Novel therapeutic enzyme fusion protein and its use

Abstract

The present invention relates to a fusion protein of a therapeutic enzyme and an immunoglobulin Fc region, a method for making the fusion protein, and a composition comprising the fusion protein.

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 in order to increase the in vivo half-life of the therapeutic enzyme, a method for preparing the same, and a composition containing the same. [Background Technology]

[0002] Lysosomes are cytoplasmic organelles whose function is to degrade macromolecules such as proteins, polynucleotides, polysaccharides, and lipids. The internal environment of lysosomes is acidic and contains hydrolases that promote the hydrolysis of biomacromolecules. Lysosomes have also been found to play a role in the uptake of molecules through endocytosis.

[0003] Lysosomal storage disease (hereinafter referred to as LSD) is a hereditary metabolic disease characterized by lysosomal dysfunction. LSD is caused by lacking enzymes that degrade materials such as lipids, proteins, polysaccharides, and their incidence rate is usually one in 100,000, and is inherited as an autosomal recessive trait. When specific degradation enzymes are insufficient or lacking, LSD will occur, and when these degradation enzymes are insufficient, the resulting excessive material will accumulate and will not be degraded, which ultimately leads to problems in cell function. Like many other genetic diseases, LSD is inherited from parents. In addition, each of these diseases occurs through any mutation of the gene that participates in the translation of different enzymes respectively. The enzymes that cause these diseases usually have similar biochemical properties, and all LSDs are caused by the abnormal accumulation of materials in lysosomes. At present, about 50 different types of LSDs are known (for example, Niemann-Pick disease, Fabry disease, Gaucher disease, Hunter syndrome, Maro-Lamy syndrome (Maroteaux-Lamysyndrome) etc.). A representative method for treating these LSDs may be enzyme replacement therapy (ERT), and many related studies are currently being conducted (Frances M. Platt et al., J Cell Biol. 2012 Nov 26; 199(5): 723-34).

[0004] Hunter syndrome, a representative of LSD, is a disease caused by the deficiency of iduronate-2-sulfatase (IDS), in which glucosaminoglycans (GAGs) are not degraded and accumulate in lysosomes due to the deficiency of iduronate-2-sulfatase. The symptoms of Hunter syndrome include unique roughness in facial features, large head, abdominal swelling due to hepatomegaly and splenomegaly, etc., and it is also accompanied by hearing loss, valvular heart disease, obstructive airway disease, sleep apnea, etc. Hunter syndrome is known to occur in 1 in 162,000 people and is inherited in an X-linked recessive form associated with the X chromosome. Currently used (recombinant IDS, Shire Pharmaceuticals Group) as enzyme replacement therapy for the treatment of Hunter syndrome.

[0005] Generally, protein such as therapeutic enzyme has low stability, and therefore is easily denatured and decomposed by the protease in the blood. Therefore, in order to maintain the blood concentration and the effectiveness of these proteins, it is necessary to frequently administer to the patient. However, when the protein drug is administered to the patient in the form of injection, the frequent injection of the blood concentration of the active polypeptide can cause significant pain to the patient. In order to address these problems, efforts are made to maintain their blood concentration at a high level to maximize the pharmacological effect by increasing the blood stability and long period of time of the therapeutic enzyme. This therapeutic enzyme long-acting preparation is needed to increase the stability of the therapeutic enzyme, and simultaneously the effectiveness of the drug itself is maintained at a sufficiently high level, and the immune response of the patient will not be caused.

[0006] Specifically, LSD is a fatal disease caused by a genetic defect in a specific enzyme that can lead to death, and replacement therapy is necessary to treat the defective enzyme. Enzyme replacement therapy is a standard therapy in LSD, and the therapy has the effect of alleviating existing symptoms or delaying disease progression by replacing the defective enzyme. However, since continuous intravenous administration lasting 2 to 6 hours once every one to two weeks is required, the daily lives of patients and their family members may be restricted.

[0007] Since the half-life of recombinases used for LSD treatment in humans is very short, ranging from 10 minutes to less than 3 hours, and the recombinase must be administered for the rest of the patient's life, it is inconvenient for the patient. Therefore, there is a high demand for extending the half-life of recombinases.

[0008] In order to stabilize proteins and prevent them from being eliminated in the kidney, fusion proteins using the Fc region of immunoglobulins are currently being actively studied.

[0009] Immunoglobulin is the main component of blood and has five different types (i.e., IgG, IgM, IgA, IgD, and IgE). The most commonly used type for fusion protein research is IgG, and it is divided into four subtypes (IgG1 to IgG4). Fusion proteins prepared using immunoglobulin Fc can increase their size and thereby prevent them from being removed in the kidneys and also bind to FcRn receptors, and thereby enter cells through endocytosis and circulation to have the effect of increasing blood half-life.

