecule and a dimeric therapeutic enzyme are fused.

[0021] An enzyme fusion protein according to any of the above specific examples, wherein the immunoglobulin Fc region is characterized in that at least one amino acid in the native immunoglobulin Fc region is subjected to a modification selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

[0022] An enzyme fusion protein according to any of the above specific examples, wherein the immunoglobulin Fc region is characterized in that the second amino acid of the immunoglobulin Fc region of SEQ ID NO: 8 is substituted with proline, or the 71st amino acid is substituted with glutamine, or the second amino acid is substituted with proline and the 71st amino acid is substituted with glutamine.

[0023] An enzyme fusion protein according to any of the above specific examples, wherein the immunoglobulin Fc region is characterized in that no chain exchange reaction occurs.

[0024] An enzyme fusion protein according to any of the above specific examples, wherein the enzyme fusion protein is characterized in that, compared with a therapeutic enzyme without an Fc region fused thereto, its stability is improved, its binding affinity for lysosomal receptors is decreased, and its tissue distribution property is improved.

[0025] An enzyme fusion protein according to any of the above specific examples, wherein the immunoglobulin Fc region is selected from the group consisting of: (a) a combination of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of at least one domain selected from the group consisting of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain and an immunoglobulin hinge region or a part of the hinge region; and (f) a dimer consisting of each domain of the heavy chain constant region and the light chain constant region.

[0026] An enzyme fusion protein according to any of the above specific examples, wherein the immunoglobulin Fc region has at least one characteristic selected from the group consisting of: (a) removal of a site forming a disulfide bond; (b) deletion of a part of the amino acids at the N-terminus from native Fc; (c) addition of a methionine residue to the N-terminus of native Fc; (d) removal of a complement binding site; and (e) removal of an ADCC (antibody dependent cell mediated cytotoxicity) site.

[0027] An enzyme fusion protein according to any of the above specific examples, wherein the immunoglobulin Fc region is non-glycosylated.

[0028] An enzyme fusion protein according to any of the above specific examples, wherein the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE, or IgM.

[0029] An enzyme fusion protein according to any of the above specific examples, wherein the immunoglobulin Fc region is a hybrid of domains having different origins derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

[0030] An enzyme fusion protein according to any of the above specific examples, wherein the immunoglobulin Fc region is an IgG4 Fc region.

[0031] An enzyme fusion protein according to any of the above specific examples, wherein the hinge region of the immunoglobulin IgG4 Fc region is replaced with an IgG1 hinge region.

[0032] Another aspect of the present invention is a pharmaceutical composition for preventing or treating lysosomal storage disorder (LSD).

[0033] In one specific example, the present invention relates to a pharmaceutical composition for preventing or treating lysosomal storage disorder (LSD) containing the enzyme fusion protein.

[0034] A composition according to the above specific example, wherein the lysosomal storage disorder is selected from the group consisting of mucopolysaccharidosis (MPS), glycogen storage disease, sphingolipidosis, Niemann-Pick disease, Fabry’s disease, Gaucher disease, Hunter syndrome, and Maroteaux-Lamy syndrome.

[0035] A composition according to any of the above specific examples, wherein the enzyme is iduronate-2-sulfatase (IDS) or arylsulfatase B (ARSB).

[0036] A composition according to any of the above specific examples, wherein the composition is characterized by reducing the binding ability of a therapeutic enzyme to a lysosomal receptor.

[0037] Still another aspect of the present invention is a polynucleotide encoding an enzyme fusion protein.

[0038] Still another aspect of the present invention is an expression vector containing a polynucleotide.

[0039] Still another aspect of the present invention is a transformant into which an expression vector has been introduced.

[0040] Still another aspect of the present invention is a method for producing an enzyme fusion protein.

[0041] In one specific example, the present invention relates to a method for producing an enzyme fusion protein, which includes culturing the transformant to obtain a culture and recovering the enzyme fusion protein from the culture.

Advantages of the Invention

[0042] The present invention relates to a sustained therapeutic enzyme fusion protein, and particularly to an enzyme fusion protein in which the stability of a therapeutic enzyme is improved and the enzyme removal mechanism by the kidney is reduced by the fusion of an immunoglobulin Fc region. The enzyme fusion protein of the present invention has a long duration and is useful for patients.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0044] Hereinafter, the mode for carrying out the present invention will be described. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present invention are included in the present invention. Further, the present invention is not limited to the following specific description.

[0045] Also, those having ordinary knowledge in the art will be able to recognize and confirm many equivalents of the specific embodiments of the present invention described in the present invention using only ordinary experiments. Furthermore, such equivalents are also intended to be included in the present invention.

[0046] Throughout this specification, not only the usual one-letter and three-letter codes for naturally occurring amino acids are used, but also three-letter codes generally accepted for other amino acids such as Aib (α-aminoisobutyric acid) and Sar (N-methylglycine) are used. Also, the amino acids referred to by abbreviations in this specification are described according to the IUPAC-IUB nomenclature. Alanine A Arginine R Asparagine N Aspartic acid D Cysteine C Glutamic acid E Glutamine Q Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V

[0047] One aspect of the present invention provides an enzyme fusion protein in which an immunoglobulin Fc region is fused to a therapeutic enzyme, and which has improved in vivo persistence compared to a therapeutic enzyme without the fused Fc region.

[0048] In the present invention, the enzyme fusion protein is one in which an immunoglobulin Fc region is fused to a therapeutic enzyme, and by the fusion of the immunoglobulin Fc region, the activity of the therapeutic enzyme is maintained, the binding affinity for lysosomal receptors is decreased, and the plasma half-life is extended, compared to a therapeutic enzyme not fused to the immunoglobulin Fc region.

[0049] In order to extend the plasma half-life of therapeutic enzymes, the inventors produced fusion proteins with the Fc region of immunoglobulin. Here, in order to suppress glycosylation, the potential glycosylation sequence was replaced in the Fc region, and further, by replacing the hinge sequence of IgG4 Fc, when an IgG4 Fc derivative in which chain exchange was suppressed was used, it was confirmed that the therapeutic enzyme fusion protein fused to the immunoglobulin Fc region significantly extended the plasma half-life and maintained equivalent activity compared to known enzymes, leading to the provision of a fusion protein structure in which a new form of therapeutic enzyme is fused to the immunoglobulin Fc region.

[0050] The therapeutic enzyme contained in the enzyme fusion protein of the present invention is not particularly limited, and any therapeutic enzyme that can obtain the advantage of an extended in-vivo persistence time compared to the therapeutic enzyme in a non-fused form may be included in the enzyme fusion protein of the present invention. The enzyme fusion protein in one embodiment of the present invention is a fusion protein of a therapeutic enzyme.

[0051] In addition, the enzyme fusion protein of the present invention may be used as a drug for enzymatic replacement therapy (ERT). The enzymatic replacement therapy can prevent or treat a disease by restoring the reduced enzyme function by supplementing the deficient or insufficient enzyme that causes the disease.

[0052] As a specific embodiment, the therapeutic enzyme is beta-glucosidase, alpha-galactosidase, beta-galactosidase, iduronidase, iduronate-2-sulfatase, Galactose-6-sulfatase, acid alpha-glucosidase, acid ceramidase, acid sphingomyelinsase, galactocerebrosidsase, arylsulfatase A, arylsulfatase B, beta-hexosaminidase A, beta-hexosaminidase B, heparin N-sulfatase, alpha-D-mannosidase, beta-glucuronidase, N-acetylgalactosamine-6 sulfatase, lysosomal acid lipase, alpha-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholinesterase, Chitinase, glutamate decarboxylase, imiglucerase, lipase, Uricase, Platelet-Activating Factor Acetylhydrolase, neutral endopeptidaseIt may be a therapeutic enzyme selected from the group consisting of endopeptidase and myeloperoxidase. However, as long as it is a therapeutic enzyme having a therapeutic effect on a disease, the origin and type of the enzyme can be anything and are included in the present invention.

[0053] The "enzyme fusion protein" in the present invention may be used interchangeably with the "enzyme sustained fusion protein".

[0054] The "therapeutic enzyme" in the present invention is an enzyme for treating diseases caused by enzyme deficiency, lack, dysfunction, etc., and means an enzyme that can treat an individual having the disease by enzyme replacement therapy, administration, etc. Specifically, it may be an enzyme for treating lysosomal storage diseases caused by deficiency, lack, etc. of lysosomal enzymes, but is not limited thereto.

[0055] Specifically, the therapeutic enzyme of the present invention may be arylsulfatase B (ARSB) or iduronate-2-sulfatase, but is not limited thereto as long as it is an enzyme that exhibits a therapeutic effect on the target disease.

[0056] "Arylsulfatase B" in the present invention refers to an arylsulfatase enzyme present in the lysosomes of the liver, pancreas, and kidneys, which plays a role in hydrolyzing sulfate by decomposing glycosaminoglycan. The arylsulfatase B is known to be related to mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). Arylsulfatase B may be used interchangeably with galsulfase. Specifically, the arylsulfatase B may contain the amino acid sequence of SEQ ID NO: 4, which may be encoded by the polynucleotide sequence of SEQ ID NO: 3, but is not limited thereto.

