Therapeutic enzyme fusion protein having novel structure and use thereof

Abstract

The present invention relates to a therapeutic enzyme fusion protein having a novel structure and a use thereof. The invention relates to a fusion protein of a dimeric therapeutic enzyme and an immunoglobulin Fc region, a preparation method thereof and a composition containing the same.

Description

[0001] This application is a divisional application. The application date of the original application is December 21, 2018, the application number is 201880083193.8, and the name of the invention is “Therapeutic enzyme fusion protein with novel structure and its use”, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to an enzyme fusion protein containing a dimeric therapeutic enzyme, a method for preparing the same, and a composition containing the same. Background Art

[0003] Lysosomes are cytoplasmic organelles that have the function of degrading macromolecules such as proteins, polynucleotides, polysaccharides, and lipids. The intracellular environment of the lysosome is acidic and contains hydrolases that promote the hydrolysis of biomacromolecules. Lysosomes have also been found to play a role in the absorption of molecules through endocytosis within the cell.

[0004] Lysosomal storage disease (hereinafter referred to as LSD) is a hereditary metabolic disorder characterized by lysosomal dysfunction. LSD is caused by the deficiency of enzymes that degrade substances (such as lipids, proteins, polysaccharides, etc.), and LSD usually occurs at an incidence of one in 100,000 and is inherited as an autosomal recessive trait. When there is a lack or lack of specific degradation enzymes, LSD will occur, and when these degradation enzymes are insufficient, the excess substances produced accumulate and are not degraded, eventually causing problems with cell function. Like many other genetic disorders, LSD is inherited from parents. In addition, each of these diseases occurs due to any mutation in the genes involved in the translation of different enzymes. The enzymes that cause these diseases usually have similar biochemical properties, and all LSDs are caused by the abnormal accumulation of substances in lysosomes. At present, about 50 different types of LSDs are known (for example, Niemann-Pick disease, Fabry disease, Gaucher disease, Hunter syndrome, Mara-La syndrome, etc.).

[0005] Fabry disease, known as LSD, is an inborn error of the metabolism of glycosphingolipids due to a deficiency or absence of the enzymatic activity of the hydrolase α-galactosidase A present in lysosomes.

[0006] α-Galactosidase A is an enzyme that hydrolyzes ceramide hexotrioside (Gb3) into lactosylsphingosine, and it is known that disorders of α-galactosidase A lead to abnormal accumulation of Gb3 in blood vessel walls and various parts of the body, thereby causing the occurrence of Fabry disease.

[0007] A representative method for treating these LSDs may be enzyme replacement therapy (ERT), and many related studies are currently underway (Frances M. Platt et al., J Cell Biol. 2012 Nov 26; 199(5): 723 to 34). In particular, since LSDs are diseases caused by genetic defects in specific enzymes, replacement therapy is essential for the treatment of defective enzymes. Enzyme replacement therapy is the standard treatment for LSDs, and the therapy has the effect of alleviating existing symptoms or delaying disease progression by replacing insufficient enzymes.

[0008] However, proteins that exhibit such therapeutic effects generally have low stability and are therefore easily denatured and degraded by proteases in the blood. Therefore, frequent administration to patients is necessary to maintain blood concentrations and efficacy of these proteins.

[0009] However, in the case of administering protein drugs to patients in the form of injections, frequent injections to maintain blood concentrations of active polypeptides may cause significant pain to patients. In order to address these problems, efforts have been made to maximize the pharmacological efficacy by improving the blood stability of therapeutic enzymes and maintaining their blood concentrations at a high level for a long period of time. Such long-acting preparations of therapeutic enzymes are needed to increase the stability of the therapeutic enzymes and at the same time maintain the efficacy of the drug itself at a sufficiently high level, and not induce an immune response in patients.

[0010] Therefore, there is a need to develop a therapeutic agent having a therapeutic effect on a target disease, in which the activity of a fusion protein is maintained while the duration of the fusion protein is stably increased in vivo. Summary of the invention

[0011] [Technical issues]

[0012] The object of the present invention is to provide an enzyme fusion protein of the following formula 1:

[0013] [Formula 1]

[0014]

[0015] wherein X and X′ are each independently the same or different types of therapeutic enzymes;

[0016] L and L' are linkers, each independently being the same or different types of linkers;

[0017] F is the immunoglobulin Fc region;

[0018] | is a covalent bond; and

[0019] : is a covalent bond or a non-covalent bond.

[0020] Another object of the present invention is to provide a pharmaceutical composition containing the enzyme fusion protein for preventing or treating LSD.

[0021] Another object of the present invention is to provide a polynucleotide encoding an enzyme fusion protein.

[0022] Another object of the present invention is to provide an expression vector containing the polynucleotide.

[0023] Still another object of the present invention is to provide a transformant into which the expression vector is introduced.

[0024] Another object of the present invention is to provide a method for preparing an enzyme fusion protein, which comprises culturing a transformant to obtain a culture; and recovering the enzyme fusion protein from the culture.

[0025] [Beneficial Effects of the Invention]

[0026] The present invention relates to a fusion protein containing a dimerized therapeutic enzyme, and more specifically, to an enzyme fusion protein in which an immunoglobulin Fc region is fused to a therapeutic enzyme so that the therapeutic enzyme has increased stability and the enzyme removal mechanism by the kidney is reduced. The enzyme fusion protein of the present invention can be effectively used by patients due to the increased duration. In addition, the enzyme fusion protein of the present invention contains a dimerized therapeutic enzyme, and therefore the preparation steps can be reduced, and production costs can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of measuring the activity of an exemplary α-galactosidase-Fc fusion protein are shown.

[0028] Figure 2 Shown are the results of comparison of in vitro enzyme activities between agalsidase-β and α-galactosidase-Fc fusion proteins.

[0029] Figure 3 A graph illustrating the results of comparison of in vitro intracellular uptake activity between agalsidase-β and α-galactosidase-Fc fusion proteins is shown.

[0030] Figure 4 Shown is a graph illustrating the results of a comparison of the pharmacokinetic behavior between agalsidase-β and α-galactosidase-Fc fusion proteins.

[0031] [Technical solution]

[0032] One aspect of the present invention provides an enzyme fusion protein in which a therapeutic enzyme and an immunoglobulin Fc region are fused together.

[0033] In a specific embodiment of the present invention, the enzyme fusion protein can be represented by the following formula 1:

[0034] [Formula 1]

[0035]

[0036] wherein X and X′ are each independently the same or different types of therapeutic enzymes;

[0037] L and L' are linkers, each independently being the same or different types of linkers;

[0038] F is the immunoglobulin Fc region;

[0039] | is a covalent bond; and

[0040] : is a covalent bond or a non-covalent bond.

[0041] In the enzyme fusion protein according to the aforementioned specific embodiments, the therapeutic enzyme can form a dimer through a non-covalent bond.

[0042] In the enzyme fusion protein according to any one of the aforementioned embodiments, the therapeutic enzyme may form a dimer in an antiparallel configuration to each other.

[0043] In the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein can increase stability and reduce binding affinity to lysosomal receptors compared to a therapeutic enzyme not fused to an immunoglobulin Fc region.

[0044] In the enzyme fusion protein according to any of the foregoing specific embodiments, the enzyme can be selected from β-glucosidase, α-galactosidase, β-galactosidase, iduronidase, iduronic acid-2-sulfatase, galactose-6-sulfatase, acid α-glucosidase, acid ceramidase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, arylsulfatase B, β-hexosaminidase A, β-hexosaminidase B ... glucosidase B, heparin N-sulfatase, α-D-mannosidase, β-glucuronidase, N-acetylgalactosamine-6-sulfatase, lysosomal acid lipase, α-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, glucosylceramide analog, lipase, urate oxidase, platelet-activating factor acetylhydrolase, neutral endopeptidase, and myeloperoxidase.

[0045] In the enzyme fusion protein according to any one of the preceding embodiments, the enzyme may be α-galactosidase A or β-galactosidase.

[0046] In the enzyme fusion protein according to any one of the aforementioned specific embodiments, the immunoglobulin Fc region may have a change selected from substitution, addition, deletion, modification and a combination thereof in at least one amino acid of the native immunoglobulin Fc region.

[0047] In the enzyme fusion protein according to any of the aforementioned specific embodiments, in the immunoglobulin Fc region having the amino acid sequence of SEQ ID NO:8, the 2nd amino acid may be substituted by proline; the 71st amino acid may be substituted by glutamine; or the 2nd amino acid may be substituted by proline and the 71st amino acid may be substituted by glutamine.

[0048] In the enzyme fusion protein according to any of the aforementioned embodiments, chain exchange may not occur in the immunoglobulin Fc region.

[0049] In the enzyme fusion protein according to any one of the foregoing embodiments, the immunoglobulin Fc region may be selected from:

[0050] (a) CH1 domain, CH2 domain, CH3 domain and CH4 domain;

[0051] (b) CH1 domain and CH2 domain;

[0052] (c) CH1 domain and CH3 domain;

[0053] (d) a CH2 domain and a CH3 domain; and

[0054] (e) A combination of one or two or more of the CH1 domain, CH2 domain, CH3 domain and CH4 domain with an immunoglobulin hinge region or a portion of the hinge region.