[0010] However, the disadvantage of the immunoglobulin Fc region is that it can lead to unexpected immune responses, thereby having effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). These functions occur through the binding of the immunoglobulin Fc region to Fc receptors or complement, or through glycosylation of the Fc region. In addition, it is likely that instability of the Fc itself can occur in vivo.

[0011] Therefore, there is a disadvantage in that the activity of the fusion protein cannot be maintained during the period of time during which the desired fusion protein is stably increased in vivo. [Summary of the invention]

[0012] [Technical issues]

[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, such that the therapeutic enzyme has an increased in vivo duration compared to the therapeutic enzyme not fused to the immunoglobulin Fc region.

[0014] Another object of the present invention is to provide a pharmaceutical composition comprising the therapeutic enzyme fusion protein.

[0015] Still another object of the present invention is to provide a polynucleotide encoding a therapeutic enzyme fusion protein, an expression vector comprising the polynucleotide, and a transformant into which the expression vector is introduced.

[0016] Still another object of the present invention is to provide a method for preparing an enzyme fusion protein, which comprises culturing a transformant.

[0017] [Technical solution]

[0018] Aspects of the invention provide enzyme fusion proteins in which a therapeutic enzyme is fused to an immunoglobulin Fc region.

[0019] In a specific embodiment, the present invention is directed to an enzyme fusion protein in which an immunoglobulin Fc region is fused to a therapeutic enzyme such that the therapeutic enzyme has an increased in vivo duration compared to the therapeutic enzyme not fused to the immunoglobulin Fc region.

[0020] The following corresponds to further embodiments of the present invention.

[0021] Specifically, as an enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the enzyme is selected from β-glucosidase, α-galactosidase, β-galactosidase, iduronidase, iduronate-2-sulfatase, galactose-6-sulfatase, acid α-glucosidase, acid ceramidase, acid sphingomyelinase (acid sphingomyelinsase), 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, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, and myeloperoxidase.

[0022] As the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the therapeutic enzyme and the immunoglobulin Fc region in the enzyme fusion protein are fused via a peptide linker.

[0023] As the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that an immunoglobulin Fc region molecule and a dimeric therapeutic enzyme are fused.

[0024] As the enzyme fusion protein according to any of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the immunoglobulin Fc region has a variation selected from substitution, addition, deletion, modification and a combination thereof in at least one amino acid in the native immunoglobulin Fc region.

[0025] As an enzyme fusion protein according to any of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the immunoglobulin Fc region has the amino sequence of SEQ ID NO: 8, and 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.

[0026] As the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that no chain exchange occurs in the immunoglobulin Fc region.

[0027] As the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that it has increased stability and reduced binding affinity to lysosomal receptors compared to a therapeutic enzyme not fused to an immunoglobulin Fc region, and thus has a high tissue distribution.

[0028] As an enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the immunoglobulin Fc region is 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; (e) a combination of one or two or more of the CH1 domain, the CH2 domain, the CH3 domain and the CH4 domain and an immunoglobulin hinge region or a portion of the hinge region; and (f) a dimer between each domain of the heavy chain constant region and the light chain constant region.

[0029] As an enzyme fusion protein according to any of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the immunoglobulin Fc region has at least one feature selected from the following: (a) removal of the region capable of forming a disulfide bond, (b) removal of certain amino acid residues at the N-terminus of the native Fc, (c) addition of a methionine residue at the N-terminus of the native Fc, (d) removal of the complement binding site or (e) deletion of the antibody-dependent cell-mediated cytotoxicity (ADCC) site.

[0030] As the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the immunoglobulin Fc region is non-glycosylated.

[0031] As the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the immunoglobulin Fc region is an Fc fragment derived from IgG, IgA, IgD, IgE or IgM.

[0032] As the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the immunoglobulin Fc region is a hybrid having domains of different origins, which is derived from an immunoglobulin selected from IgG, IgA, IgD, IgE and IgM.

[0033] As the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the immunoglobulin Fc region is an IgG4 Fc region.

[0034] As the enzyme fusion protein according to any one of the aforementioned specific embodiments, the enzyme fusion protein is characterized in that the hinge region of the IgG4 Fc region is replaced by the IgG1 hinge region.

[0035] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating lysosomal storage diseases (LSDs).

[0036] In a specific embodiment, the present invention relates to a pharmaceutical composition comprising an enzyme fusion protein for preventing or treating LSD.

[0037] As a composition according to any of the aforementioned specific embodiments, the composition is characterized in that the LSD is selected from mucopolysaccharidosis (MPS), glycogen storage disease, sphingolipid metabolism disorder, Niemann-Pick disease, Fabry disease, Gaucher disease, Hunter syndrome, and Mara-La syndrome.