[0057] "Iduronate-2-sulfatase" in the present invention refers to a sulfatase enzyme related to Hunter syndrome (MPS-II), which is an enzyme necessary for the lysosomal degradation of heparin sulfate and dermatan sulfate. The "iduronate-2-sulfatase" in the present invention may be used interchangeably with "idursulfase". The idursulfase may be idursulfase alpha or idursulfase beta, but is not limited thereto. Specifically, the iduronate-2-sulfatase may contain the amino acid sequence of SEQ ID NO: 2, which may be encoded by the polynucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

[0058] The therapeutic enzyme can be prepared or produced by methods well known in the art. Specifically, animal cells transfected with an animal cell expression vector can be cultured and purified from the culture, or commercially available enzymes can be purchased and used, but are not limited thereto.

[0059] The enzyme fusion protein of the present invention may be in a form in which one or two enzymes are bound to one Fc region in a dimeric form consisting of two chains, but is not limited thereto. Specifically, a monomeric Fc region and an enzyme may be fused and expressed, and then two monomeric Fc regions may form a dimeric Fc region of one molecule by a disulfide bond, and the two enzymes may be linked to the two Fc regions respectively, but is not limited thereto. The enzymes may be linked by covalent or non-covalent bonds, or may be independent of each other, but is not limited thereto.

[0060] Specifically, in one embodiment of the present invention, it was confirmed that a sustained enzyme fusion protein in which a dimer of iduronate-2-sulfatase or arylsulfatase B is fused with one Fc region has higher in vitro enzyme activity than an enzyme without an Fc region fused thereto. It was confirmed that this is due to the structural characteristics of the enzyme fusion protein containing a dimer of a therapeutic enzyme (Example 5).

[0061] Also, as another specific aspect, the enzyme fusion protein of the present invention may be one in which an immunoglobulin Fc region is fused to a therapeutic enzyme via a peptide linker.

[0062] The peptide linker may contain one or more amino acids, for example, may contain 1 to 1000 amino acids, but is not particularly limited thereto. Any peptide linker known in the art, such as [GS]x linker, [GGGS]x linker, [GGGGS]x linker, etc. may be mentioned, where x may be a natural number of 1 or more (for example, 1, 2, 3, 4, 5 or more). More specifically, it may be the amino acid sequence of SEQ ID NO: 6, but is not limited thereto.

[0063] For the purpose of the present invention, as long as it is possible to link a therapeutic enzyme and an immunoglobulin Fc region while maintaining the activity of the therapeutic enzyme, the position where the peptide linker is fused to the therapeutic enzyme and the immunoglobulin Fc may be any position. Specifically, it may be both ends of the therapeutic enzyme and the immunoglobulin Fc region. More specifically, it may be the C-terminus of the therapeutic enzyme and the N-terminus of the immunoglobulin Fc region, but is not limited thereto.

[0064] In the present invention, "N-terminus" or "C-terminus" means the amino terminus or carboxyl terminus of a protein, respectively. For example, but not limited thereto, it may include not only the outermost amino acid residue of the N-terminus or C-terminus, but also all amino acid residues around the N-terminus or C-terminus. Specifically, the 1st to 20th amino acid residues from the outermost end may be included.

[0065] In one embodiment of the present invention, a fusion protein (SEQ ID NO: 23 or 25) in which the N-terminus of IgG4 is fused to the C-terminus of a therapeutic enzyme was prepared by overlapping PCR of the therapeutic enzyme and linker (SEQ ID NO: 6)-IgG4, and it was confirmed that it was expressed in a transformant (Examples 1 to 3).

[0066] The therapeutic enzyme contained in the enzyme fusion protein of the present invention may be a natural one, or a derivative (analog) of a therapeutic enzyme in which a modification selected from the group consisting of a fragment composed of a part, or substitution, addition, deletion, modification of some amino acids, and combinations thereof is performed, as long as it has the same activity as the natural therapeutic enzyme, it is all included in the present invention.

[0067] In addition, the derivatives of the therapeutic enzyme all include those in which at least one amino acid is added to the N and / or C terminus of the natural therapeutic enzyme.

[0068] As the amino acids to be replaced or added, not only the 20 types of amino acids commonly observed in human proteins but also abnormal or non-natural amino acids can be used. Commercially available sources of abnormal amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized and purchased from private peptide synthesis companies such as the American peptide company and Bachem in the United States, or Anygen in South Korea, but are not particularly limited to these.

[0069] The "fragment" in the present invention means a form in which at least one amino acid at the N-terminus or C-terminus of a natural therapeutic enzyme or a derivative of a natural therapeutic enzyme is deleted. As long as it has the activity of a therapeutic enzyme, it is included in the present invention regardless of the size of the fragment or the type of amino acid deleted.

[0070] The therapeutic enzyme derivatives include biosimilar and bio-better forms of the therapeutic enzyme. As an example of a biosimilar, differences in the expression host from a known therapeutic enzyme, differences in the pattern and degree of glycosylation, and differences in the degree of substitution when the residues at specific positions are not 100% substitution with respect to the reference sequence of the enzyme also correspond to biosimilar enzymes that can be used in the enzyme fusion protein of the present invention. The therapeutic enzyme can be prepared or produced by methods well known in the art. Specifically, it can be produced from animal cells, Escherichia coli, yeast, insect cells, plant cells, living animals, etc. by genetic recombination. The production method is not limited to these, and commercially available therapeutic enzymes may be purchased and used, but are not limited to these.

[0071] In addition, it may contain an amino acid sequence having 80% or more, specifically 90% or more, more specifically 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more homology with the therapeutic enzyme or its derivative. The therapeutic enzyme may be obtained from a microorganism by recombinant technology or may be a commercially available product, but is not limited thereto.

[0072] In the present invention, "homology" indicates the degree of similarity with a wild-type amino acid sequence or a wild-type nucleic acid sequence. The comparison of homology can be performed visually or using a comparison program that is easily available for purchase. Commercially available computer programs can calculate the homology between two or more sequences as a percentage (%). Homology (%) may be calculated for adjacent sequences.

[0073] Information on the sequence of the therapeutic enzyme or its derivative and the nucleotide sequence encoding the same can be obtained from known databases such as NCBI.

[0074] In the present invention, the "immunoglobulin Fc region" refers to the region excluding the variable regions of the heavy and light chains of an immunoglobulin, and means a site containing the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3) portion. For the purpose of the present invention, such an Fc region may include a mutated hinge region, but is not limited thereto.

[0075] Such an immunoglobulin Fc region may include a hinge region in the heavy chain constant region, but is not limited thereto. In addition, as long as the immunoglobulin Fc region of the present invention has an effect substantially equivalent to or improved over that of the natural one, it may be an extended Fc region excluding the variable regions of the heavy and light chains of the immunoglobulin and including a part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1). Furthermore, it may be a region in which a very long part of the amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0076] As a further specific alternative aspect, the immunoglobulin Fc region of the present invention may be 1) a combination of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; 5) a combination of at least one domain of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain and an immunoglobulin hinge region (or a part of the hinge region); 6) each domain of the heavy chain constant region and a dimer of the light chain constant region. However, it is not limited thereto.

[0077] In the present invention, "chain exchange" means that when IgG4 Fc is used as a carrier of a protein fusion, it forms a hybrid with IgG4 present in the living body or exists as a monomer, and thus has a problem of changing the original structure to a therapeutically inactive structure. It has been reported that it is very difficult to use a protein fusion, which is a fusion protein with a protein, for therapeutic purposes (Non-Patent Document 2).

[0078] In the present invention, an attempt was made to solve the above problem by substituting the sequence of the hinge region within the immunoglobulin Fc region. Specifically, the immunoglobulin Fc region of the present invention may be one in which a potential glycosylation sequence has been substituted to regulate glycosylation, one in which a sequence involved in chain exchange has been substituted, or one corresponding to both of them.

[0079] As a specific embodiment, the immunoglobulin Fc region of the present invention may have the second amino acid and / or the 71st amino acid of the immunoglobulin Fc region of SEQ ID NO: 8 substituted with other amino acids in order to prevent chain exchange and N-glycosylation. More specifically, 1) the second amino acid (serine) of the immunoglobulin Fc region of SEQ ID NO: 8 is substituted with proline, 2) the 71st amino acid (asparagine) is substituted with glutamine, or 3) the second amino acid is substituted with proline and the 71st amino acid is substituted with glutamine, but is not limited thereto. In addition to the mutations described above, mutations suitable as drug carriers that improve the stability of therapeutic enzymes may also be included.

[0080] Specifically, the immunoglobulin Fc region may have the hinge region of immunoglobulin IgG4 Fc replaced with the IgG1 hinge region, but is not limited thereto.

[0081] In one example of the present invention, by substituting the second amino acid of the immunoglobulin Fc represented by SEQ ID NO: 8 with proline and the 71st amino acid with glutamine, chain exchange and N-glycosylation were reduced. The sequence of the prepared immunoglobulin Fc has the amino acid sequence of SEQ ID NO: 9 (Example 1).