[0055] In the enzyme fusion protein according to any one of the aforementioned specific embodiments, the immunoglobulin Fc region may be in the form of a monomer.

[0056] In the enzyme fusion protein according to any one of the aforementioned specific embodiments, the immunoglobulin Fc region may consist of a hinge region, a CH2 domain and a CH3 domain.

[0057] In the enzyme fusion protein according to any one of the preceding embodiments, in the immunoglobulin Fc region:

[0058] Regions capable of forming disulfide bonds can be removed;

[0059] A certain amino acid residue can be removed from the N-terminus of the native Fc;

[0060] A methionine residue can be added to the N-terminus of the native Fc form;

[0061] The complement binding sites may be removed; or

[0062] Antibody-dependent cell-mediated cytotoxicity (ADCC) sites may be deleted.

[0063] In the enzyme fusion protein according to any of the aforementioned embodiments, the immunoglobulin Fc region may be aglycosylated.

[0064] In the enzyme fusion protein according to any one of the aforementioned specific embodiments, the immunoglobulin Fc region may be an Fc fragment derived from IgG, IgA, IgD, IgE or IgM.

[0065] In the enzyme fusion protein according to any one of the aforementioned specific embodiments, the immunoglobulin Fc region may be a hybrid having domains derived from different sources of immunoglobulins selected from IgG, IgA, IgD, IgE and IgM.

[0066] In the enzyme fusion protein according to any one of the aforementioned specific embodiments, the immunoglobulin Fc region may be an IgG4 Fc region.

[0067] In the enzyme fusion protein according to any one of the aforementioned embodiments, the hinge region of the IgG4 Fc region may be replaced by the IgG1 hinge region.

[0068] In the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein may include the amino acid sequence of SEQ ID NO:13.

[0069] Another aspect of the present invention provides a pharmaceutical composition containing the enzyme fusion protein for preventing or treating LSD.

[0070] In the composition according to a specific embodiment, the LSD may be selected from mucopolysaccharidosis (MPS), glycogen storage disease, sphingolipidosis, Niemann-Pick disease, Fabry disease, Gaucher disease, Hunter syndrome, and Mara-La syndrome.

[0071] In the composition according to the aforementioned specific embodiments, the composition can reduce the binding affinity of the enzyme to the lysosomal receptor.

[0072] Another aspect of the present invention provides a polynucleotide encoding an enzyme fusion protein.

[0073] Another aspect of the present invention provides an expression vector containing the polynucleotide.

[0074] Still another aspect of the present invention provides a transformant into which the expression vector is introduced.

[0075] Another aspect of the present invention provides a method for preparing an enzyme fusion protein.

[0076] In the method of preparing an enzyme fusion protein according to a specific embodiment, the method may include culturing the transformant to obtain a culture; and recovering the enzyme fusion protein from the culture. DETAILED DESCRIPTION

[0077] The preferred embodiments are described in detail below. Meanwhile, each description and embodiment disclosed in the present disclosure may also be applied to other descriptions and embodiments. That is to say, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present invention. Further, the scope of the present invention is not limited by the following specific description.

[0078] Additionally, those skilled in the art will be able to recognize or ascertain based on routine experimentation many equivalents to the specific embodiments of the invention described in this application, and such equivalents are intended to be encompassed by the present invention.

[0079] Throughout the specification, not only the conventional one-letter or three-letter codes for naturally occurring amino acids are used, but also those three-letter codes generally permitted for other amino acids, such as α-aminoisobutyric acid (Aib), Sar (N-methylglycine), α-methyl-glutamic acid, etc. In addition, the amino acids referred to herein by abbreviations are described according to IUPAC-IUB rules as follows:

[0080] Alanine A; Arginine R;

[0081] Asparagine N; Aspartic acid D;

[0082] CysteineC; GlutamateE;

[0083] Glutamine Q; Glycine G;

[0084] Histidine H; Isoleucine I;

[0085] Leucine L; Lysine K;

[0086] Methionine M; Phenylalanine F;

[0087] Proline P; Serine S;

[0088] Threonine T; Tryptophan W;

[0089] Tyrosine, Y; and Valine, V.

[0090] One aspect of the present invention provides an enzyme fusion protein of Formula 1;

[0091] [Formula 1]

[0092]

[0093] wherein X and X′ are each independently the same or different types of therapeutic enzymes;

[0094] L and L' are linkers, each independently being the same or different types of linkers;

[0095] F is the immunoglobulin Fc region;

[0096] | is a covalent bond; and

[0097] : is a covalent bond or a non-covalent bond.

[0098] As used herein, the term "enzyme fusion protein" may refer to a protein in which an immunoglobulin Fc region is fused to a therapeutic enzyme, such that the therapeutic enzyme can maintain its activity while reducing its binding affinity to lysosomal receptors compared to a therapeutic enzyme not fused to the immunoglobulin Fc region, thereby increasing its blood half-life. The enzyme fusion protein of the present invention can be used as a drug for enzyme replacement therapy (ERT). Enzyme replacement therapy can prevent or treat a disease by restoring the function of a degraded enzyme by supplementing a defective enzyme or insufficient enzyme that causes the disease.

[0099] The present inventors have prepared fusion proteins with immunoglobulin Fc regions to increase the blood half-life of therapeutic enzymes. In particular, as the Fc region, an IgG4 Fc analog was used, in which a potential glycosylation sequence was substituted to inhibit glycosylation, and additionally the hinge sequence of IgG4 Fc was substituted to inhibit chain exchange.

[0100] Additionally, it was confirmed that when preparing the enzyme fusion protein of the present invention, when each therapeutic enzyme fused to the immunoglobulin Fc region forms a dimer, particularly a dimer connected in an anti-parallel configuration, the in vivo duration of the enzyme fusion protein can be increased while maintaining the activity of the therapeutic enzyme, and further, the preparation steps of the enzyme fusion protein can be reduced, thereby enabling efficient production.

[0101] Therefore, compared with the therapeutic enzyme not fused with the immunoglobulin Fc region, the enzyme fusion protein of the present invention has the advantages of increasing the stability of the enzyme while maintaining the activity of the enzyme and reducing the production cost.

[0102] As used herein, the term "therapeutic enzyme" refers to an enzyme used to treat a disease that occurs due to a lack, deficiency, dysfunction, etc. of the enzyme, and the enzyme can be used to treat a subject suffering from the disease through enzyme replacement therapy, administration, etc. Specifically, the enzyme can be an enzyme used to treat LSD that may occur due to a lack, deficiency, etc. of a lysosomal enzyme, but the enzyme is not limited thereto.

[0103] The therapeutic enzyme included in the enzyme fusion protein of the present invention is not particularly limited, but may include any therapeutic enzyme having the advantage of prolonged in vivo duration compared to the therapeutic enzyme type not fused to the immunoglobulin Fc region. In an exemplary embodiment of the present invention, the enzyme fusion protein is a fusion protein of a therapeutic enzyme.

[0104] The therapeutic enzyme included in the enzyme fusion protein of the present invention can be an enzyme that forms a dimer through a non-covalent bond, but the therapeutic enzyme is not limited thereto. Specifically, when the fusion protein is expressed in a transformant and the immunoglobulin Fc region forms a dimer, the therapeutic enzyme can form a dimer.

[0105] The dimer of the therapeutic enzyme may be a dimer formed by two enzymes identical to each other, or may be a dimer formed by two different enzymes. As long as these enzymes have the desired activity in vivo, the specific type of enzyme constituting the dimer is not limited.

[0106] Meanwhile, these therapeutic enzymes constituting dimers may be in the form of parallel dimers or antiparallel dimers depending on the direction in which they are linked, but the therapeutic enzyme is not limited thereto.

[0107] In an exemplary embodiment of the present invention, a fusion protein in which α-galactosidase (i.e., a therapeutic enzyme) was fused to an immunoglobulin Fc region was prepared, and it was confirmed that α-galactosidase formed an antiparallel dimer through a non-covalent bond, while the immunoglobulin Fc region of the fusion protein formed a dimer. In addition, it was confirmed that the fusion protein could maintain the activity of the enzyme even in a state where the fusion protein was fused to the immunoglobulin Fc region (Example 2).

[0108] In another exemplary embodiment of the present invention, a fusion protein in which α-galactosidase was fused to an immunoglobulin Fc region and agalsidase-β not fused to an immunoglobulin Fc region were compared. As a result, it was confirmed that the fusion protein maintained in vitro enzyme activity and intracellular absorption activity even when fused to the Fc region (Examples 2 and 3), and it was also confirmed that these fusion proteins had excellent pharmacokinetic behavior due to fusion with the Fc region (Example 4).