[0038] As a composition according to the aforementioned specific embodiment, the composition is characterized in that the enzyme is iduronate-2-sulfatase (IDS) or aromatic sulfatase B (ARSB).

[0039] As a composition according to any of the preceding embodiments, the composition is characterized in that it reduces the binding affinity of the therapeutic enzyme to the lysosomal receptor.

[0040] Still another aspect of the present invention provides a polynucleotide encoding an enzyme fusion protein.

[0041] Still another aspect of the present invention provides an expression vector comprising the polynucleotide.

[0042] Still another aspect of the present invention provides a transformant into which the expression vector is introduced.

[0043] Still another aspect of the present invention provides a method for preparing an enzyme fusion protein.

[0044] In a specific embodiment, the present invention relates to 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.

[0045] [Advantageous Effects of the Invention]

[0046] The present invention relates to a long-acting therapeutic enzyme fusion protein, and in particular, 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 reduces the mechanism of enzyme removal by the kidney. Due to the increased duration, the enzyme fusion protein of the present invention can be effectively used by patients. [Drawings]

[0047] Figure 1 Shown are graphs demonstrating expression of IDS-Fc fusion protein.

[0048] Figure 2 Shown are graphs demonstrating expression of ARSB-Fc fusion protein.

[0049] Figure 3 A diagram showing the results of experiments demonstrating the pharmacokinetics of the IDS-Fc fusion protein of the present invention.

[0050] Figure 4 A diagram showing the results of experiments demonstrating the pharmacokinetics of the ARSB-Fc fusion protein of the present invention.

[0051] Figure 5 Shown are graphs demonstrating the in vitro enzymatic activity of the IDS-Fc fusion protein of the present invention.

[0052] Figure 6 Shown are graphs demonstrating the in vitro enzymatic activity of the ARSB-Fc fusion protein of the present invention.

[0053] Figure 7Shown are graphs illustrating the results of measuring the level of glycosaminoglycan (GAG) in urine after intravenous or subcutaneous injection of the IDS-Fc fusion protein of the present invention into IDS knockout mice.

[0054] Figure 8 Shown are graphs illustrating the results of measuring glycosaminoglycan (GAG) levels in tissues after intravenous or subcutaneous injection of the IDS-Fc fusion protein of the present invention into IDS knockout mice.

[0055] Fig. 9 Shown are graphs demonstrating the extent of tissue distribution of the ARSB-Fc fusion protein of the present invention. [Specific implementation method]

[0056] Hereinafter, exemplary embodiments of the present invention will be described in detail. Meanwhile, each explanation and exemplary embodiment disclosed herein can be applied to other explanations and exemplary embodiments. That is, all combinations of various elements disclosed herein belong to the scope of the present invention. In addition, the scope of the present invention should not be limited by the specific disclosure provided below.

[0057] In addition, 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.

[0058] 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, e.g., α-aminoisobutyric acid (Aib), Sar (N-methylglycine), α-methyl-glutamic acid, etc. In addition, according to IUPAC-IUB rules, amino acids referred to herein by abbreviations are described as follows:

[0059] Alanine A; Arginine R;

[0060] Asparagine N; Aspartic acid D;

[0061] CysteineC; GlutamateE;

[0062] Glutamine Q; Glycine G;

[0063] Histidine H; Isoleucine I;

[0064] Leucine L; Lysine K;

[0065] Methionine M; Phenylalanine F;

[0066] Proline P; Serine S;

[0067] Threonine T; Tryptophan W;

[0068] Tyrosine, Y; and Valine, V.

[0069] Aspects of the invention provide enzyme fusion proteins in which an immunoglobulin Fc region is fused to a therapeutic enzyme, such that the therapeutic enzyme has an increased duration in vivo compared to the therapeutic enzyme not fused to the immunoglobulin Fc region.

[0070] In the present invention, the enzyme fusion protein can be a protein in which the immunoglobulin Fc region is fused to the therapeutic enzyme, so that compared with the therapeutic enzyme not fused with the immunoglobulin Fc region, the therapeutic enzyme can maintain its activity while reducing its binding affinity to lysosomal receptors, thereby increasing its blood half-life.

[0071] The present inventors have prepared fusion proteins with immunoglobulin Fc regions to increase the blood half-life of therapeutic enzymes. Specifically, as Fc regions, IgG4 Fc analogs were used in which potential glycosylation sequences were substituted to inhibit glycosylation, and in addition, the hinge sequence of IgG4 Fc was substituted to inhibit chain exchange. As a result, the present inventors have confirmed that the blood half-life of the therapeutic enzyme fusion protein fused to the immunoglobulin Fc region has a significantly increased blood half-life and can maintain an activity similar to that of a known enzyme, thereby providing a new form of fusion protein structure in which the therapeutic enzyme and the immunoglobulin Fc region are fused.