[0082] As an example, the hinge region may have a part of the hinge sequence having the following amino acid sequence deleted or mutated. Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 26)

[0083] Specifically, a part of the hinge region may be deleted and mutated to contain only one cysteine (Cys) residue, or a serine (Ser) residue involved in chain exchange may be replaced with a proline (Pro) residue. More specifically, the second serine residue of the hinge sequence may be replaced with a proline residue, but is not limited thereto.

[0084] In the present invention, since the immunoglobulin Fc region contains a natural hinge region or a mutated hinge region, not only does chain exchange and monomer formation not occur in the Fc region, but the stability of the fused therapeutic enzyme can be improved.

[0085] As another specific aspect, the immunoglobulin Fc region of the present invention includes not only the natural amino acid sequence but also sequence derivatives thereof. An amino acid sequence derivative means one in which a mutation selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof has occurred in at least one amino acid residue of the natural amino acid sequence.

[0086] For example, in the case of IgG Fc, the 214-238th, 297-299th, 318-322nd, or 327-331st amino acid residues, which are known to be important for binding, are used as sites suitable for modification.

[0087] In addition, various types of derivatives are used, such as a derivative in which the site forming a disulfide bond is removed, a derivative in which several amino acids at the N-terminus are deleted from the natural Fc, and a derivative in which a methionine residue is added to the N-terminus of the natural Fc. Furthermore, in order to eliminate effector functions, a complement binding site, such as a C1q binding site, may be removed, or an ADCC (antibody dependent cell mediated cytotoxicity) site may be removed. Techniques for producing such sequence derivatives of the immunoglobulin Fc region are disclosed in Patent Documents 1, 2, etc.

[0088] Amino acid exchanges in proteins and peptides that do not globally change the activity of the molecule are known in the art (Non-Patent Document 3). The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0089] In addition, the Fc derivative described above may exhibit biological activity equivalent to that of the Fc region of the present invention and may improve the structural stability of the Fc region against heat, pH, etc.

[0090] In addition, such an Fc region may be obtained from a natural one isolated from the living body of an animal such as a human, and cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or may be a recombinant one or a derivative thereof obtained from transformed animal cells or microorganisms. Here, the method of obtaining from a natural one may be a method of separating whole immunoglobulin from a human or animal living body and then treating it with a proteolytic enzyme. Treatment with papain cleaves it into Fab and Fc, and treatment with pepsin cleaves it into pF'c and F(ab)2. These can separate Fc or pF'c using size-exclusion chromatography, etc. In a more specific embodiment, the Fc region derived from a human is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0091] In addition, the immunoglobulin Fc region may be in the form of a natural sugar chain, a sugar chain increased compared to the natural one, a sugar chain decreased compared to the natural one, or a form in which the sugar chain is removed. For such increase, decrease, or removal of the immunoglobulin Fc sugar chain, ordinary methods such as chemical methods, enzymological methods, and genetic engineering methods using microorganisms may be used. Here, the immunoglobulin Fc region from which the sugar chain has been removed from Fc has a significantly reduced binding ability to complement (c1q), and antibody-dependent cell cytotoxicity or complement-dependent cell cytotoxicity is reduced or removed, so that it does not induce an unnecessary immune reaction in vivo. For this reason, the immunoglobulin Fc region from which the sugar chain has been removed or non-glycosylated is suitable for the original purpose as a drug carrier.

[0092] "Deglycosylation" in the present invention means an Fc region from which sugar has been removed by an enzyme, and "aglycosylation" means an Fc region produced in a prokaryote, more specifically in Escherichia coli and not glycosylated.

[0093] On the other hand, the immunoglobulin Fc region may be of human origin or of animal origin such as bovine, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., and in a more specific embodiment, it is of human origin.

[0094] In addition, the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which is the most abundant in human blood. In a further specific embodiment, it is derived from IgG, which is known to extend the half-life of the ligand-binding protein. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region. In an even more specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4. In the most specific embodiment, the immunoglobulin Fc region contains a mutation in which the second amino acid of the immunoglobulin Fc region having the amino acid sequence of SEQ ID NO: 8 is substituted with proline, and / or a mutation in which the 71st amino acid is substituted with glutamine, or the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO: 9, and the polynucleotide sequence encoding the same is SEQ ID NO: 7, but is not limited thereto.

[0095] On the other hand, the "combination" in the present invention means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin binds to a single-chain polypeptide of a different origin. That is, a dimer or multimer can be produced from at least two fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0096] In addition, the protein of the present invention may have an unmodified N-terminus and / or C-terminus. However, in order to protect it from proteolytic enzymes in vivo and improve stability, the N-terminus and / or C-terminus thereof may be in a chemically modified form, a form protected by an organic group, or a form modified by adding an amino acid to the peptide terminus or the like, and is also included in the protein according to the present invention. When the C-terminus is not modified, the terminus of the protein according to the present invention has a carboxyl group, but is not particularly limited thereto.

[0097] In particular, in the case of a chemically synthesized protein, since the N- and C-termini are charged, N-terminal acetylation and / or C-terminal amidation may be performed to remove such charges, but is not particularly limited thereto.

[0098] In this specification, unless otherwise specified, the detailed description and claims of the "enzyme" or "fusion protein" according to the present invention clearly apply not only to the enzyme or fusion protein itself, but also to all forms including salts (e.g., pharmaceutically acceptable salts of the fusion protein) or solvates thereof. Therefore, even if only "enzyme" or "fusion protein" is described in the specification, the description also applies equally to its specific salts, its specific solvates, and specific solvates of its specific salts. These salt forms may be, for example, forms using any pharmaceutically acceptable salt. The types of the salts are not particularly limited. However, it is preferably in a form that is safe and effective for an individual, such as a mammal, but is not particularly limited thereto.

[0099] The term "pharmaceutically acceptable" means a substance that can be effectively used for a desired use without inducing excessive toxicity, irritation, allergic reaction, etc. within the scope of pharmaceutical judgment.

[0100] The "pharmaceutically acceptable salts" in the present invention include salts derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, ammonium, and the like.

[0101] In addition, the "solvate" in the present invention means a complex formed by the enzyme, fusion protein or salt thereof according to the present invention and a solvent molecule.

[0102] The enzyme fusion protein of the present invention can be produced by a method known in the art.

[0103] In one embodiment of the present invention, a recombinant vector expressing iduronate-2-sulfatase (IDS) and arylsulfatase B (ARSB), which are therapeutic enzymes, in a form fused to a peptide linker-immunoglobulin Fc, respectively, was prepared and expressed in a CHO cell line for production (Examples 1 to 3).

[0104] However, the enzyme fusion protein of the present invention can also be produced by other known methods in addition to the methods described in the above examples. The enzyme fusion protein of the present invention may contain the amino acid sequence of SEQ ID NO: 23 or 25, but is not limited thereto.

[0105] The enzyme fusion protein according to the present invention can extend the half-life of a therapeutic enzyme while maintaining the activity of the therapeutic enzyme by fusing a therapeutic enzyme that exerts a therapeutic effect on lysosomal storage disease with an immunoglobulin Fc region. In particular, a therapeutic enzyme fused to a mutated immunoglobulin Fc region has reduced chain exchange and glycosylation, and compared to a therapeutic enzyme not fused to the Fc region, has a reduced binding affinity for lysosomal receptors and improved persistence, thus exerting a useful effect in the treatment of lysosomal storage diseases.

[0106] In one embodiment of the present invention, the enzyme fusion protein according to the present invention has a half-life (T 1 / 2 ) and a maximum plasma drug concentration (C max) The bioavailability (AUC) is significantly excellent (Example 4), it was confirmed that the in vitro enzyme activity was maintained (Example 5), and it was thereby confirmed that a therapeutic effect can be exhibited even with a lower dosing frequency as compared with conventional drugs (Example 6).

[0107] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating lysosomal storage disorder (LSD) containing, as an active ingredient, the enzyme fusion protein or the enzyme fusion protein produced by the method for producing the enzyme fusion protein.

[0108] The composition according to the present invention is characterized in that the in vivo persistence and stability of a therapeutic enzyme are improved.

[0109] In a specific aspect, the enzyme fusion protein of the pharmaceutical composition of the present invention may be one in which iduronate-2-sulfatase (IDS) or arylsulfatase B (ARSB) is fused to the immunoglobulin Fc region, but is not limited thereto.

[0110] The "lysosome" in the present invention is one of the organelles present in the cytoplasm, contains many hydrolytic enzymes, decomposes foreign substances such as macromolecules and bacteria in the body, and helps the decomposed products to be recycled in other parts of the cell. The function of the lysosome is carried out by a plurality of enzymes, but when a specific enzyme loses its function due to mutation, deficiency, etc., the decomposition function of the lysosome is lost, and eventually macromolecules that should be decomposed accumulate in the cell, inducing cell damage, etc., and a disease caused thereby occurs.

[0111] The "lysosomal storage disease (LSD)" in the present invention refers to a rare genetic disease caused by such loss of lysosomal function, and enzymatic replacement therapy to replenish the defective enzyme is essential. The lysosomal storage disease is classified into mucopolysaccharidosis (MPS), glycogen storage disease, sphingolipidosis, Niemann-Pick disease, Fabry's disease, Gaucher disease, Hunter syndrome, Maroteaux-Lamy syndrome, etc. according to the deficient enzyme.