[0109] As used herein, the term "parallel dimer" means that when each monomer forms a dimer, the N-terminus and C-terminus of the amino acid sequence of each monomer form a dimer in the same direction. In particular, the dimer can be formed by a non-covalent bond or a covalent bond, but the method of parallel dimer formation is not limited thereto.

[0110] As used herein, the term "antiparallel dimer" means that when each monomer forms a dimer, the N-terminus and the C-terminus of the amino acid sequence of each monomer form a dimer in different directions from each other. In particular, the dimer can be formed by a non-covalent bond or a covalent bond, but the method of antiparallel dimer formation is not limited thereto.

[0111] In other words, in the enzyme fusion protein of the present invention, it is possible that (i) the N-terminus of one therapeutic enzyme (X) and the N-terminus of another therapeutic enzyme (X') can form a dimer in the same direction; (ii) the C-terminus of one therapeutic enzyme (X) and the C-terminus of another therapeutic enzyme (X') can form a dimer in the same direction; (iii) the N-terminus of one therapeutic enzyme (X) and the C-terminus of another therapeutic enzyme (X') can form a dimer in the same direction; or (iv) the C-terminus of one therapeutic enzyme (X) and the N-terminus of another therapeutic enzyme (X') can form a dimer in the same direction. The dimers in cases (i) and (ii) are called parallel dimers, and the dimers in cases (iii) and (iv) are called antiparallel dimers. The formation of these dimers can be by covalent bonds or non-covalent bonds, but the formation of dimers is not limited to this.

[0112] Specifically, in the formation of the above-mentioned dimer, the formation of the parallel dimer or the antiparallel dimer may be such that as the immunoglobulin Fc region forms a dimer, the α-galactosidase A of the monomer linked thereto forms a dimer through a covalent bond or a non-covalent bond.

[0113] Regarding the fusion protein containing the antiparallel configuration of the dimeric therapeutic enzyme of the present invention, the excellent advantage of the process for preparing the fusion protein is that it can reduce the number of preparation steps, thereby reducing production costs, and can maintain enzyme activity while reducing the instability of the dimer type caused by changes in pH in vivo.

[0114] The therapeutic enzyme of the present invention can be selected from β-glucosidase, α-galactosidase, β-galactosidase, iduronidase, iduronic acid-2-sulfatase, galactose-6-sulfatase, acid α-glucosidase, acid ceramidase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, arylsulfatase B, β-hexosaminidase A, β-hexosaminidase B, heparin N-sulfate The therapeutic enzymes of the present invention include esterase, α-D-mannosidase, β-glucuronidase, N-acetylgalactosamine-6-sulfatase, lysosomal acid lipase, α-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, glucosylceramide analog, lipase, uricase, platelet activating factor acetylhydrolase, neutral endopeptidase, and myeloperoxidase. The therapeutic enzymes of the present invention may be human-derived enzymes, but any therapeutic enzyme having a therapeutic effect on LSD may be included in the present invention without limitation, regardless of the source or type of the enzyme.

[0115] More specifically, the enzyme may be α-galactosidase A or β-galactosidase.

[0116] As used herein, the term "α-galactosidase A" is an enzyme present in lysosomes of the spleen, brain, liver, etc., which hydrolyzes the terminal α-galactosyl moiety in glycolipids and glycoproteins, and is a homodimeric glycoprotein. Specifically, α-galactosidase A is known to hydrolyze ceramide trihexosyl, catalyze the hydrolysis of melibiose into galactose and glucose, and, in particular, is known to be associated with Fabry disease (LSD).

[0117] In the present invention, α-galactosidase A can be used interchangeably with agalsidase-α or agalsidase-β (i.e., recombinant), and any enzyme having equivalent activity and exhibiting a therapeutic effect on LSD can be included in the scope of the present invention without limitation, regardless of its sequence, source, preparation method, etc. Specifically, α-galactosidase can be encoded by the polynucleotide sequence of SEQ ID NO: 5, and can include the amino acid sequence of SEQ ID NO: 6, but the sequence of α-galactosidase is not limited thereto.

[0118] The therapeutic enzyme included in the enzyme fusion protein of the present invention can be a naturally occurring type and a fragment consisting of a portion of the therapeutic enzyme, or an analog of the therapeutic enzyme in which changes selected from substitutions, additions, deletions, and modifications of some amino acids and combinations thereof have occurred, and can be included in the present invention without limitation as long as it has an activity equivalent to that of the naturally occurring type of the therapeutic enzyme.

[0119] Furthermore, analogs of therapeutic enzymes include all those in which one or more amino acids are added to the amino terminus and / or carboxyl terminus of the naturally occurring type of the therapeutic enzyme.

[0120] For the replacement or addition of amino acids, not only the 20 common amino acids in human proteins can be used, but also atypical or non-natural amino acids can be used. Commercial sources of atypical amino acids can include Sigma-Aldrich, ChemPep Inc., Genzyme Pharmaceuticals, etc. Peptides including these amino acids and atypical peptide sequences can be synthesized and purchased from commercial suppliers (e.g., American Peptide Company, Bachem (U.S.), or Anygen (Korea)), but commercial sources are not limited thereto.

[0121] As used herein, the term "fragment" refers to a form in which one or more amino acids at the amino or carboxyl terminus of a natural therapeutic enzyme or an analog of a natural therapeutic enzyme are removed. Regardless of the size of the fragment or the type of amino acid, as long as they have the activity of a therapeutic enzyme, the natural therapeutic enzyme or its analog belongs to the scope of the present invention.

[0122] Therapeutic enzyme analogs may include biosimilars and biobetters of the corresponding therapeutic enzymes. For example, regarding biosimilars, considering the differences between known therapeutic enzymes and the host expressing them, the differences in glycosylation characteristics and their degree, and according to the standard sequence, when the degree of substitution is not 100% substituted, the difference in the degree of substitution in the specific amino acid residues of the corresponding enzyme, they belong to the biological similar enzymes used as enzyme fusion proteins of the present invention. Therapeutic enzymes can be produced by methods known in the art, specifically by genetic recombination in animal cells, Escherichia coli (E.coli), yeast, insect cells, plant cells, living animals, etc., and the preparation method is not limited thereto, and commercially available enzymes can be purchased and used, but the enzymes are not limited thereto.

[0123] In addition, the therapeutic enzyme may include an amino acid sequence having at least 60%, 70% or 80%, more specifically 90%, and even more specifically 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or higher homology with the above-mentioned enzyme or its analogs, and the therapeutic enzyme can be obtained from a microorganism by recombinant technology or is a commercially available enzyme, but the therapeutic enzyme is not limited thereto.

[0124] As used herein, the term "homology" means the degree of similarity to a wild-type amino acid sequence or a wild-type nucleotide sequence, and homology comparisons can be performed by the naked eye or using readily commercially available comparison programs. The homology between two or more sequences can be calculated as a percentage (%) using a commercial computer program. Homology (%) can be calculated for adjacent sequences.

[0125] Information on the sequence of the therapeutic enzyme or its analogs and the nucleotide sequence encoding the same can be obtained from known databases (eg, NCBI, etc.).

[0126] The therapeutic enzyme can be prepared or manufactured by a method known in the art, and specifically, the enzyme can be purified from the culture after culturing animal cells into which the animal expression vector is inserted, or can be used after purchasing a commercially available enzyme, but the enzyme is not limited thereto.

[0127] The enzyme fusion protein of the present invention may be a protein in which the therapeutic enzyme and the immunoglobulin Fc region are fused via a peptide linker. That is, L or L' of Formula 1 may be a peptide linker, but as long as they can fuse the immunoglobulin Fc region with the therapeutic enzyme, L or L' is not particularly limited.

[0128] The peptide linker may include at least one amino acid, such as 1 to 1,000 amino acids, any peptide linker known in the art (e.g., including [GS] x Connector, [GGGS] x Connector, and [GGGGS] xLinker, etc., wherein x is a natural number of 1 or more (e.g., 1, 2, 3, 4, 5 or more), but the peptide linker is not particularly limited thereto. Specifically, the peptide linker of the present invention can be composed of 10 to 50 amino acid sequences, and more specifically, can be composed of 20 to 40 amino acid sequences, and can be composed of the amino acid sequence of SEQ ID NO: 11.

[0129] For the purpose of the present invention, as long as the peptide linker can connect the therapeutic enzyme and the immunoglobulin Fc while maintaining the activity of the therapeutic enzyme, the position of the peptide linker fused to the therapeutic enzyme and the immunoglobulin Fc is not limited, specifically, the two ends of the therapeutic enzyme and the immunoglobulin Fc region, and more specifically, the C-terminus of the therapeutic enzyme and the N-terminus of the immunoglobulin Fc region, but the position is not limited thereto.

[0130] As used herein, the terms "N-terminus" and "C-terminus" refer to the amino terminus and carboxyl terminus of a protein, respectively. For example, "N-terminus" or "C-terminus" may include not only the terminal amino acid residue of the N-terminus or C-terminus, but also the amino acid residues adjacent to the amino acid residue of the N-terminus or C-terminus, and specifically, the 1st to 20th amino acid residues starting from the terminal itself, but the N-terminus or C-terminus is not limited thereto.