[0072] The therapeutic enzyme included in the enzyme fusion protein of the present invention can include any therapeutic enzyme having an advantage of prolonged in vivo duration compared to the therapeutic enzyme type not fused to the immunoglobulin Fc region, but the therapeutic enzyme is not specifically limited thereto. In an exemplary embodiment of the present invention, the enzyme fusion protein is a fusion protein of a therapeutic enzyme.

[0073] In addition, 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 diseases by supplementing defective or insufficient enzymes that cause diseases and restoring the functions of deteriorated enzymes.

[0074] In a specific embodiment, the therapeutic enzyme can be selected from 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-mannosyltransferase, Therapeutic enzymes include glycosidase, β-glucuronidase, N-acetylgalactosamine-6-sulfatase, lysosomal acid lipase, α-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet activating factor acetylhydrolase, neutral endopeptidase and myeloperoxidase, but regardless of its source or type, any therapeutic enzyme that has a therapeutic effect on the disease can be included in the present invention.

[0075] In the present invention, the term "enzyme fusion protein" can be used interchangeably with "long-acting enzyme fusion protein".

[0076] As used herein, the term "therapeutic enzyme" refers to an enzyme used to treat a disease that occurs due to deficiency, lack, ineffectiveness, etc., 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 deficiency, lack, etc. of lysosomal enzymes, but the enzyme is not limited thereto.

[0077] Specifically, the therapeutic enzyme of the present invention may be aromatic sulfatase B (ARSB) or iduronate-2-sulfatase, but the therapeutic enzyme is not limited thereto as long as it is an enzyme that exhibits a therapeutic effect on a target disease.

[0078] As used herein, the term "arylsulfatase B (ARSB)" refers to an arylsulfatase present in the lysosomes of the liver, pancreas and kidney, and the enzyme has the function of hydrolyzing sulfate by decomposing glucosaminoglycans. It is known that arylsulfatase B is associated with mucopolysaccharidosis type VI (Mar-La syndrome). In the present invention, the term arylsulfatase B can be used interchangeably with galsulfase. Specifically, arylsulfatase B may include the amino acid sequence of SEQ ID NO: 4, which may be encoded by the polynucleotide sequence of SEQ ID NO: 3, but arylsulfatase B is not limited thereto.

[0079] As used herein, the term "iduronic acid-2-sulfatase" is a sulfatase associated with Hunter syndrome (MPS-II), and it is an enzyme essential for lysosomal degradation of heparan sulfate and dermatan sulfate. In the present invention, the term iduronic acid-2-sulfatase can be used interchangeably with idursulfase. Idursulfatase can be idursulfatase α or idursulfatase β, but idursulfatase is not limited thereto. Specifically, iduronic acid-2-sulfatase can include the amino acid sequence of SEQ ID NO: 2, which can be encoded by the polynucleotide sequence of SEQ ID NO: 1, but iduronic acid-2-sulfatase is not limited thereto.

[0080] 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 inserted with an animal expression vector, or can be used after purchasing a commercially available enzyme, but the enzyme is not limited thereto.

[0081] Enzyme fusion protein of the present invention can be the form of wherein one or two Fc districts in conjunction with one molecule, and this Fc district has two immunoglobulin chains of dimerization form, but enzyme fusion protein is not limited to this.Particularly, enzyme fusion protein of the present invention can be such form, wherein monomer Fc district is fused with enzyme and expressed, and then two monomer Fc districts form the dimerization Fc district of one molecule by disulfide bond, and each of two enzymes is connected to each of two Fc districts, but enzyme fusion protein is not limited to this.Enzyme can be connected to each other by covalent bond or non-covalent bond, or can be independent of each other, but enzyme fusion protein is not limited to this.

[0082] Specifically, in an embodiment of the present invention, it was confirmed that a long-acting enzyme fusion protein in which a dimer of iduronate-2-sulfatase or aromatic sulfatase B is fused to a molecule of Fc region showed higher in vitro enzyme activity compared to an enzyme not fused to an Fc region, and it was confirmed that this was due to the structural properties of the enzyme fusion protein comprising a dimer of a therapeutic enzyme (Example 5).

[0083] Furthermore, in another aspect, the enzyme fusion protein of the present invention may be an enzyme fusion protein in which an immunoglobulin Fc region is fused to a therapeutic enzyme via a peptide linker.

[0084] The peptide linker may include one or more amino acids, such as 1 to 1,000 amino acids, but the peptide linker is not specifically limited thereto. In the present invention, any known peptide linker (e.g., including [GS] x Connector, [GGGS] x Connector and [GGGGS] xlinker, etc., wherein x is a natural number of 1 or more (eg, 1, 2, 3, 4, 5 or more), and more specifically, the amino acid sequence of SEQ ID NO: 6, but the peptide linker is not limited thereto.