[0112] Hereinafter, the lysosomal storage disease will be described in detail according to its classification.

[0113] The "Maroteaux-Lamy syndrome" in the present invention is a type VI mucopolysaccharidosis (MPS) disease, which is an autosomal recessive genetic disease caused by the deficiency of Arylsulfatase B (N-acetylgalactosamine-4-sulfatase) required for the degradation of glycosaminoglycan. It is a disease in which dermatan sulfate that has not been degraded due to the deficiency of the enzyme is deposited in bones, heart valves, spleen, liver, cornea, etc.

[0114] "Arylsulfatase B" in the present invention refers to an arylsulfatase enzyme present in the lysosomes of the liver, pancreas, and kidney, which plays a role in hydrolyzing sulfate by decomposing glycosaminoglycan. The arylsulfatase B is known to be related to mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). Arylsulfatase B may be used interchangeably with galsulfase.

[0115] "Hunter syndrome" in the present invention is an X-linked recessive genetic disorder that is caused by a deficiency of iduronate 2-sulfatase (IDS), and it is known that the deficiency of this enzyme causes the accumulation of heparan sulfate and dermatan sulfate. It shows symptoms such as functional decline, progressive hearing loss, pigmentary retinopathy, papilledema, and hydrocephalus. In the present invention, "mucopolysaccharidosis II type" and "Hunter syndrome" may be used interchangeably.

[0116] "Iduronate-2-sulfatase" in the present invention is a sulfatase enzyme related to Hunter syndrome (MPS-II), and it is an enzyme necessary for the lysosomal degradation of heparin sulfate and dermatan sulfate. The "iduronate-2-sulfatase" in the present invention may be used interchangeably with "idursulfase". The idursulfase may be idursulfase alpha or idursulfase beta, but is not limited thereto.

[0117] The therapeutic enzyme can be prepared or produced by methods well-known in the art. Specifically, it can be cultured from animal cells into which an animal cell expression vector has been inserted and purified from the culture, or commercially available therapeutic enzymes can be purchased and used, but it is not limited thereto.

[0118] The enzyme fusion protein contained in the composition of the present invention can extend the half-life of the therapeutic enzyme while maintaining its activity by fusing a therapeutic enzyme that exerts a therapeutic effect on lysosomal storage diseases with the immunoglobulin Fc region. In particular, the therapeutic enzyme fused to the mutated immunoglobulin Fc region has reduced chain exchange and glycosylation, and compared to the therapeutic enzyme not fused to the Fc region, has a reduced binding affinity for lysosomal receptors and improved persistence, thus exerting a useful effect in the treatment of lysosomal storage diseases.

[0119] In one embodiment of the present invention, it was confirmed that the enzyme fusion protein of the present invention decreased the numerical value of glycosaminoglycan (GAG) in IDS knockout mice (Example 6) despite a lower administration frequency compared to the enzyme not fused to the Fc region. Also, in another embodiment of the present invention, the enzyme fusion protein of the present invention not only had a higher distribution in the bone marrow and spleen compared to the native enzyme not fused to the Fc region, but while the distribution of the native enzyme was not confirmed in the lungs, kidneys, heart, etc., the enzyme fusion protein was confirmed to be distributed in these tissues (Example 7).

[0120] This suggests that the enzyme fusion protein of the present invention, based on its high stability, not only reduces the administration frequency during drug administration to improve patient convenience, but also enables subcutaneous administration due to its high tissue distribution.

[0121] As used herein, "prevention" means any act of suppressing or delaying lysosomal storage disease, which is the target disease, by administering the enzyme fusion protein or a composition containing the same, and "treatment" means any act of improving or favorably changing the symptoms of the target disease, for example, lysosomal storage disease, by administering the enzyme fusion protein or a composition containing the same.

[0122] As used herein, "administration" means introducing a predetermined substance into a patient by any suitable method, and the administration route of the composition is not particularly limited to these, but can be administered by any general route as long as the composition can reach the in vivo target. Examples include intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intratracheal administration, rectal administration, and the like. However, in the case of oral administration, since the peptide is digested, it is preferable that the oral composition is formulated to coat the active drug or to be protected from degradation in the stomach. Specifically, it is preferable to administer in the form of an injection. In addition, the pharmaceutical composition can be administered by any device capable of delivering the active ingredient to the target cells.

[0123] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or may be administered by a fractionated treatment protocol with multiple doses over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of the disease. Specifically, the total dosage of the fusion protein of the present invention is preferably about 0.0001 mg to 500 mg per kg of body weight per day. However, the dosage of the conjugate is determined by various factors such as the administration route and the number of treatments of the pharmaceutical composition, as well as the age, weight, health status, gender, severity of the disease, diet, excretion rate, etc. of the patient. Considering these factors, a person with ordinary knowledge in the art will be able to determine an appropriate effective dosage according to the specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its dosage form, administration route, and administration method as long as it exhibits the effects of the present invention.

[0124] The actual dosage of the enzyme fusion protein of the present invention is determined by the type of the therapeutic enzyme as the active ingredient, together with various relevant factors such as the disease to be treated, the administration route, the age, gender and weight of the patient, and the severity of the disease. Since the enzyme fusion protein of the present invention has excellent blood persistence and in vivo activity, the dosage, the number of administrations, and the frequency of the pharmaceutical composition containing the enzyme fusion protein of the present invention can be significantly reduced.

[0125] The pharmaceutical composition according to the present invention may further contain a pharmaceutically acceptable carrier, excipient, or diluent. Such a carrier may be non-naturally occurring.

[0126] "Pharmaceutically acceptable" in the present invention means an amount sufficient to exhibit a therapeutic effect and no side effects, and can be easily determined by those skilled in the art based on known elements in the medical field such as the type of disease, the age, weight, health status, gender, sensitivity to drugs, administration route, administration method, number of administrations, treatment period, formulation, and drugs used simultaneously.

[0127] For oral administration, pharmaceutically acceptable carriers can include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. For injections, buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used. For topical administration, bases, excipients, lubricants, preservatives, etc. can be used.

[0128] The dosage form of the pharmaceutical composition of the present invention can be manufactured in various forms by mixing with pharmaceutically acceptable carriers as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. For injections, it can be manufactured in disposable ampoules or multiple-dose forms. In addition, it can be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0129] Examples of carriers, excipients, and diluents suitable for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. Further, fillers, anticoagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc. may also be included.

[0130] In addition, the enzyme fusion protein can be used by mixing with various carriers acceptable to the drug, such as physiological saline and organic solvents. Carbohydrates such as glucose, sucrose, dextran, antioxidants such as ascorbic acid and glutathione, chelating agents, low molecular weight proteins, and other stabilizers can be used as drugs to improve stability and absorbability.

[0131] Although not limited thereto, the pharmaceutical composition of the present invention may contain the above component (active ingredient) in an amount of 0.01 to 99% (w / v).

[0132] Still another aspect of the present invention provides a polynucleotide encoding an enzyme fusion protein according to the present invention.

[0133] The polynucleotide encoding the enzyme fusion protein of the present invention is not limited thereto, and may be a polynucleotide in which a portion encoding a therapeutic enzyme and a portion encoding a peptide linker - immunoglobulin Fc region are linked. Specifically, although not limited thereto, it may be a polynucleotide encoding a fusion protein in which the N - terminus of the immunoglobulin Fc region is linked to the C - terminus of a therapeutic enzyme via a GGGGS linker. More specifically, the polynucleotide of the present invention may contain the sequence of SEQ ID NO: 1 or 3, but may be any polynucleotide capable of encoding a fusion protein of a therapeutic enzyme and an immunoglobulin Fc region.

[0134] Still another aspect of the present invention provides a recombinant expression vector containing the above polynucleotide.

[0135] The "recombinant vector" in the present invention means a DNA product in which a target peptide, for example, an enzyme fusion protein, is operably linked to a suitable regulatory sequence so that the target peptide, for example, the enzyme fusion protein can be expressed in a suitable host. The recombinant vector according to the present invention may typically be constructed as a vector for cloning or a vector for expression, and may be constructed using a prokaryotic cell or a eukaryotic cell as a host cell.

[0136] The regulatory sequences include a promoter that initiates transcription, any operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that regulate the termination of transcription and translation. When the recombinant vector is transformed into a suitable host cell, it can replicate and function regardless of the host genome and may be integrated into the genome itself.

[0137] The recombinant vector used in the present invention is not particularly limited as long as it can replicate in a host cell, and can be prepared using any vector known in the art. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant states. The vector that can be used in the present invention is not particularly limited, and known expression vectors can be used.

[0138] The recombinant vector is used for the transformation of a host cell to produce the enzyme fusion protein of the present invention. Such transformed cells included in the present invention may be cultured cells or cell lines that have been used for the propagation of the nucleic acid fragment and vector of the present invention or for the recombinant production of the enzyme fusion protein of the present invention.

[0139] "Transformation" in the present invention means introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell to express the protein encoded by the polynucleotide in the host cell. As long as the transformed polynucleotide is expressed in the host cell, it includes all of them regardless of whether they are inserted and located within the host cell chromosome or located extrachromosomally.