[0131] In one embodiment of the present invention, a fusion protein (SEQ ID NO: 13) was prepared by synthesis, in which the N-terminus of IgG4 was fused to the C-terminus of the therapeutic enzyme, so that α-galactosidase (therapeutic enzyme) was fused to linker-IgG4 at the genetic level, and it was confirmed that the fusion protein was expressed in the transformant into which the fusion protein was transformed (Example 2).

[0132] In the present invention, the peptide linker may be a linker linked to each immunoglobulin Fc region of a dimer formed by immunoglobulin Fc regions of monomers, and the linker linked to each immunoglobulin Fc region may be the same or different species.

[0133] The immunoglobulin Fc region as a part of the enzyme fusion protein of the present invention may be an immunoglobulin Fc region in which a dimer is formed between the immunoglobulin Fc regions of the respective monomers.

[0134] As used herein, the term "immunoglobulin Fc region" refers to a region of an immunoglobulin molecule that includes a heavy chain constant region 2 (CH2) and / or a heavy chain constant region 3 (CH3), excluding the variable regions of the heavy chain and the light chain. For the purposes of the present invention, such an immunoglobulin Fc region may include a modified hinge region, but is not limited thereto. Specifically, an immunoglobulin Fc region may be an immunoglobulin Fc region having a change selected from substitution, addition, deletion, modification, and a combination thereof in at least one amino acid in a native immunoglobulin Fc region, but the immunoglobulin Fc region is not limited thereto.

[0135] Immunoglobulin Fc region is the material used as carrier in the preparation of medicine, and in order to stabilize protein and prevent it from being removed from kidney, the fusion protein research utilizing immunoglobulin Fc region is being actively carried out recently.Immunoglobulin is the main component of blood, and there are five different types (i.e., IgG, IgM, IgA, IgD and IgE).The most commonly used type in fusion protein research is IgG, and it is divided into four subtypes.The fusion protein utilizing immunoglobulin Fc preparation can increase the size of protein, and thereby prevent their removal in kidney, and can also bind to FcRn receptors, and thereby enter cells by endocytosis and circulation and have the effect of increasing blood half-life.

[0136] Such an immunoglobulin Fc region may include a hinge region in a heavy chain constant region, and a monomeric immunoglobulin Fc region may form a dimer due to the hinge region, but the immunoglobulin Fc region is not limited thereto. In addition, as long as the immunoglobulin Fc region has the same or better effect as its natural type, the immunoglobulin Fc region of the present invention may be an extended Fc region including all or part of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1), excluding the variable regions of the heavy and light chains of the immunoglobulin. In addition, the immunoglobulin Fc region of the present invention may be a region in which a portion of a significantly long amino acid sequence corresponding to CH2 and / or CH3 is removed.

[0137] Specifically, the immunoglobulin Fc region of the present invention can be an immunoglobulin Fc region selected from (a) 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; and (e) a combination of one or two or more domains of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain and an immunoglobulin hinge region or a part of the hinge region, but the immunoglobulin Fc region is not limited thereto. More specifically, the immunoglobulin Fc region can be an immunoglobulin Fc region composed of a hinge region, a CH2 domain, and a CH3 domain, but the immunoglobulin Fc region is not limited thereto.

[0138] In an embodiment, the hinge region may be one in which a portion of the hinge sequence having the following amino acid sequence is deleted or modified.

[0139] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 14)

[0140] Specifically, the hinge region can be a hinge region with a variation in which a portion of the hinge region is deleted to include only one cysteine ​​(Cys) residue; or it can be a hinge region in which a serine (Ser) residue participating in chain exchange is substituted by a proline (Pro) residue, and more specifically, a hinge region in which the second serine of the hinge sequence is substituted by a proline residue, but the hinge region is not limited thereto.

[0141] In the present invention, the immunoglobulin Fc region can prevent chain exchange and monomer formation in the Fc region while increasing the stability of the fused therapeutic enzyme by including a hinge region in its native form or modifying the hinge region.

[0142] In the present invention, the immunoglobulin Fc region may be in the form of a monomer, but the immunoglobulin Fc region is not limited thereto. Specifically, the immunoglobulin Fc region represented by F in the above formula 1 may be in the form of a monomer, and is expressed by fusing the therapeutic enzyme of the monomer with a peptide linker. As the monomer of the immunoglobulin Fc region forms a dimer, the therapeutic enzyme fused to the immunoglobulin Fc region may form a dimer through a non-covalent bond, but is not limited thereto.

[0143] The disadvantage of immunoglobulin Fc regions used as drug carriers is that they can cause unexpected immune responses, such as having effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). These functions occur through the binding of immunoglobulin Fc regions to Fc receptors or complement, or glycosylation of the Fc region. In addition, it is very likely that the instability of Fc itself will occur in vivo.

[0144] In the present invention, the inventors have endeavored to solve the above-mentioned problems by replacing the sequence of the hinge region in the immunoglobulin Fc region. Specifically, the immunoglobulin Fc region of the present invention may be an immunoglobulin Fc region in which an effective glycosylation sequence is replaced for regulation of glycosylation or a sequence involved in chain exchange is replaced, or may correspond to both cases. More specifically, the immunoglobulin Fc region of the enzyme fusion protein of the present invention may be an immunoglobulin Fc region in which chain exchange does not occur.

[0145] Specifically, the immunoglobulin Fc region of the present invention may be an immunoglobulin Fc region in which the second amino acid and / or the 71st amino acid of the immunoglobulin Fc region of SEQ ID NO: 8 are replaced by different amino acids in order to prevent chain exchange and N-glycosylation. More specifically, the immunoglobulin Fc region of the present invention may be 1) an immunoglobulin Fc region in which the second amino acid (i.e., serine) is replaced by proline, 2) an immunoglobulin Fc region in which the 71st amino acid (i.e., asparagine) is replaced by glutamine, or 3) an immunoglobulin Fc region in which the second amino acid is replaced by proline and the 71st amino acid is replaced by glutamine, and specifically an immunoglobulin Fc region represented by the amino acid sequence of SEQ ID NO: 9, but the immunoglobulin Fc region is not limited thereto. In addition to the changes described above, the immunoglobulin Fc region as a drug carrier for increasing the stability of the therapeutic enzyme may include appropriate changes.

[0146] Specifically, the immunoglobulin Fc region may be an immunoglobulin Fc region in which the hinge region of immunoglobulin IgG4 Fc is replaced with an IgG1 hinge region, but the immunoglobulin Fc region is not limited thereto.

[0147] In an embodiment of the present invention, the 2nd amino acid of immunoglobulin Fc is substituted with proline, and the 71st amino acid of immunoglobulin Fc is substituted with glutamine, and thereby chain exchange and N-glycosylation are reduced (Example 1).

[0148] As used herein, the term "chain exchange" refers to a problem in which, when IgG4 Fc is used as a carrier of a protein fusion, IgG4 Fc forms a hybrid with IgG4 existing in the body or exists as a monomer and changes the original structure to a structure with low therapeutic activity, and it has been previously reported that when a protein fusion in which proteins are fused is used for therapeutic purposes, there are significant difficulties (van der Neut Kolfschoten, et al., Science, 317: 1554-1557.2007).

[0149] In addition, in another embodiment, the immunoglobulin Fc region of the present invention includes not only the natural amino acid sequence, but also its sequence analogs. Amino acid analogs refer to changes selected from substitution, addition, deletion, modification and combinations thereof in at least one amino acid residue of the natural amino acid sequence.

[0150] For example, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 in IgG Fc known to be important for ligation can be used as suitable sites for variation.

[0151] In addition, various types of analogs are possible, for example, analogs in which the site capable of forming a disulfide bond is moved; analogs in which several N-terminal amino acids are removed from native Fc; analogs in which a methionine residue is added to the N-terminus of native Fc, etc. In addition, the complement binding site (e.g., C1q binding site) or antibody-dependent cell-mediated cytotoxicity (ADCC) site can be removed to remove the effector function. The technology for preparing sequence analogs of the immunoglobulin Fc region is disclosed in International Publication Nos. WO 97 / 34631, WO 96 / 32478, etc.

[0152] Amino acid substitutions in protein or peptide molecules that do not alter the overall activity of the molecule are well known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common substitutions occur between amino acid residues of 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, and Asp / Gly. In some cases, amino acids may be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0153] Furthermore, the Fc analogs described above may be those that exhibit the same biological activity as the Fc region of the present invention, but have increased structural stability of the Fc region to heat, pH, and the like.

[0154] In addition, such Fc regions can be obtained from natural types isolated from humans or animals (e.g., cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc.), or can be recombinants or their analogs obtained from transformed animal cells or microorganisms. Herein, the Fc region can be obtained from natural Fc by separating complete immunoglobulins from human or animal organisms and treating them with proteases. Papain digests the natural Fc region into Fab and Fc regions, and pepsin treatment results in the production of pF'c and F(ab)2 fragments. These fragments can be subjected to size exclusion chromatography to separate Fc or pF'c. In a more specific embodiment, the Fc region can be a recombinant immunoglobulin Fc region, in which the humanized Fc region is obtained from a microorganism.