[0085] For the purpose of the present invention, there is no limitation on the position where the peptide linker is fused to the therapeutic enzyme and the immunoglobulin Fc, as long as the peptide linker can connect the therapeutic enzyme and the immunoglobulin Fc while maintaining the activity of the therapeutic enzyme, specifically, connecting the therapeutic enzyme and the two ends of the immunoglobulin Fc region, and more specifically, connecting the C-terminus of the therapeutic enzyme and the N-terminus of the immunoglobulin Fc region, but the position is not limited thereto.

[0086] 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, from the 1st amino acid residue to the 20th amino acid residue of the terminal itself, but the N-terminus or C-terminus is not specifically limited thereto.

[0087] In an embodiment of the present invention, a fusion protein (SEQ ID NO: 23 or 25) was prepared by overlapping PCR using a therapeutic enzyme and a linker (SEQ ID NO: 6)-IgG4, wherein the N-terminus of IgG4 was fused to the C-terminus of the therapeutic enzyme, and the expression of the fusion protein was confirmed (Examples 1 to 3).

[0088] The therapeutic enzyme contained in the enzyme fusion protein of the present invention can be a naturally occurring type and a fragment consisting of a portion of a therapeutic enzyme or a therapeutic enzyme analog, in which variations selected from substitutions, additions, deletions and modifications of some amino acids and combinations thereof occur, and as long as its activity is equivalent to that of the naturally occurring type of therapeutic enzyme, it can be included in the present invention without limitation.

[0089] Furthermore, analogs of therapeutic enzymes include all therapeutic enzyme analogs having one or more amino acids added to the amino and / or carboxyl termini of the naturally occurring type of the therapeutic enzyme.

[0090] For the substitution or addition of amino acids, not only the 20 amino acids commonly found in human proteins can be used, but also atypical or non-naturally occurring amino acids can be used. Commercial sources of atypical amino acids may 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, for example, American Peptide Company, Bachem (USA) or Anygen (Korea), but commercial sources are not limited thereto.

[0091] 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. Natural therapeutic enzymes or their analogs, regardless of the size of the fragment or the type of amino acids, fall within the scope of the present invention as long as they have the activity of a therapeutic enzyme.

[0092] Therapeutic enzyme analogs may include biosimilars and biobetters of the corresponding therapeutic enzymes. For example, for biosimilars, differences in expression of the host compared to known therapeutic enzymes, differences in glycosylation characteristics and their extent, and differences in the degree of substitution in the specific amino acid residues of the corresponding enzymes according to the standard sequence of non-100% substitution degree, belong to the biosimilar 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, 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.

[0093] In addition, the therapeutic enzyme may include an amino acid sequence that has at least 80%, more specifically 90%, even more specifically 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more homology to the above-mentioned enzymes or their analogs, and the therapeutic enzyme can be obtained from a microorganism by recombinant technology or is commercially available, but the therapeutic enzyme is not limited to this.

[0094] As used herein, the term "homology" means the 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. Homology between two or more sequences can be calculated as a percentage (%) using commercial computer programs. Homology (%) can be calculated for adjacent sequences.

[0095] 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.).

[0096] As used herein, the term "immunoglobulin Fc region" refers to a region of an immunoglobulin molecule that includes the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3), excluding the variable regions of the heavy and light chains. For the purposes of the present invention, such immunoglobulin Fc regions may include a modified hinge region in the heavy chain constant region, but are not limited thereto.

[0097] Such immunoglobulin Fc regions may include a hinge region in the heavy chain constant region, but are not limited thereto. In addition, the immunoglobulin Fc region of the present invention may be an extended Fc region, which includes part or all of the heavy chain constant region 1 (CH1) and / or the light constant region 1 (CL1), excluding the variable regions of the heavy and light chains of the immunoglobulin, as long as the immunoglobulin Fc region has the same or equivalent function as its natural type. In addition, the immunoglobulin Fc region of the present invention may be such a region, in which a portion of a significantly long amino acid sequence corresponding to CH2 and / or CH3 is removed.

[0098] On the other hand, the present invention provides an immunoglobulin Fc region, which can be selected from: 1) 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 one or two or more of the CH1 domain, the CH2 domain, the CH3 domain and the CH4 domain and an immunoglobulin hinge region (or part of the hinge region); 6) a dimer between each domain of the heavy chain constant region and the light chain constant region, but the immunoglobulin Fc region is not limited thereto.

[0099] As used herein, the term "chain exchange" refers to the following problem, in which when IgG4 Fc is used as a carrier of a fusion protein, IgG4 Fc forms a hybrid with IgG4 existing in the body or exists as a monomer and changes the original structure to have a structure with low therapeutic activity, and it has been previously reported that there are significant difficulties when the fusion protein of the fusion protein is used for therapeutic purposes (van der Neut Kolfschoten et al., Science, 317: 1554-1557.2007).