[0140] In addition, the polynucleotide includes DNA or RNA encoding a target protein. The polynucleotide may be introduced into a host cell in any form as long as it can be introduced into the host cell and expressed therein. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its own expression. Usually, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicable expression vector. Further, the polynucleotide may be introduced into a host cell in its own form and be operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0141] Furthermore, the term "operably linked" means that a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target peptide of the present invention is functionally linked to the gene sequence.

[0142] The host suitable for the present invention is not particularly limited as long as it can express the polynucleotide of the present invention. Specific examples of the host used in the present invention include bacteria of the genus Escherichia such as Escherichia coli, bacteria of the genus Bacillus such as Bacillus subtilis, bacteria of the genus Pseudomonas such as Pseudomonas putida, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, insect cells such as Spodoptera frugiperda (Sf9), and animal cells such as CHO, COS, and BSC.

[0143] Still another aspect of the present invention provides a transformant into which the expression vector has been introduced.

[0144] The transformant into which the expression vector of the present invention has been introduced is not limited as long as it expresses and produces an enzyme fusion protein for the purpose of the present invention, and includes bacteria belonging to the genus Escherichia such as Escherichia coli, bacteria belonging to the genus Bacillus such as Bacillus subtilis, bacteria belonging to the genus Pseudomonas such as Pseudomonas putida, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, insect cells such as Sf9, and animal cells such as CHO, COS, and BSC.

[0145] Still another aspect of the present invention provides a method for producing an enzyme fusion protein according to the present invention.

[0146] Specifically, the production method includes, but is not limited to, (a) culturing a transformant to obtain a culture, and (b) recovering the enzyme fusion protein from the culture.

[0147] In the present invention, the medium used for culturing the transformant must satisfy the requirements for host cell culture in a suitable manner. The carbon source contained in the medium for the growth of the host cell may be appropriately selected by those skilled in the art according to the type of the transformant to be produced, and suitable culture conditions may be adopted to adjust the timing and amount of the culture.

[0148] Examples of sugar sources that can be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerin and ethanol, and organic acids such as acetic acid. These substances can be used alone or as a mixture.

[0149] Examples of nitrogen sources that can be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, yellow powder, and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The nitrogen source can also be used alone or as a mixture.

[0150] Examples of phosphorus sources that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or sodium-containing salts corresponding thereto. The culture medium may also contain metal salts such as magnesium sulfate and iron sulfate necessary for growth.

[0151] Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins may be used. A suitable precursor may also be used in the culture medium. The above-described raw materials may be added batchwise or continuously to the culture in a suitable manner during the culture process. The pH of the culture can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, or ammonia, or an acidic compound such as phosphoric acid or sulfuric acid in a suitable manner. An antifoaming agent such as a fatty acid polyglycol ester may be used to suppress bubble formation. To maintain an aerobic state, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture.

[0152] The culture of the transformant according to the present invention is usually carried out at a temperature of 20°C to 45°C, specifically 25°C to 40°C. The culture is continued until the maximum production amount of the desired insulin analog is obtained, and for these purposes, it is usually continued for 10 to 160 hours.

[0153] As described above, when appropriate culture conditions are adjusted according to the host cell, insulin analogs are produced by the transformant according to the present invention, and depending on the vector configuration and the characteristics of the host cell, the produced insulin analogs are secreted into the cytoplasm of the host cell, the periplasmic space or extracellularly.

[0154] Proteins expressed inside and outside the host cell can be purified by ordinary methods. Examples of purification methods include salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (e.g., protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, chromatography such as reverse phase column chromatography, and ultrafiltration, which can be used alone or in combination.

[0155] Yet another aspect of the present invention provides a method for preventing or treating lysosomal storage disease, comprising the step of administering to an individual the enzyme fusion protein or a composition containing the same.

[0156] Since the enzyme fusion protein of the present invention contains a therapeutic enzyme capable of preventing or treating lysosomal storage disease, administration of the enzyme fusion protein containing the same or a pharmaceutical composition containing the enzyme fusion protein can prevent or treat an individual suspected of having lysosomal storage disease.

[0157] The "individual" in the present invention refers to an individual suspected of having a lysosomal storage disorder (LSD), and the individual suspected of having the lysosomal storage disorder means a mammal including humans, mice, livestock, etc., which has developed or is at risk of developing the disease, but any individual that can be treated with the enzyme fusion protein of the present invention or the composition containing the same may be used.

[0158] The method of the present invention may include administering a pharmaceutical composition containing an enzyme fusion protein in a pharmaceutically effective amount. A suitable total daily dosage may be determined by the attending physician within the scope of sound medical judgment and can be administered once or in several divided doses. However, for the purposes of the present invention, the specific therapeutically effective amount for a particular patient will depend on the type and degree of response to be achieved, whether other formulations are used in some cases, the specific composition, the patient's age, weight, general health, sex, diet, time of administration, route of administration, rate of secretion of the composition, duration of treatment, various factors including drugs administered with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical field, and preferably will be in different amounts according to these factors.

[0159] On the other hand, although not limited thereto, the method for preventing or treating the lysosomal storage disorder may be a combination therapy further including administering a compound or substance having therapeutic activity against at least one lysosomal storage disorder.

[0160] "Combination" in the present invention should be understood to mean simultaneous, sequential or separate administration. When the administration is sequential or separate, the interval between the administrations of the secondary component must not lose the advantageous effects of the combination.

[0161] The dosage of the enzyme fusion protein having therapeutic activity against the lysosomal storage disorder may be about 0.0001 μg to 500 mg per kg of the patient's body weight, but is not particularly limited thereto.

[0162] Still another aspect of the present invention provides the use of the enzyme fusion protein or a composition containing the same in the manufacture of a medicament (or pharmaceutical composition) for preventing or treating lysosomal storage disorders.

[0163] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for explaining the present invention more specifically, and the present invention is not limited to these examples.

Examples

[0164] Preparation of Fusion Protein Expression Vector To produce the enzyme fusion protein, an expression vector (IDS cDNA, Cat No. EX-C0003-M02, Gencopoeia; ARSB cDNA, Cat No. EX-C0073-M02, Genecopoeia) into which natural iduronate-2-sulfatase (Iduronate-2-sulfatase, IDS, SEQ ID NO: 1) and arylsulfatase B (Arylsulfatase B, ARSB, SEQ ID NO: 3) were inserted respectively, a synthesized linker (linker, SEQ ID NO: 5), and the IgG4 Fc region (SEQ ID NO: 7) were used to prepare a fusion protein expression vector by overlap PCR. In the overlap PCR method, when each enzyme and linker-Fc are PCR amplified respectively, the primers contain sequences that overlap with each other, so the produced PCR products contain overlapping sequences. The PCR conditions for the amplification of the fusion protein were as follows: in the first step, the process of 95°C for 1 minute, 57°C for 30 seconds, and 68°C for 3 minutes was repeated 25 times, and in the second step, the process of 95°C for 1 minute, 57°C for 30 seconds, and 68°C for 4 minutes was repeated 25 times for amplification.

[0165] Specifically, IDS was subjected to PCR using the primers of SEQ ID NOs: 10 and 11, and Linker-Fc was subjected to PCR using the primers of SEQ ID NOs: 12 and 13, so that the IDS PCR product contained the linker-Fc sequence on the 3' side, and the Linker-Fc PCR product contained the IDS sequence on the 5' side.

[0166] Using the two PCR products obtained in the first PCR as templates, secondary PCR was performed using the primers of SEQ ID NOs: 10 and 13. When a PCR product with the IDS-Fc sequence was obtained, the overlapping sequence was cleaved with KpnI and XhoI restriction enzymes, and the PCR product obtained as described above was inserted into the X0GC vector to construct an IDS-Fc fusion protein expression vector (pX0GC-Enzyme-Fc).

[0167] When a PCR product having an ARSB-Fc sequence is obtained using the primers of SEQ ID NOs: 14, 15, 16, and 17 in the same manner, it is digested with KpnI and XhoI restriction enzymes and inserted into an X0GC vector digested with the same restriction enzymes to construct a fusion protein expression vector.

[0168] [Table 1]

[0169] To remove chain exchange and N-glycosylation sites from the Fc region of the constructed fusion protein sequence, a site-directed mutagenesis PCR method was used.

[0170] Specifically, using the primers of SEQ ID NOs: 18 and 19, serine, the second amino acid of the Fc region (SEQ ID NO: 8) involved in chain exchange, was replaced with proline, and using the primers of SEQ ID NOs: 20 and 21, asparagine, the 71st amino acid of the Fc region where N-glycosylation occurs, was replaced with glutamine. In the protein sequence of Table 3, bold indicates the portion where the amino acid was substituted, and italic indicates the linker.

[0171] [Table 2]

[0172] The enzyme fusion protein expression vectors prepared in the above examples were named IDS-Fc vector and ARSB-Fc vector. Alternatively, the vector may be used interchangeably with pX0GC-Enzyme-Fc.