[0155] In addition, the immunoglobulin Fc region can be in the form of native polysaccharides, in the form of polysaccharides increased or decreased compared to its native type, or in the form of deglycosylation or non-glycosylation. The increase, reduction or removal of immunoglobulin Fc polysaccharides can be achieved by conventional methods (such as chemical methods, enzymatic methods and genetic engineering methods using microorganisms). In particular, the immunoglobulin Fc region from which polysaccharides are removed from the Fc region shows a significant reduction in binding affinity to complement (C1q), as well as a reduction or removal of antibody-dependent cellular toxicity or complement-dependent cellular toxicity, and therefore it will not induce unnecessary immune responses in vivo. In this regard, the immunoglobulin Fc region in the deglycosylated or non-glycosylated immunoglobulin Fc region can be a more suitable form to meet the original purpose of the present invention as a drug carrier.

[0156] As used herein, the term "deglycosylation" refers to the removal of sugar moieties from an Fc region by an enzyme, and the term "aglycosylated" refers to an unglycosylated Fc region produced in a prokaryote (more specifically, E. coli).

[0157] Meanwhile, the immunoglobulin Fc region may be derived from humans or animals (eg, cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc.), and in a more specific embodiment, it may be derived from humans.

[0158] In addition, the immunoglobulin Fc region can be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof. In a more specific embodiment, it can be derived from IgG or IgM, which are the most abundant proteins in human blood, and in an even more specific embodiment, it can be derived from IgG, which is known to enhance the half-life of ligand-binding proteins. In a more specific embodiment, the immunoglobulin Fc region can be an IgG4 Fc region, in an even more specific embodiment, it can be a non-glycosylated Fc region derived from human IgG4, and in the most specific embodiment, the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO: 9, and the polynucleotide sequence encoding the amino acid sequence can be SEQ ID NO: 7, but the immunoglobulin Fc region is not limited thereto.

[0159] As used herein, the term "combination" means that a polypeptide encoding a single-chain immunoglobulin Fc region of the same source is connected to a single-chain polypeptide of a different source to form a dimer or multimer. That is, the dimer or multimer can be prepared by two or more fragments of the Fc fragment selected from IgG Fc, IgAFc, IgM Fc, IgD Fc, and IgE Fc.

[0160] In addition, the therapeutic enzyme or enzyme fusion protein of the present invention may be those in which the N-terminus and / or C-terminus of the protein are not modified, but in order to protect the therapeutic enzyme from the influence of proteases and to improve the stability of the therapeutic enzyme in vivo, the N-terminus and / or C-terminus of the therapeutic enzyme are chemically modified or protected by organic groups, or the amino terminus of the therapeutic enzyme is modified by the addition of amino acids, etc. Those therapeutic enzymes or enzyme fusion proteins are also included in the scope of the protein according to the present invention. When the C-terminus of the therapeutic enzyme is not modified, the end of the therapeutic enzyme or enzyme fusion protein according to the present invention may have a carboxyl terminus, but the therapeutic enzyme or enzyme fusion protein of the present invention is not particularly limited thereto.

[0161] In particular, since the N-terminus and C-terminus of the chemically synthesized protein have charges, the N-terminus may be acetylated and / or the C-terminus may be amidated to remove these charges, but the method is not particularly limited thereto.

[0162] Unless otherwise specified in this specification, the technology of " enzyme " or " fusion protein " described in the specific description of the present invention or claim according to the present invention will not only be applied to the relevant enzyme or fusion protein, and be applied to all salts (for example, the pharmaceutically acceptable salt of fusion protein) including the relevant enzyme or fusion protein, or the scope of its solvate. Therefore, although simply described as " enzyme " or " fusion protein " in the specification, the relevant description will be applied to the specific solvate of specific salt, specific solvate and specific salt equally. Such salt form can be the form of, for example, using any pharmaceutically acceptable salt, but the kind of salt is not particularly limited. Those salt forms can be, for example, safe and effective salt forms for mammals, but salt forms are not particularly limited thereto.

[0163] As used herein, the term "pharmaceutically acceptable" refers to substances that are effective for their intended use without causing excessive toxicity, irritation, or allergic response, etc., within the scope of pharmaceutical discretion.

[0164] As used herein, the term "pharmaceutically acceptable salt" refers to a salt derived from a pharmaceutically acceptable inorganic salt, organic salt, or base. Examples of suitable acids may 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, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Examples of salts derived from suitable bases may include alkali metals such as sodium, potassium, and the like; alkaline earth metals such as magnesium, and the like; ammonium, and the like.

[0165] Furthermore, as used herein, the term "solvate" refers to a complex formed between a solvent molecule and the enzyme, fusion protein, or a salt thereof according to the present invention.

[0166] The enzyme fusion protein of the present invention can be prepared by methods known in the art.

[0167] In an embodiment of the present invention, a recombinant vector in which α-galactosidase (ie, therapeutic enzyme) can be expressed in the form of fusion to a peptide linker-immunoglobulin Fc was prepared, and the therapeutic enzyme was prepared by expressing it in a CHO cell line (Examples 1 and 2).

[0168] However, the enzyme fusion protein of the present invention may be prepared by methods other than those described in the above embodiments.The enzyme fusion protein of the present invention may include the amino acid sequence of SEQ ID NO: 13, but the amino acid sequence is not limited thereto.

[0169] The enzyme fusion protein according to the present invention can increase the half-life of the therapeutic enzyme that exhibits a therapeutic effect on LSD by improving the in vivo stability of the therapeutic enzyme while maintaining the activity of the therapeutic enzyme through fusion of the therapeutic enzyme with the immunoglobulin Fc region. In particular, the therapeutic enzyme fused to the modified immunoglobulin Fc region has reduced chain exchange and glycosylation, and thus can have a lower binding affinity for lysosomal receptors than the Fc-unfused therapeutic enzyme, and thus can have a high duration, confirming that such a therapeutic enzyme is effective for the treatment of LSD.

[0170] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating LSD, which contains the enzyme fusion protein as an active ingredient.

[0171] The composition according to the invention is characterized by increased in vivo duration and stability of the therapeutic enzyme.

[0172] Specifically, the enzyme fusion protein of the pharmaceutical composition of the present invention may be an enzyme fusion protein in which α-galactosidase A and an immunoglobulin Fc region are fused together, but the enzyme fusion protein is not limited thereto.

[0173] In addition, the composition according to the present invention can be a composition in which when the immunoglobulin Fc region forms a dimer, the α-galactosidase A (i.e., the therapeutic enzyme) in the fusion protein can also form a dimer, and more specifically, the dimer of α-galactosidase A is a dimer formed by non-covalent bonds with each other, and in particular, is characterized in that the dimer is formed in an anti-parallel orientation.

[0174] As used herein, the term "lysosome" is one of the organelles present in the cytoplasm, contains many hydrolases, and thus decomposes unwanted substances in the body (such as macromolecules, bacteria, etc.), and helps to utilize the decomposition products in other parts of the cell. The function of the lysosome can be performed by many enzymes. When a specific enzyme loses its function due to changes, deficiencies, etc., it causes the loss of the decomposition function of the lysosome, and causes the accumulation of macromolecules that must be decomposed, etc. in the cell, and induces cell damage, etc., thereby causing disease.

[0175] As used herein, the term "lysosomal storage disease (LSD)" refers to a rare genetic disease caused by loss of lysosomal function as described above, and enzyme replacement therapy using the defective enzyme is necessary. LSD may include mucopolysaccharidosis (MPS), glycogen storage disease, sphingolipid storage disease, Niemann-Pick disease, Fabry disease, Gaucher disease, Hunter syndrome, and Mara-La syndrome, among others.

[0176] As used herein, the term "Fabry disease" is a LSD that is inherited as a recessive form associated with sex chromosomes and is caused by X-inactivation. Fabry disease is an inborn error of glycosphingolipid metabolism that occurs due to a lack or deficiency in the activity of the hydrolase α-galactosidase A present in lysosomes. It is known that a defect in α-galactosidase A can lead to abnormal accumulation of ceramide hexotrioside (Gb3) in the walls of blood vessels and various parts of the body (e.g., skin, kidneys, heart, nervous system, etc.), and thereby affect blood circulation and reduce nutrient supply. Symptoms such as anhidrosis (lack of sweating), paresthesia, severe pain, angina keratomas, corneal opacities, cardiac ischemia, myocardial infarction, renal failure, etc. may manifest, leading to renal failure, and ultimately death.

[0177] As used herein, the term "prevention" refers to all actions that inhibit or delay the occurrence of LSD by administering an enzyme fusion protein or a composition containing the enzyme fusion protein, and the term "treatment" refers to all actions that improve or favorably change the symptoms of LSD by administering an enzyme fusion protein or a composition containing the enzyme fusion protein.