[0100] In the present invention, the inventors have made efforts to solve the above 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 a region in which an effective glycosylation sequence is replaced for regulating glycosylation, or a sequence involved in chain exchange is replaced, or may correspond to both cases.

[0101] In a specific embodiment, the immunoglobulin Fc region of the present invention may be a region in which the second amino acid and / or the 71st amino acid of the immunoglobulin Fc region of SEQ ID NO: 8 are substituted with different amino acids to prevent chain exchange and N-glycosylation. More specifically, the immunoglobulin Fc region of the present invention may be 1) a region in which the second amino acid (i.e., serine) is substituted with proline, 2) a region in which the 71st amino acid (i.e., asparagine) is substituted with glutamine, but the immunoglobulin Fc region is not limited thereto. In addition to the above changes, the immunoglobulin Fc region may include appropriate variations as a drug carrier that increases the stability of the therapeutic enzyme.

[0102] Specifically, the immunoglobulin Fc region may be a 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.

[0103] In an embodiment of the present invention, the second amino acid in the immunoglobulin Fc region of SEQ ID NO: 8 is substituted with proline, and the 71st amino acid in the immunoglobulin Fc region of SEQ ID NO: 8 is substituted with glutamine, thereby reducing chain exchange and N-glycosylation. The sequence of the prepared immunoglobulin Fc has the amino acid sequence of SEQ ID NO: 9 (Example 1).

[0104] In an embodiment, the hinge region may be a region in which a portion of a hinge sequence having the following amino acid sequence is deleted or modified.

[0105] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 26)

[0106] Specifically, the hinge region may be a region having a variation in which a portion of the hinge region is deleted so that only one cysteine ​​(Cys) residue is included; or the region may be a region in which a serine (Ser) residue participating in chain exchange is substituted by a proline (Pro) residue, and more specifically, a region in which the second serine of the hinge sequence is substituted by a proline residue, but the hinge region is not limited thereto.

[0107] In the present invention, the immunoglobulin Fc region can increase the stability of the fused therapeutic enzyme by including its native form of hinge region or a modified hinge region while preventing chain exchange and monomer formation in the Fc region.

[0108] 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 a variation selected from substitution, addition, deletion, modification and a combination thereof in at least one amino acid residue of the natural amino acid sequence.

[0109] 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 attachment can be used as sites suitable for variation. In addition, various types of analogs are possible, for example, analogs in which sites capable of forming disulfide bonds are removed, analogs in which several N-terminal amino acids in native Fc are removed, analogs in which methionine residues are added to the N-terminus of native Fc, etc. In addition, complement binding sites (e.g., C1q binding sites) or antibody-dependent cell-mediated cytotoxicity (ADCC) sites can be removed to remove effector functions. Techniques for preparing sequence analogs of immunoglobulin Fc regions are disclosed in International Publication Nos. WO97 / 34631, WO 96 / 32478, etc.

[0110] Amino acid substitutions in proteins or peptide molecules that do not change 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 can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0111] Furthermore, the above-mentioned Fc analogs may be analogs that exhibit the same biological activity as the Fc region of the present invention and have increased structural stability of the Fc region against heat, pH, etc.

[0112] In addition, this type of Fc region can be obtained by separating from the natural type of humans or animals such as cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or can be its recombinant or the like obtained from the transformed animal cells or microorganisms. In this article, they can be obtained from natural Fc by separating complete immunoglobulin from humans or animal organisms and treating it with protease. Papain digests the natural Fc region into Fab and Fc region, and pepsin treatment causes the generation 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 the recombinant immunoglobulin Fc region obtained from a microorganism, which is a human Fc region.

[0113] In addition, the immunoglobulin Fc region can be in the form of native polysaccharides, polysaccharides increased or decreased compared to its natural type, or deglycosylated forms. 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. Specifically, 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 does 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.

[0114] 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 a non-glycosylated Fc region produced in a prokaryote, more specifically, E. coli.

[0115] Meanwhile, the immunoglobulin Fc region may be derived from humans or animals including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, and more specifically, it may be derived from humans.

[0116] In addition, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination or a mixture thereof. In a more specific embodiment, it may be derived from IgG or IgM, which are one of the most abundant proteins in human blood, and in an even more specific embodiment, it may be derived from IgG, which is known to increase the half-life of ligand-binding proteins. In a more specific embodiment, the immunoglobulin Fc region may be an IgG4 Fc region, in an even more specific embodiment, it may be a non-glycosylated Fc region derived from human IgG4, and in the most specific embodiment, the immunoglobulin Fc region may be an IgG4 Fc region, which includes a variation in which the second amino acid of the amino acid sequence of SEQ ID NO: 8 of the immunoglobulin Fc region is substituted with proline and / or 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 encoding the amino acid sequence is SEQ ID NO: 7, but the immunoglobulin Fc region is not limited thereto.