[0173] [Table 3] TIFF0007697784000004.tif248144 TIFF0007697784000005.tif250150 TIFF0007697784000006.tif249147 TIFF0007697784000007.tif249147 TIFF0007697784000008.tif218157

Example

[0174] Transformation of CHO cell line using a fusion protein expression vector The recombinant expression vector pX0GC-Enzyme-Fc prepared in Example 1 was introduced into a DG44 / CHO cell line (CHO / dhfr-) (Non-Patent Document 4) in which the DHFR gene was disrupted and the nucleic acid biosynthesis process was incomplete to obtain a transformant, and an enzyme fusion protein (Enzyme-Fc) was expressed from the transformant.

[0175] Specifically, the DG44 / CHO cell line was cultured to such an extent that the bottom of the culture vessel was covered by about 80 to 90%, and then the cells were washed 3 times with Opti-MEM (Gibco, cat. No. 51985034).

[0176] On the other hand, a mixture of 3 ml of Opti-MEM and 5 μg of the expression vector pX0GC-Enzyme-Fc and a mixture of 3 ml of Opti-MEM and 20 μl of Lipofectamine 2000 (Gibco, cat no. 11668-019) were each allowed to stand at room temperature for 30 minutes. Next, the respective mixtures were mixed and added to the cultured DG44 / CHO cell line, and cultured at 37°C and 5% CO2 for about 18 hours to introduce the expression vector pX0GC-Enzyme-Fc into the DG44 / CHO cell line.

[0177] Next, the cultured cells were washed three times with DMEM-F12 (Gibco, cat no. 11330) medium containing 10% FBS, and then the medium was added and the cells were cultured for an additional 48 hours. Trypsin was added to the cultured cells to separate the cultured cells, and they were inoculated into a selection medium (α-MEM medium (WELGENE, cat no. LM008-02) containing no HT supplement (Hypoxanthine-Thymidine), 10% FBS, and 1 mg / ml of G418 (Cellgro, cat no. 61-234-RG)). The transformed cells were selected from the separated cells by culturing with the selection medium replaced every two or three days until only the transformed cells survived and formed colonies. Here, in order to improve the expression level of the enzyme fusion protein in the selected transformed cells, 10 nM MTX (Sigma, cat no. M8407) was added to the selection medium, and its concentration was gradually increased, and the MTX content was increased to 20 nM after one to two weeks.

Example

[0178] Confirmation of the expression of IDS-Fc and ARSB-Fc fusion proteins using enzyme immunoassay (ELISA) A part of the cells transfected in Example 2 was transferred to a 175-T cell culture flask at a concentration of 1×10 7 cell number and cultured until the bottom of the culture vessel was almost covered. Then, 15 mL of serum-free medium Ex-cell media (custom-made by Sigma, cat no. 14360C) supplemented with 1 mM sodium butyrate (Sigma, cat no. B5887) was added, and the cells were cultured at 33°C for 48 hours in a 5% CO2 incubator. The cell culture solution was transferred to a 50 mL tube, and then only the supernatant was collected again by centrifugation to measure the expression levels of IDS-Fc and ARSB-Fc fusion proteins.

[0179] First, the measurement of IDS-Fc expression level was performed using the indirect ELISA method. Human α-IDS antibody (R&D systems, cat no. AF2449) diluted in PBS at 1 μg / mL was added at 100 μL per well to a 96-well ELISA plate (Nunk, cat no. 44-2404-21), and the reaction was allowed to proceed overnight in a refrigerator at 4°C. The next day, the plate was washed 5 times with PBS-T buffer, and then 100 μL each of culture fluid samples and IDS standards (Shire, Elaprase®, lot no. TEPE09A17) diluted to various concentrations were dispensed per well and reacted at room temperature for 1 hour. After 1 hour, the plate was washed and a biotin-conjugated human α-IDS antibody (R&D systems, cat no. BAF 2449) was added, followed by reaction at room temperature for 1 hour. Finally, streptavidin-HRP (GE healthcare, cat no. RPN440IV) diluted 1:30,000 was added at 100 μL per well, and the reaction was allowed to proceed for 1 hour. After washing, the substrate solution was added and the reaction was allowed to proceed for approximately 10 minutes, and the reaction was stopped with a reaction stop solution. Then, the absorbance at 450 nm was measured. Using the concentration of the human IDS standard and the obtained absorbance values, a standard curve and function were determined, and thereby the amount of the human IDS-Fc fusion protein was quantified. As a result, it was confirmed that the cells transfected and selected expressed a predetermined amount of the human IDS-Fc fusion protein (Figure 1).

[0180] In addition, the expression level of the ARSB-Fc fusion protein was measured using an enzyme immunoassay (Bethyl, cat no. E80-104) that quantifies human IgG. Human IgG-Fc antibody (Bethyl, cat no. A80-104A-9) diluted to 10 μg / mL in carbonate buffer (0.05 M carbonate-bicarbonate, pH 9.6) was added to a 96-well ELISA plate (Nunk, cat no. 44-2404-21) at 100 μL per well and reacted at room temperature for 1 hour. After 1 hour, the ELISA plate was washed 5 times repeatedly with the washing solution, and the culture fluid samples and human IgG standards (Bethyl, cat no. RS10-110-4) contained in the human IgG quantification kit were diluted to various concentrations and dispensed at 100 μL per well respectively, and reacted at room temperature for 1 hour. After 1 hour, the plate was washed and the human IgG-Fc antibody (Bethyl, cat no. A80-104P-87) conjugated with HRP was diluted 1:150,000 and added, and then reacted at room temperature for 1 hour. Finally, streptavidin-HRP (GE healthcare, cat no. RPN440IV) was diluted 1:30,000 and added at 100 μL per well, and then reacted for 1 hour. After washing, the substrate solution was added and reacted for about 15 minutes, the reaction was stopped with the reaction stop solution, and then measured at an absorbance of 450 nm.

[0181] Using the concentration of the human IgG standard and the obtained absorbance values, a standard curve and function were obtained, and thereby the amount of the human ARSB-Fc fusion protein was quantified. As a result, it was confirmed that the cells transfected and selected expressed a predetermined amount of the human ARSB-Fc fusion protein (Figure 2).

Example

[0182] Pharmacokinetics confirmation of the enzyme-sustained fusion protein The pharmacokinetics of the enzyme-sustained fusion protein produced as described above and the enzyme not fused to the Fc region were investigated, and the effects of the production of the fusion protein were compared.

[0183] Example 4-1: Pharmacokinetic Experiment of Idursulfase-2 Sulfatase Sustained Fusion Protein The inventors investigated the pharmacokinetics of the idursulfase-2 sulfatase sustained fusion protein produced in the above examples, and confirmed the sustained efficacy effect of the fusion protein of the present invention.

[0184] Therefore, three ICR mice were administered with idursulfase-2 sulfatase (Idursulfase, control group) and idursulfase-2 sulfatase sustained fusion protein (IDS-Fc fusion protein, experimental group), respectively, and the blood stability and pharmacokinetic parameters at each blood sampling time point of each group were compared.

[0185] Specifically, the control group and the experimental group were intravenously and subcutaneously injected with 0.5 mg / kg and 1.0 mg / kg, respectively, in terms of idursulfase-2 sulfatase. After that, the intravenous injection group was bled at 0, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 144, and 168 hours after injection, and the subcutaneous injection group was bled at 0, 1, 4, 8, 24, 48, 72, 96, 120, 144, 168, 192, and 216 hours after injection. The protein amount in the serum was measured by ELISA using a human-specific anti-idursulfase-2 sulfatase antibody. The analysis results are shown in FIG. 3 and Table 4.

[0186] [Table 4]

[0187] As can be seen from these results, the idursulfase-2 sulfatase sustained fusion protein according to the present invention exhibits significantly superior pharmacokinetic characteristics compared to the control group. These results suggest that in actual drug administration, the idursulfase-2 sulfatase sustained fusion protein of the present invention has the advantage of shortening the drug administration interval due to the effect of showing a sustained efficacy compared to an enzyme that is not a fusion protein.

[0188] As can be seen from the pharmacokinetic results in Figure 3 and Table 4, in the iduronate-2-sulfatase sustained-release fusion protein, compared with the control group that is not a fusion protein, the half-life (T 1 / 2 ), the maximum blood drug concentration (C max ), and the bioavailability (AUC) all increased. In particular, the bioavailability of the iduronate-2-sulfatase sustained-release fusion protein was 64.9%, and it was confirmed that it showed excellent bioavailability compared with the enzyme that is not a fusion protein.

[0189] Example 4-2: Pharmacokinetic Experiment of Arylsulfatase B Sustained-Release Fusion Protein In order to investigate the pharmacokinetics of the arylsulfatase B sustained-release fusion protein (ARSB-Fc fusion protein) produced in the above example, the present inventors measured the pharmacokinetics of the arylsulfatase B sustained-release fusion protein and compared it with arylsulfatase B.

[0190] Specifically, in a control group administered with natural arylsulfatase B (Naglazyme, Biomarin) and an experimental group administered with the arylsulfatase B sustained-release fusion protein, ICR mice were intravenously and subcutaneously injected with 5.0 mg / kg each in terms of arylsulfatase B. Then, in the control group, regardless of the administration method, blood was collected 0, 0.25, 0.5, 0.75, 1, 1.5, 4, 8, and 24 hours after injection. Among the experimental groups, the intravenous injection group had blood collected 0, 0.25, 0.5, 1, 1.5, 2, 4, 8, 24, 48, 96, and 168 hours after injection, and the subcutaneous injection group among the experimental groups had blood collected 0, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 hours after injection.