[0178] As used herein, the term "administer" refers to the introduction of a specific substance into a patient by any appropriate method, and the administration route of the composition can be any conventional route capable of delivering the composition to a target in vivo, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. However, since peptides are digested when administered orally, the active ingredient of the composition for oral administration is preferably coated or formulated to prevent degradation in the stomach, and specifically, can be administered in an injectable form. In addition, the pharmaceutical composition can be administered using a device capable of delivering the active ingredient to the target cell.

[0179] The total effective dose of the pharmaceutical composition of the present invention can be given to the patient in a single dose, or can be given in multiple doses (multiple doses) for a long period of time according to a fractionated treatment protocol. In the pharmaceutical composition of the present invention, the content of the active ingredient can vary according to the severity of the disease. Specifically, the total daily dose of the fusion protein of the present invention can be about 0.0001mg to 500mg per 1 kg of body weight of the patient. However, in addition to considering the administration route and treatment frequency of the pharmaceutical composition, it is also considered that the effective dose of the fusion protein is determined by various factors including the patient's age, weight, health status, sex, severity of disease, diet, excretion rate. In this regard, those skilled in the art can easily determine the effective dose suitable for the specific use of the pharmaceutical composition of the present invention. As long as it shows the effect of the present invention, the pharmaceutical composition according to the present invention is not particularly limited to preparations, administration routes and methods.

[0180] In the present invention, the actual dosage of the enzyme fusion protein used as a carrier can be determined based on the type of therapeutic enzyme used as an active ingredient together with various factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, the severity of the disease, etc. Since the enzyme fusion protein of the present invention has a significantly excellent in vivo duration, the dosage, number and frequency of administration of the pharmaceutical preparation containing the enzyme fusion protein of the present invention can be significantly reduced.

[0181] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient or diluent. The pharmaceutically acceptable carrier may be non-naturally occurring.

[0182] As used herein, the term "pharmaceutically acceptable" refers to a property of having a sufficient amount to exhibit a therapeutic effect without causing adverse effects, and can be easily determined by those skilled in the art based on factors well known in the medical field (such as the type of disease, the patient's age, weight, health status, sex, drug sensitivity, administration route, administration method, administration frequency, treatment duration, drug (one or more) to be mixed or administered simultaneously, etc.).

[0183] Pharmaceutically acceptable carriers may include: for oral administration, binders, glidants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc.; for injection, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., which may be used in combination; and for topical administration, bases, excipients, lubricants, preservatives, etc.

[0184] The formulation types of the composition of the present invention can be prepared in various ways by combining with the pharmaceutically acceptable carriers described above. For example, for oral administration, the composition can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc.; for injection, the composition can be formulated into unit dose ampoules or multi-dose containers. In addition, the composition can also be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0185] Meanwhile, examples of suitable carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the composition may further contain fillers, anticoagulants, lubricants, wetting agents, flavoring agents, preservatives, etc.

[0186] In addition, the enzyme fusion protein can be used by mixing with various pharmaceutically acceptable carriers (such as physiological saline or organic solvents) approved as drugs. In order to increase stability or absorbability, carbohydrates (such as glucose, sucrose or dextran), and antioxidants (such as ascorbic acid and glutathione), chelating agents, low molecular weight proteins, or other stabilizers can be used as drugs.

[0187] The pharmaceutical composition may contain the above-mentioned ingredient (active ingredient) in an amount of 0.01% to 99% (w / v), but the amount is not limited thereto.

[0188] Another aspect of the present invention provides a polynucleotide encoding the enzyme fusion protein according to the present invention.

[0189] The polynucleotide encoding the enzyme fusion protein according to the present invention may be a polynucleotide in which a portion encoding a therapeutic enzyme and a portion encoding a peptide linker-immunoglobulin Fc region are connected, and specifically, a polynucleotide encoding a fusion protein in which the N-terminus of the immunoglobulin Fc region is connected to the C-terminus of the therapeutic enzyme via a GGGGS linker, but the polynucleotide is not limited thereto. More specifically, the polynucleotide of the present invention may include the sequence of SEQ ID NO: 12, but as long as the polynucleotide can encode a fusion protein of a therapeutic enzyme and an immunoglobulin Fc region, the sequence is not limited thereto.

[0190] Another aspect of the present invention provides a recombinant expression vector comprising the polynucleotide.

[0191] As used herein, the term "recombinant vector" refers to a DNA construct wherein a target peptide (e.g., an enzyme fusion protein) is operably linked to an appropriate control sequence so that the target peptide (e.g., an enzyme fusion protein) can be expressed in an appropriate host. Recombinant vectors according to the present invention can be constructed as vectors for typical cloning or for expression, and can be constructed to use prokaryotic or eukaryotic cells as host cells.

[0192] Control sequences include promoters capable of initiating transcription, any operator sequences for controlling transcription, sequences encoding appropriate mRNA ribosome binding domains, and sequences controlling transcription and translation termination. After transformation into a suitable host cell, the recombinant vector can be replicated or function independently of the host genome, or can be integrated into the host genome itself.

[0193] The recombinant vector used in the present invention is not particularly limited, as long as the vector can replicate in a host cell, and it can be constructed using any vector known in the art. Examples of vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. The vector that can be used in the present invention is not particularly limited, but any known expression vector can be used.

[0194] Recombinant vectors are used for transformation of host cells to produce enzyme fusion proteins of the present invention. In addition, as a part of the present invention, these transformed cells can be used for the amplification of nucleic acid fragments and vectors, or they can be cultured cells or cell lines used in the recombinant production of enzyme fusion proteins of the present invention.

[0195] As used herein, the term "transformation" refers to a process in which a recombinant vector comprising a polynucleotide encoding a target protein (e.g., a therapeutic enzyme, an enzyme fusion protein) is introduced into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. For the transformed polynucleotide, as long as it can be expressed in the host cell, it is not important whether it is inserted into the chromosome of the host cell and located therein or located outside the chromosome, and both cases are included.

[0196] In addition, polynucleotides include DNA and RNA encoding target proteins. As long as the polynucleotides can be introduced into the host cell and expressed therein, they can be inserted in any form. For example, the polynucleotides can be introduced into the host cell in the form of an expression cassette, which is a gene construct comprising all necessary elements required for self-expression. The expression cassette can routinely include a promoter, a transcription termination signal, a ribosome binding domain, and a translation termination signal that are operably connected to the polynucleotides. The expression cassette can be in the form of an expression vector that can self-replicate. In addition, the polynucleotides can be introduced into the host cell as is and be operably connected to the sequence necessary for its expression in the host cell, but the polynucleotides are not limited thereto.

[0197] In addition, as used herein, the term "operably linked" refers to a functional connection between a promoter sequence (which initiates and mediates the transcription of a polynucleotide encoding a target peptide of the present invention) and the above-mentioned gene sequence.

[0198] As long as it can express the polynucleotide of the present invention, the appropriate host for the present invention will not be specifically limited. Examples of appropriate hosts may include 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; yeast, such as Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces pombe; insect cells, such as Spodoptera frugiperda (Sf9); and animal cells, such as CHO, COS, BSC, etc.

[0199] Still another aspect of the present invention provides a transformant into which the expression vector is introduced.

[0200] For the purpose of the present invention, as long as the transformant is capable of expressing and producing the enzyme fusion protein, the transformant into which the expression vector of the present invention is introduced may not be limited, but the transformant may be a bacterium belonging to the genus Escherichia, such as Escherichia coli; a bacterium belonging to the genus Bacillus, such as Bacillus subtilis; a bacterium belonging to the genus Pseudomonas, such as Pseudomonas putida; yeast, such as Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces pombe; insect cells, such as Spodoptera frugiperda (Sf9); and animal cells, such as CHO, COS, BSC, etc.

[0201] Another aspect of the present invention provides a method for preparing the enzyme fusion protein according to the present invention.

[0202] Specifically, the method may include (a) culturing the transformant to obtain a culture; and

[0203] (b) The enzyme fusion protein is recovered from the culture, but the method is not limited thereto.

[0204] In the present invention, the culture medium for culturing the transformant must meet the requirements of host cell culture in an appropriate manner. For host cell growth, the carbon source that can be included in the culture medium can be appropriately selected according to the type of transformant prepared therefrom by those skilled in the art, and appropriate culture conditions can be selected to control the cycle and amount of cultivation.

[0205] Examples of sugar sources used in the culture medium may 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 glycerol and ethanol); and organic acids (such as acetic acid). These substances may be used alone or in combination.

[0206] Examples of the nitrogen source used may include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used alone or in combination.

[0207] Examples of phosphorus sources used may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts. In addition, the culture medium may contain metal salts necessary for growth (such as magnesium sulfate or iron sulfate).