[0117] As used herein, the term "combination" refers to a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin and a single-chain polypeptide of a different origin connected to form a dimer or multimer. That is, a dimer or multimer can be prepared from two or more fragments of the Fc fragment selected from IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc.

[0118] In addition, the protein of the present invention may be a protein in which the N-terminus and / or C-terminus of the protein are not modified, but in order to protect the therapeutic enzyme from destruction by proteolytic enzymes in the body and to increase the stability of the therapeutic enzyme, the protein according to the present invention also includes within its scope proteins in which the N-terminus and / or C-terminus of the therapeutic enzyme are chemically modified or protected by an organic group, or the amino terminus of the therapeutic enzyme is modified by adding amino acids, etc. When the C-terminus of the therapeutic enzyme is not modified, the terminal of the protein according to the present invention may have a carboxyl terminus, but the protein of the present invention is not specifically limited thereto.

[0119] Specifically, 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 in order to remove these charges, but the method is not particularly limited thereto.

[0120] Unless otherwise specified in this specification, according to the present invention described in the specific embodiments of the present invention or the claims, the technology of "enzyme" or "fusion protein" will not only be applied to the target enzyme or fusion protein, but also to the scope of all salts (e.g., pharmaceutically acceptable salts of fusion protein) or solvates thereof including the target enzyme or fusion protein. Therefore, although it is simply described as "enzyme" or "fusion protein" in the specification, this subject description will also be applied to specific salts, specific solvates, and specific solvates of specific salts. For example, such salt forms can be forms using any pharmaceutically acceptable salt, but the type of salt is not specifically limited. For example, those salt forms can be salt forms that are safe and effective for mammals, but the salt forms are not specifically limited thereto.

[0121] As used herein, the term "pharmaceutically acceptable" refers to materials that are effective for their intended use within the scope of pharmaceutical discretion without causing undue toxicity, irritation, or allergic response, and the like.

[0122] 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 salts 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; ammonium, and the like.

[0123] Furthermore, as used herein, the term "solvate" refers to a complex formed between an enzyme, a fusion protein according to the present invention, or a salt thereof and solvent molecules.

[0124] The enzyme fusion protein of the present invention can be prepared by methods known in the art.

[0125] In an embodiment of the present invention, a recombinant vector was prepared in which each of iduronate-2-sulfatase (IDS) and arylsulfatase B (ARSB) (i.e., therapeutic enzymes) can be expressed in the form of fusion to a peptide linker-immunoglobulin Fc, and these therapeutic enzymes were prepared by expressing them in a CHO cell line (Examples 1 to 3).

[0126] 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 may include the amino acid sequence of SEQ ID NO: 23 or 25, but the amino acid sequence is not limited thereto.

[0127] By fusing the therapeutic enzyme to the immunoglobulin Fc region, the enzyme fusion protein according to the present invention can increase the half-life of the therapeutic enzyme showing a therapeutic effect on LSD while maintaining the activity of the therapeutic enzyme. Specifically, the therapeutic enzyme fused to the modified immunoglobulin Fc region reduces chain exchange and glycosylation, and thus can have a lower binding affinity to lysosomal receptors than the therapeutic enzyme not fused to Fc, and can thus have a high duration, which confirms that the therapeutic enzyme is effective in the treatment of LSD.

[0128] In an embodiment of the present invention, it was confirmed that the enzyme fusion protein according to the present invention can maintain in vitro enzyme activity (Example 5) and has a significantly superior half-life (T 1 / 2 ), the maximum drug concentration in blood (C max ) and in vivo availability (AUC) (Example 4), and from these results it was confirmed that the drug can be used even at a low dose compared with conventional drugs (Example 6).

[0129] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating lysosomal storage diseases (LSDs), the pharmaceutical composition comprising an enzyme fusion protein prepared according to the method for preparing an enzyme fusion protein or the enzyme fusion protein.

[0130] The composition according to the invention is characterized by increased in vivo duration and stability of the therapeutic enzyme.

[0131] In a specific embodiment, the enzyme fusion protein of the pharmaceutical composition of the present invention can be an enzyme fusion protein in which iduronate-2-sulfatase (IDS) or aromatic sulfatase B (ARSB) is fused to the Fc region of an immunoglobulin, but the enzyme fusion protein is not limited thereto.

[0132] As used herein, the term "lysosome" is one of the organelles present in the cytoplasm, which contains many hydrolases, and thus decomposes unwanted substances in the body, such as macromolecules, bacteria, etc., and helps the decomposed products to be used in other parts of the cell. Many enzymes can perform the functions of lysosomes. When a specific enzyme loses its function due to mutation, deficiency, etc., it will cause the decomposition function of the lysosome to be lost, and cause the macromolecules that must be decomposed, etc. to accumulate in the cell and induce cell damage, etc., thereby causing disease.