[0191] The blood of each group collected was centrifuged to separate the serum, and the amounts of the arylsulfatase B sustained-release fusion protein and natural arylsulfatase B in the blood were quantified by an enzyme activity measurement method. The analysis results are shown in Figure 4 and Table 5.

[0192] [Table 5]

[0193] As can be seen from these results, the arylsulfatase B persistent fusion protein according to the present invention exhibits significantly superior pharmacokinetic properties compared to nagrazym, which is natural arylsulfatase B. These results suggest the advantage of shortening the drug administration interval due to the effect that the arylsulfatase B persistent fusion protein exhibits a persistent efficacy compared to natural arylsulfatase B in actual drug administration.

[0194] As can be seen from the pharmacokinetic results in FIG. 4 and Table 5, in the arylsulfatase B persistent fusion protein, the half-life (T 1 / 2 ), the maximum blood drug concentration (C max ), and the bioavailability (AUC) all increased compared to the control group that is not a fusion protein. In particular, the bioavailability of the arylsulfatase B persistent fusion protein was 65.8%, and it was confirmed that it exhibited superior bioavailability compared to the enzyme that is not a fusion protein.

[0195] As a result of investigating the pharmacokinetics of the enzyme fusion protein in Examples 4-1 and 4-2, it was confirmed that the enzyme fusion protein of the present invention can be expected to have a persistent drug efficacy because the half-life, bioavailability, etc. are significantly increased compared to the enzyme not fused to the Fc region.

Example

[0196] Confirmation of Enzyme Activity of Enzyme Persistent Fusion Protein The activity of the enzyme contained in the enzyme fusion protein produced as described above was compared with the enzyme not fused to the Fc region.

[0197] Example 5-1: In Vitro Enzyme Activity of Iduronate-2-Sulfatase Persistent Fusion Protein In order to measure the change in enzyme activity due to the production of the sustained-release fusion protein of iduronate-2-sulfatase produced in the above example, in vitro enzyme activity measurement was performed.

[0198] Specifically, 4MU-α-IdopyraA-2 (4-Methylumbelliferyl a-L-Idopyranosiduronic Acid-2-sulfate Sodium Salt), which is known as an enzyme substrate, was reacted with iduronate-2-sulfatase and the sustained-release fusion protein of iduronate-2-sulfatase at 37°C for 4 hours, and then further reacted with α-iduronidase, which is a secondary reaction enzyme, at 37°C for 24 hours. Then, the enzyme activity of the substance was confirmed by measuring the fluorescence of the finally produced 4MU (4-Methylumbelliferone).

[0199] As a result, it was confirmed that the enzyme activities (specific activities) of iduronate-2-sulfatase and the sustained-release fusion protein of iduronate-2-sulfatase were 32.0±1.58 nmol / min / mM and 87.3±6.49 nmol / min / mM, respectively. Since the sustained-release fusion protein of iduronate-2-sulfatase has a structure composed of one Fc molecule in the dimeric form of two Fc chains and two iduronate-2-sulfatases, an in vitro enzyme activity about 2.7 times higher was measured compared to iduronate-2-sulfatase that is not a fusion protein. This suggests that the structural characteristics of the sustained-release fusion protein of iduronate-2-sulfatase having two iduronate-2-sulfatases have advantages over iduronate-2-sulfatase that is not a fusion protein in terms of enzyme activity (Figure 5).

[0200] Example 5-2: In Vitro Enzyme Activity of Arylsulfatase B Sustained-Release Fusion Protein The inventors measured and compared the enzyme activity of the arylsulfatase B persistent fusion protein produced in the above examples with that of arylsulfatase B (Naglazyme, Biomarin), which is a natural enzyme.

[0201] Specifically, the in vitro enzyme activity of the arylsulfatase B persistent fusion protein was measured by reacting the arylsulfatase B persistent fusion protein and arylsulfatase B with 4-methylumbelliferyl sulfate at 37°C for 20 minutes and then measuring the fluorescence of 4-methylumbelliferyl produced by cleavage of the sulfate group.

[0202] As a result, it was confirmed that the enzyme activities (specific activities) of arylsulfatase B and the arylsulfatase B persistent fusion protein were 438.5 ± 29.4 nmol / min / μM and 823.8 ± 37.0 nmol / min / μM, respectively. The arylsulfatase B persistent fusion protein, which is composed of one Fc molecule in the dimeric form of two Fc chains and two arylsulfatase Bs, was confirmed to have an in vitro enzyme activity approximately 1.9 times higher than that of natural arylsulfatase B. As can be seen from these results, due to the structural characteristics of the arylsulfatase B persistent fusion protein, which is differentiated from natural arylsulfatase B at the molecular unit level, it exhibits excellent enzyme activity (Figure 6).

[0203] As a result of confirming the enzyme activity of the enzyme fusion protein in Examples 5-1 and 5-2, it was confirmed that the enzyme fusion protein of the present invention has a higher in vitro enzyme activity than the enzyme not fused to the Fc region.

Example

[0204] Confirmation of the administration efficacy of iduronate-2-sulfatase persistent fusion protein The efficacy of administration of the enzyme fusion protein of the present invention was confirmed by administering a drug to iduronate 2-sulfatase (IDS) knockout mice and investigating the changes in the content of glycosaminoglycan (GAG) in tissues and urine.

[0205] Specifically, in addition to the normal mouse group as the negative control group, IDS knockout mice at 7 to 14 weeks of age were divided into a total of 4 groups with 4 mice per group based on the GAG content in urine. Iduronate 2-sulfatase (Elaprase, Genzyme) 0.5 mg / kg was administered intravenously into the tail 4 times (on days 0, 7, 14, and 21) (control group), and the sustained-release fusion protein of iduronate 2-sulfatase was divided into a group administered 2.0 mg / kg intravenously into the tail once (on day 0) and a group administered 4.0 mg / kg subcutaneously once (on day 0).

[0206] Urine was collected from the mice in each group before drug administration and 7 days, 14 days, 21 days, and 28 days after administration, and the liver, spleen, heart, and bone marrow were all collected 28 days after drug administration. Tissue disruption buffer (PBS containing 1 μg / mL aprotinin, 1 mM PMSF, and 2 mM EDTA) at a volume 5 times the tissue weight (9 times for bone marrow) was added and disrupted with an ultrasonic disrupter, and then centrifuged. The supernatant obtained was used to analyze the GAG content.

[0207] Thereafter, 50 μl of the collected urine and the supernatant obtained after disruption of each tissue were added to a 96-well plate, 250 μl of dimethylmethylene blue solution was added and stirred, and the GAG content was quantified at a wavelength of 525 nm. The GAG content in urine was corrected with the creatinine content in urine to calculate the value. Statistical analysis between the control group and the test group was performed using one-way ANOVA based on the calculated values. The measured GAG contents in urine and each tissue are shown in FIGS. 7 and 8, respectively.

[0208] As can be seen from Figures 7 and 8, the iduronate-2-sulfatase long-acting fusion protein, even when administered intravenously or subcutaneously once a month, significantly reduced GAG levels in the urine and each tissue compared to IDS knockout mice, to the same extent as the once-weekly intravenous administration of an enzyme not fused to the Fc region (Elaphrase).

[0209] This example confirmed that the iduronate-2-sulfatase long-acting fusion protein of the present invention, based on its extended half-life in blood, shows the same efficacy when administered once a month as the conventional drug administration method of once a week. In addition, the results showed efficacy in reducing GAG levels even in the group administered subcutaneously once a month, indicating the possibility of subcutaneous injection as an administration route for the fusion protein of the present invention. Thus, the iduronate-2-sulfatase long-acting fusion protein of the present invention suggests the possibility of monthly and subcutaneous administration to Hunter syndrome patients. EXAMPLES

[0210] Confirmation of tissue distribution of arylsulfatase B persistent fusion protein The present inventors confirmed the tissue distribution of the enzyme fusion protein of the present invention prepared in the above Example.

[0211] To this end, arylsulfatase B (control group) and a long-acting arylsulfatase B fusion protein (experimental group) were administered to three ICR mice, and the distribution of arylsulfatase B in tissues and organs at each blood sampling time point of each group was compared.

[0212] Specifically, the control and experimental groups were intravenously injected with 5.0 mg / kg of arylsulfatase B. The organs of the mice were removed 1, 4, 8, and 24 hours after drug administration of natural arylsulfatase B (Naglazyme) in the control group and the arylsulfatase B long-acting fusion protein in the experimental group, and the concentrations of each substance in the tissues (bone marrow, liver, spleen, lungs, kidneys, and heart) were measured and compared using an enzyme activity measurement method.

[0213] As a result, compared with the native arylsulfatase B that was not fused to the Fc region and used as a control group, the arylsulfatase B persistent fusion protein showed high tissue distribution results or longer tissue distribution results in all tissues at the same time point.