[0208] Finally, essential growth substances (such as amino acids and vitamins) can be used. In addition, suitable culture medium precursors can also be used. During the cultivation period by batch culture or continuous culture, the above-mentioned source can be appropriately added to the culture. The pH of the culture can be appropriately adjusted using alkaline compounds (such as sodium hydroxide, potassium hydroxide and ammonia), or acidic compounds (such as phosphoric acid or sulfuric acid). In addition, defoamers such as fatty acid polyethylene glycol esters can be added to prevent the generation of foam. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas (for example, air) can be injected into the culture.

[0209] The transformant of the present invention can be cultured at 20° C. to 45° C., and particularly at 25° C. to 40° C. Furthermore, the culture is continued until the maximum production of the desired therapeutic enzyme or enzyme fusion protein is obtained, and in this regard, the culture can generally be continued for 10 to 160 hours.

[0210] As described above, when appropriate culture conditions are provided according to the host cells, the transformant of the present invention can produce a therapeutic enzyme or an enzyme fusion protein, and the therapeutic enzyme or enzyme fusion protein produced according to the vector construction and characteristics of the host cells can be secreted in the cytoplasm of the host cells or secreted into the periplasmic space or outside the cells.

[0211] Proteins expressed in or outside host cells can be purified by conventional methods. Examples of purification methods may include salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (e.g., protein fractionation precipitation using acetone or ethanol, etc.), dialysis, gel filtration, ion exchange or chromatography (e.g., reverse column chromatography, ultrafiltration, etc.), and these methods may be used alone or in combination.

[0212] Still another aspect of the present invention provides a method comprising administering to a subject an enzyme fusion protein or a composition containing the enzyme fusion protein for preventing or treating LSD.

[0213] Since the enzyme fusion protein of the present invention contains a therapeutic enzyme capable of preventing or treating LSD, LSD can be prevented or treated by administering the enzyme fusion protein containing the therapeutic enzyme or a pharmaceutical composition containing the enzyme fusion protein to a subject suspected of having LSD.

[0214] As used herein, the term "subject" refers to a subject suspected of having LSD, and a subject suspected of having LSD refers to a mammal suffering from LSD or at risk of developing LSD, including humans, rats, livestock, etc., but includes without limitation any subject that can be treated with the enzyme fusion protein of the present invention or a composition containing the enzyme fusion protein.

[0215] The method of the present invention may include administering a pharmaceutical composition containing an enzyme fusion protein in a pharmaceutically effective amount. The appropriate daily total dose of the composition may be determined by a physician within the scope of correct medical judgment, and the composition may be administered once a day or in divided doses for multiple administrations. However, for purposes of the present invention, preferably, according to various factors, the specific therapeutically effective dose of the composition for any particular patient is applied differently, the various factors including the type and degree of the response to be achieved, including whether the specific composition occasionally used together with other medicaments, the patient's age, body weight, health status, sex and diet, administration time, administration route, excretion rate of the composition, treatment duration, other drugs used in combination with the specific composition or used simultaneously, and similar factors known in the medical field.

[0216] Meanwhile, the preventive or therapeutic method for LSD may be a combination therapy further comprising administering a compound or substance having a therapeutic effect on at least one LSD, but the method is not limited thereto.

[0217] As used herein, the term "combination" must be understood to mean simultaneous, separate or sequential administration. When the administration is sequential or separate, the interval allowing the administration of the second component must be such that the beneficial effects of the combination should not be lost.

[0218] The enzyme fusion protein having therapeutic activity for LSD may be administered at a dose of about 0.0001 μg to 500 mg per 1 kg of patient body weight, but the dose is not particularly limited.

[0219] Another aspect of the present invention provides use of an enzyme fusion protein, or a composition containing the enzyme fusion protein, in the preparation of a medicament (or a pharmaceutical composition) for preventing or treating LSD.

[0220] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

[0221] Example 1: Preparation of enzyme fusion protein

[0222] In order to produce an enzyme fusion protein comprising a therapeutic enzyme in the form of an antiparallel dimer, the present inventors have fused native α-galactosidase, a linker (SEQ ID NO: 10), and an Fc immunoglobulin region (SEQ ID NO: 7) at the genetic level and have inserted the fusion product into an expression vector.

[0223] In order to remove the chain exchange and N-glycosylation sites in the Fc region of the constructed fusion protein, site-directed mutagenesis PCR technology was used.

[0224] Specifically, the 2nd amino acid (i.e., serine) of the Fc region involved in chain exchange was substituted with proline using primers (SEQ ID NOs: 1 and 2), and the 71st amino acid (i.e., asparagine) of the Fc region involved in N-glycosylation was substituted with glutamine using primers (SEQ ID NOs: 3 and 4) from the Fc region (SEQ ID NO: 8).

[0225] [Table 1] Primers used for mutagenesis

[0226]

[0227] The polynucleotide encoding the synthetic α-galactosidase-Fc fusion protein was inserted into the expression vector (XOGC vector) using restriction endonucleases. Both BamHI and XhoI are restriction endonucleases that do not cut α-galactosidase and Fc immunoglobulin regions. The α-galactosidase-Fc cut with the above restriction endonucleases was inserted into the XOGC vector cut with the same restriction endonucleases, and the vector capable of expressing the α-galactosidase-Fc fusion protein was completed. When the immunoglobulin Fc region forms a dimer, the α-galactase forms an antiparallel dimer.

[0228] The DNA and protein sequences of α-galactosidase-Fc are shown in Table 2 below, wherein the underlined portion represents the signal sequence, the bold portion represents the substituted amino acid, and the italic portion represents the linker.

[0229] [Table 2] DNA sequence and protein sequence of enzyme fusion protein

[0230]

[0231]

[0232]

[0233]

[0234] The enzyme fusion protein expression vector prepared in Example 1 was named α-galactosidase-Fc.

[0235] Example 2: Confirmation of in vitro enzyme activity of enzyme fusion protein

[0236] Example 2-1: Preparation of α-galactosidase-Fc fusion protein

[0237] The α-galactosidase-Fc expression vector (pX0GC-α-galactosidase-Fc) prepared in Example 1 was transformed into a CHO-S cell line, and thereby a cell line capable of mass-producing α-galactosidase-Fc fusion protein was prepared.

[0238] Specifically, serum-free medium (FreeStyle TM CHO expression medium (Expression Medium, Thermo Fisher Scientific, Cat. No. 12651014) was used to proliferate CHO-S cells by suspension culture in a 1L conical flask (Corning Inc., Cat. No. 431147). When the number of cells in the culture container reached 5×10 8 When cells are confluent, use FreeStyle TM MAX (Thermo Fisher Scientific, Cat. No. 16447-100) to transform them. That is, each of the two tubes contained 10 mL of OptiPro TM SFM (Thermo Fisher Scientific, Cat. No. 12309-019), then one tube with 500 μg of DNA and another tube with 500 μL of FreeStyle TM The two solutions in each tube were mixed, left at room temperature for 10 minutes, and added to the tubes pre-washed with fresh FreeStyle TM The cells were cultured in an incubator (37° C., 5% CO 2 ) at 125 rpm for about 96 hours after the culture medium was replaced with CHO expression medium (Thermo Fisher Scientific, Cat. No. 12651014).

[0239] Then, the culture supernatant was recovered by centrifugation on day 4, and the in vitro enzyme activity was measured to examine changes in the enzyme activity according to the production of α-galactosidase-Fc.

[0240] Example 2-2: Confirmation of in vitro enzyme activity of α-galactosidase-Fc

[0241] In order to examine the in vitro enzymatic activity of α-galactosidase-Fc, the generated α-galactosidase-Fc fusion protein was allowed to establish temperature equilibrium with pNP-gal (p-nitrophenyl-α-D-galactopyranoside), a known substrate of the enzyme, at 37° C. for 30 minutes. Then, the substrate solution was reacted with α-galactosidase-Fc at 37° C. for 30 minutes, and the absorbance of the product was measured to measure the in vitro enzymatic activity of α-galactosidase-Fc.

[0242] As a result, from the in vitro enzyme activity measurement, it was confirmed that the expression level of α-galactosidase-Fc was 1,649 ng / mL ( Figure 1 ). These results suggest that the α-galactosidase of the enzyme fusion protein of the present invention forms an antiparallel dimer, and the α-galactosidase of the antiparallel dimer maintains the enzyme activity at a significant level.

[0243] Example 2-3: Comparison of in vitro enzyme activity between α-galactosidase and α-galactosidase-Fc

[0244] Meanwhile, the in vitro enzyme activity of the α-galactosidase-Fc fusion protein prepared in Example 1 was compared with that of agalsidase-β not linked to the Fc region.

[0245] Specifically, α-galactosidase-Fc and agalsidase-β were subjected to temperature equilibrium with the enzyme substrate pNP-gal (p-nitrophenyl-α-D-galactopyranoside) at 37° C. for 30 minutes. Then, the substrate solution was reacted with the α-galactosidase-Fc fusion protein at 37° C. for 20 minutes. Then, the absorbance of the finally produced 4-nitrophenol was measured to compare the enzyme activities of α-galactosidase-Fc and agalsidase-β.