[0214] In particular, it was confirmed that the arylsulfatase B persistent fusion protein had a very high distribution in the bone marrow and spleen compared with native arylsulfatase B. While arylsulfatase B was not measured in the lung, kidney, and heart, it was confirmed that the arylsulfatase B persistent fusion protein was distributed in these tissues (Figure 9).

[0215] From these experimental results, the arylsulfatase B persistent fusion protein according to the present invention exhibits excellent pharmacokinetic properties compared to naglazyme, which is native arylsulfatase B. In particular, it suggests that the quality of life of patients can be improved because the once-weekly intravenous administration used in conventional drugs can be changed to once-monthly administration or even subcutaneous administration.

[0216] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above embodiments are merely illustrative and not restrictive. The present invention should be construed as including all changes and modifications derived from the meaning and scope of the claims and their equivalent concepts, rather than the description. Next, another preferred embodiment of the present invention is shown. 1. An enzyme fusion protein in which an immunoglobulin Fc region is fused to a therapeutic enzyme and which has improved in vivo persistence compared to a therapeutic enzyme to which the Fc region is not fused. 2. The enzyme is beta-glucosidase, alpha-galactosidase, beta-galactosidase, iduronidase, iduronate-2-sulfatase, Galactose-6-sulfatase, acid alpha-glucosidase, acid ceramidase, acid sphingomyelinsase, galactocerebrosidsase, arylsulfatase A, arylsulfatase B, beta-hexosaminidase A, beta-hexosaminidase B, heparin N-sulfatase, alpha-D-mannosidase, beta-glucuronidase, N-acetylgalactosamine-6 sulfatase, lysosomal acid lipase, alpha-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholinesterase, Chitinase, glutamate decarboxylase, imiglucerase, lipase, Uricase, Platelet-Activating Factor Acetylhydrolase, neutral endopeptidaseThe enzyme fusion protein according to 1 above, selected from the group consisting of endopeptidase) and myeloperoxidase). 3. The enzyme fusion protein according to 1 above, wherein the enzyme fusion protein is one in which a therapeutic enzyme and an immunoglobulin Fc region are fused with a peptide linker. 4. The enzyme fusion protein according to 1 above, wherein the enzyme fusion protein is one in which one molecule of an immunoglobulin Fc region and a dimer of a therapeutic enzyme are fused. 5. The enzyme fusion protein according to 1 above, wherein the immunoglobulin Fc region has been modified by at least one substitution, addition, deletion, modification, and combinations thereof selected from the group consisting of substitutions, additions, deletions, modifications, and combinations thereof in the native immunoglobulin Fc region. 6. The enzyme fusion protein according to 5 above, wherein the immunoglobulin Fc region has the second amino acid of the immunoglobulin Fc region having the amino acid sequence of SEQ ID NO: 8 substituted with proline, the 71st amino acid substituted with glutamine, or the second amino acid substituted with proline and the 71st amino acid substituted with glutamine. 7. The enzyme fusion protein according to 6 above, wherein the immunoglobulin Fc region does not undergo chain exchange. 8. The enzyme fusion protein according to 1 above, wherein the enzyme fusion protein has improved stability, reduced binding affinity for lysosomal receptors, and improved tissue distribution compared to a therapeutic enzyme to which the Fc region is not fused. 9. The enzyme fusion protein according to 1 above, wherein the immunoglobulin Fc region is selected from the group consisting of (a) CH1 domain, CH2 domain, CH3 domain, and CH4 domain, (b) CH1 domain and CH2 domain, (c) CH1 domain and CH3 domain, (d) CH2 domain and CH3 domain, (e) a combination of at least one domain of CH1 domain, CH2 domain, CH3 domain, and CH4 domain and an immunoglobulin hinge region or a part of the hinge region, and (f) a dimer of each domain of the heavy chain constant region and the light chain constant region. 10. The immunoglobulin Fc region of the enzyme fusion protein according to item 1 above has at least one characteristic selected from the following: (a) the site for forming a disulfide bond is removed; (b) a part of the amino acids at the N-terminus is deleted from the native Fc; (c) a methionine residue is added to the N-terminus of the native Fc; (d) the complement binding site is removed; (e) the ADCC (antibody dependent cell mediated cytotoxicity) site is removed. 11. The enzyme fusion protein according to any one of items 1 to 10 above, wherein the immunoglobulin Fc region is non-glycosylated. 12. The enzyme fusion protein according to any one of items 1 to 10 above, wherein the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE or IgM. 13. The enzyme fusion protein according to item 12 above, wherein the immunoglobulin Fc region is a hybrid of domains having different origins derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. 14. The enzyme fusion protein according to item 13 above, wherein the immunoglobulin Fc region is an IgG4 Fc region. 15. The enzyme fusion protein according to item 14 above, wherein the hinge region of the immunoglobulin IgG4 Fc region is replaced with an IgG1 hinge region. 16. A pharmaceutical composition for preventing or treating lysosomal storage disorder (LSD) comprising the enzyme fusion protein according to any one of items 1 to 10 above. 17. The pharmaceutical composition for preventing or treating lysosomal storage disorder according to item 16 above, wherein the lysosomal storage disorder is selected from the group consisting of mucopolysaccharidosis (MPS), glycogen storage disease, sphingolipidosis, Niemann-Pick disease, Fabry’s disease, Gaucher disease, Hunter syndrome, and Maroteaux-Lamy syndrome. 18. The pharmaceutical composition for preventing or treating the lysosomal storage disease according to item 16 above, wherein the enzyme is iduronate-2-sulfatase (IDS) or arylsulfatase B (ARSB). 19. The pharmaceutical composition for preventing or treating the lysosomal storage disease according to item 16 above, wherein the composition reduces the binding ability of the enzyme to the lysosomal receptor. 20. A polynucleotide encoding the enzyme fusion protein according to any one of items 1 to 10 above. 21. An expression vector containing the polynucleotide according to item 20 above. 22. A transformant into which the expression vector according to item 21 above has been introduced. 23. (a) A step of culturing the transformant according to item 22 above to obtain a culture, and (b) A method for producing an enzyme fusion protein, comprising a step of recovering the enzyme fusion protein from the culture.

Claims

**Claim 1** An enzyme fusion protein in which an immunoglobulin Fc region is fused to the C-terminus of a therapeutic enzyme, and which has improved in vivo persistence compared to a therapeutic enzyme to which the immunoglobulin Fc region is not fused, wherein the immunoglobulin Fc region does not undergo chain exchange, the immunoglobulin Fc region is derived from an IgG4 Fc region, the second amino acid of the immunoglobulin Fc region having the amino acid sequence of SEQ ID NO: 8 is substituted with proline, the therapeutic enzyme and the immunoglobulin Fc region are fused with a peptide linker, an enzyme fusion protein having improved stability and high tissue distribution compared to a therapeutic enzyme to which the immunoglobulin Fc region is not fused. **Claim 2** The therapeutic enzyme is selected from the group consisting of beta-glucosidase, alpha-galactosidase, beta-galactosidase, iduronidase, iduronate-2-sulfatase, Galactose-6-sulfatase, acid alpha-glucosidase, acid ceramidase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, arylsulfatase B, beta-hexosaminidase A, beta-hexosaminidase B, heparin N-sulfatase, alpha-D-mannosidase, N-acetylgalactosamine-6 sulfatase, lysosomal acid lipase, alpha-N-acetyl-glucosaminidase, butyrylcholinesterase, Chitinase, glutamate decarboxylase, imiglucerase, lipase, Uricase, Platelet-Activating Factor Acetylhydrolase, neutral endopeptidase, and myeloperoxidase, the enzyme fusion protein according to claim 1.

3. The enzyme fusion protein according to claim 1, wherein the enzyme fusion protein is a fusion of one molecule of the immunoglobulin Fc region and a dimer of a therapeutic enzyme.

4. The enzyme fusion protein according to claim 1, wherein the immunoglobulin Fc region has the amino acid sequence of SEQ ID NO: 8, with the second amino acid substituted with proline and the 71st amino acid substituted with glutamine.

5. A pharmaceutical composition for preventing or treating lysosomal storage disorder (LSD) comprising the enzyme fusion protein according to any one of claims 1 to 4.

6. The pharmaceutical composition for preventing or treating lysosomal storage disorder according to claim 5, wherein the lysosomal storage disorder is selected from the group consisting of mucopolysaccharidosis (MPS), glycogen storage disease, sphingolipidosis, Niemann-Pick disease, Fabry’s disease, Hunter syndrome, and Maroteaux-Lamy syndrome.

7. The pharmaceutical composition for preventing or treating lysosomal storage disorder according to claim 5, wherein the enzyme is iduronate-2-sulfatase (IDS) or arylsulfatase B (ARSB).

8. A polynucleotide encoding the enzyme fusion protein according to any one of claims 1 to 4.

9. An expression vector comprising the polynucleotide according to claim 8.

10. A transformant into which the expression vector according to claim 9 has been introduced.

11. A method for producing an enzyme fusion protein, comprising: (a) culturing the transformant according to claim 10 to obtain a culture; and (b) recovering the enzyme fusion protein from the culture. ​

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