[0246] As a result, it was confirmed that the enzyme activities (specific activities) of α-galactosidase-Fc and agalsidase-β were 70.9 μmol / min / mg and 67.6 μmol / min / mg, respectively. Figure 2 ). From these results, it was confirmed that the α-galactosidase-Fc fusion protein had 95.4% enzyme activity, which was similar to the level of the enzyme not linked to the Fc region. This means that the α-galactosidase-Fc fusion protein of the present invention does not lose its enzyme activity even when the Fc region is linked thereto.

[0247] Example 3: Confirmation of in vitro intracellular uptake activity of α-galactosidase-Fc

[0248] It is known that α-galactosidase acts via the mannose-6-phosphate receptor (M6PR) after being taken up into cells. Therefore, the present inventors have confirmed that the cell uptake activity exhibited by the α-galactosidase-Fc fusion protein is as follows.

[0249] Specifically, CCD986SK cells (human skin fibroblasts) known to express M6PR were treated with agalsidase-β and α-galactosidase-Fc fusion proteins, respectively, and intracellular uptake was induced at 37° C. After 24 hours, the presence of agalsidase-β and α-galactosidase-Fc fusion proteins was confirmed by enzyme activity measurement.

[0250] As a result of analyzing the correlation between the concentration of α-galactosidase-Fc fusion protein and its intracellular absorption, the Michaelis-Menten constant K representing half of the maximum absorption rate was found. m The value was 11.55 nM, thus showing similarity to agalsidase-β (K m =10.92 nM) with similar intracellular uptake activity levels ( Figure 3 ). From these results, it was confirmed that the α-galactosidase-Fc fusion protein of the present invention did not lose its intracellular uptake activity even when the Fc region was linked thereto.

[0251] Example 5: Confirmation of the pharmacodynamic behavior of α-galactosidase-Fc fusion protein in ICR mice

[0252] In ICR mice (3 mice / group), at each time point of blood sample collection, the blood stability and pharmacokinetic coefficients of agalsidase-β (control group) and the α-galactosidase-Fc fusion protein prepared in the example (test group) were compared according to each blood sample collection of each group.

[0253] Specifically, based on agalsidase, agalsidase-β (control group) and α-galactosidase-Fc fusion protein (test group) were administered to ICR mice of the control group and the experimental group at a concentration of 1.0 mg / kg by intravenous and subcutaneous injection, respectively. Blood samples were collected from the group administered by intravenous injection at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, and 4 hours after injection, and from the group administered by subcutaneous injection at 0, 1, 4, 8, 24, 48, 72, 96, 120, 144, 168, 192, and 216 hours after injection. The amount of protein in serum was quantified by measurement of in vitro enzyme activity.

[0254] As a result, the α-galactosidase-Fc fusion protein showed a half-life (T 1 / 2), the maximum drug concentration in blood (C max ) and the area under the curve (AUC). AUC represents the level of exposure to the drug molecule in the body ( Figure 4 , Table 3). Based on these results, it was confirmed that the α-galactosidase-Fc fusion protein of the present invention showed a high blood half-life and a maximum drug concentration in the blood (C max ) and in vivo bioavailability (AUC).

[0255] [Table 3] Pharmacodynamic parameters of agalsidase-β and α-galactosidase-Fc fusion proteins

[0256]

[0257] *NA = Not Applicable

[0258] These results suggest that the novel fusion proteins of the present invention that increase the half-life and in vivo bioavailability of α-galactosidase (an example of a therapeutic enzyme known to have therapeutic effects on LSD) can be used as therapeutic agents for LSD while maintaining enzyme activity.

[0259] According to the above, those skilled in the art to which the present invention belongs will be able to understand that, without modifying the technical concept or essential features of the present invention, the present invention can be embodied in other specific forms. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be interpreted as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments, but also various substitutions, modifications, equivalents and other embodiments that may be included in the spirit and scope of the present invention as defined by the appended claims.

Claims

1. An enzyme fusion protein of the following formula 1; [Formula 1] wherein X and X′ are each independently the same or different types of therapeutic enzymes; L and L' are linkers, each independently being the same or different types of linkers; F is the immunoglobulin Fc region; | is a covalent bond; and : is a covalent bond or a non-covalent bond.

2. The enzyme fusion protein of claim 1, wherein the therapeutic enzyme forms a dimer through a non-covalent bond.

3. The enzyme fusion protein of claim 1, wherein the therapeutic enzyme forms a dimer in an antiparallel configuration to each other.

4. The enzyme fusion protein of claim 1, wherein the enzyme fusion protein has increased stability and decreased binding affinity to lysosomal receptors compared to a therapeutic enzyme to which the immunoglobulin Fc region is not fused.

5. The enzyme fusion protein of claim 1, wherein the enzyme is selected from the group consisting of β-glucosidase, α-galactosidase, β-galactosidase, iduronidase, iduronate-2-sulfatase, galactose-6-sulfatase, acid α-glucosidase, acid ceramidase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, arylsulfatase B, β-hexosaminidase A, β-hexosaminidase B, heparin N-sulfatase, α-D-mannosidase, β-glucuronidase, N-acetylgalactosamine-6-sulfatase, lysosomal acid lipase, α-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, glucosylceramide analog, lipase, urate oxidase, platelet-activating factor acetylhydrolase, neutral endopeptidase, and myeloperoxidase. The enzyme fusion protein of claim 5 , wherein the enzyme is α-galactosidase A or β-galactosidase.

7. The enzyme fusion protein of claim 1, wherein the immunoglobulin Fc region has a change selected from substitution, addition, deletion, modification, and a combination thereof in at least one amino acid of a native immunoglobulin Fc region.

8. The enzyme fusion protein of claim 7, wherein in the immunoglobulin Fc region having the amino acid sequence of SEQ ID NO: 8, the 2nd amino acid is substituted by proline; the 71st amino acid is substituted by glutamine; or the 2nd amino acid is substituted by proline and the 71st amino acid is substituted by glutamine.

9. The enzyme fusion protein of claim 8, wherein chain exchange does not occur in the immunoglobulin Fc region.

10. The enzyme fusion protein of claim 1, wherein the immunoglobulin Fc region is selected from (a) CH1 domain, CH2 domain, CH3 domain and CH4 domain; (b) CH1 domain and CH2 domain; (c) CH1 domain and CH3 domain; (d) a CH2 domain and a CH3 domain; and (e) A combination of one or two or more of the CH1 domain, CH2 domain, CH3 domain and CH4 domain with an immunoglobulin hinge region or a portion of the hinge region. The enzyme fusion protein of claim 1 , wherein the immunoglobulin Fc region is in a monomeric form.

12. The enzyme fusion protein of claim 1, wherein the immunoglobulin Fc region consists of a hinge region, a CH2 domain, and a CH3 domain.

13. The enzyme fusion protein of claim 1, wherein in the immunoglobulin Fc region, (a) removing regions capable of forming disulfide bonds; (b) removing a certain amino acid residue from the N-terminus of native Fc; (c) adding a methionine residue to the N-terminus of the native Fc form; (d) removal of complement binding sites; or (e) Absence of antibody-dependent cell-mediated cytotoxicity (ADCC) site. The enzyme fusion protein of claim 1 , wherein the immunoglobulin Fc region is non-glycosylated. The enzyme fusion protein of claim 1 , wherein the immunoglobulin Fc region is an Fc fragment derived from IgG, IgA, IgD, IgE or IgM. The enzyme fusion protein of claim 1 , wherein the immunoglobulin Fc region is a hybrid having domains derived from different sources of immunoglobulins selected from IgG, IgA, IgD, IgE and IgM. The enzyme fusion protein of claim 16 , wherein the immunoglobulin Fc region is an IgG4 Fc region. The enzyme fusion protein of claim 17 , wherein the hinge region of the IgG4 Fc region is replaced by an IgG1 hinge region. The enzyme fusion protein of claim 1 , wherein the enzyme fusion protein comprises the amino acid sequence of SEQ ID NO:

6.

20. A pharmaceutical composition for preventing or treating lysosomal storage diseases (LSDs), comprising the enzyme fusion protein according to any one of claims 1 to 19.

21. The pharmaceutical composition of claim 20, wherein the lysosomal storage disease (LSD) is selected from mucopolysaccharidosis (MPS), glycogen storage disease, sphingolipid storage disease, Niemann-Pick disease, Fabry disease, Gaucher disease, Hunter syndrome, and Mara-La syndrome.

22. The pharmaceutical composition of claim 19, wherein the composition reduces the binding affinity of an enzyme to a lysosomal receptor.

23. A polynucleotide encoding the enzyme fusion protein of any one of claims 1 to 19.

24. An expression vector comprising the polynucleotide of claim 21.

25. A transformant into which the expression vector according to claim 22 is introduced.

26. A method for preparing an enzyme fusion protein, the method comprising: (a) culturing the transformant according to claim 25 to obtain a culture; as well as (b) recovering the enzyme fusion protein from the culture.

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