[0133] As used herein, the term "lysosomal storage disease (LSD)" refers to a rare genetic disease caused by the loss of lysosomal function as described above, and enzyme replacement therapy using the defective enzyme is necessary. Depending on the enzyme that is deficient, LSD may include mucopolysaccharidosis (MPS), glycogen storage disease, sphingolipid metabolism disorder, Niemann-Pick disease, Fabry disease, Gaucher disease, Hunter syndrome, Mara-La syndrome, etc.

[0134] Hereinafter, LSD will be described in detail according to its classification.

[0135] As used herein, the term "Maroteaux-Lamy syndrome" pertaining to the type VI mucopolysaccharidosis (MPS) disease is an autosomal recessive genetic disease that occurs due to a deficiency in the enzyme arylsulfatase B (N-acetylgalactosamine-4-sulfatase) necessary for the breakdown of glucosaminoglycans. Maroteaux-Lamy syndrome is a disease that occurs due to the deposition of dermatan sulfate—dermatan sulfate is not broken down due to a deficiency in the enzyme in bones, heart valves, spleen, liver, cornea, etc.

[0136] As used herein, the term "arylsulfatase B (ARSB)" refers to an arylsulfatase present in the lysosomes of the liver, pancreas and kidneys, and the enzyme has the function of hydrolyzing sulfate by decomposing glucosaminoglycans. It is known that arylsulfatase B is associated with mucopolysaccharidosis type VI (Mar-La syndrome). In the present invention, the term arylsulfatase B can be used interchangeably with galsulfase.

[0137] As used herein, the term "Hunter syndrome (Hunter disease)" is an X-linked recessive genetic disease that occurs due to a deficiency of iduronate-2-sulfatase (IDS), and is known to cause accumulation of heparan sulfate and dermatan sulfate due to the deficiency of this enzyme. The symptoms of Hunter syndrome include functional degeneration, progressive hearing loss, retinitis pigmentosa, papilledema, hydrocephalus, etc. In the present invention, the term "mucopolysaccharidosis type II" can be used interchangeably with "Hunter syndrome".

[0138] As used herein, the term "iduronic acid-2-sulfatase" is an enzyme necessary for the lysosomal degradation of heparan sulfate and dermatan sulfate, which is a sulfatase associated with Hunter syndrome (MPS-II). In the present invention, the term "iduronic acid-2-sulfatase" can be used interchangeably with "iduronic acid sulfatase". For example, iduronic acid sulfatase can be iduronic acid sulfatase α or iduronic acid sulfatase β, but iduronic acid sulfatase is not limited thereto.

[0139] 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 inserted with an animal expression vector, or can be used after purchasing a commercially available enzyme, but the enzyme is not limited thereto.

[0140] By fusing the therapeutic enzyme to the immunoglobulin Fc region, the enzyme fusion protein contained in the composition of the present invention can increase the half-life of the therapeutic enzyme, which exhibits a therapeutic effect on LSD while maintaining the activity of the therapeutic enzyme. Specifically, the therapeutic enzyme fused to the modified immunoglobulin Fc region reduces chain exchange and glycosylation, and thus can have a lower binding affinity to lysosomal receptors than the therapeutic enzyme not fused to Fc, and thus can have a high duration, which confirms that this therapeutic enzyme is effective in the treatment of LSD.

[0141] In an embodiment of the present invention, it has been confirmed that the enzyme fusion protein of the present invention reduces the value of glycosaminoglycan (GAG) in IDS knockout mice even at a lower administration frequency compared to the enzyme not fused with the Fc region (Example 6).

[0142] Furthermore, in another embodiment of the present invention, it has been confirmed that the enzyme fusion protein of the present invention shows a high distribution not only in the bone marrow and spleen, but also in the lung, kidney, heart, etc., compared with the native enzyme not fused with the Fc region, although the distribution of the native enzyme was not confirmed in the subject's tissues (Example 7).

[0143] These results indicate that the enzyme fusion protein of the present invention can not only increase patient convenience by reducing the frequency of administration when administered based on high stability, but also allow subcutaneous administration due to its high distribution in tissues.

[0144] As used herein, the term "prevent" refers to all activities that inhibit or delay the occurrence of LSD by administering an enzyme fusion protein or a composition comprising the enzyme fusion protein, and the term "treat" refers to all activities that improve or favorably alter the symptoms of LSD by administering an enzyme fusion protein or a composition comprising the enzyme fusion protein.

[0145] As used herein, the term "administer" refers to the introduction of a specific substance into a patient by any appropriate method, and the route of administration 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, intrarectal administration, etc. However, since the peptide is 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 the form of an injection. In addition, a certain device capable of transporting the active ingredient into the target cell can be used to administer the pharmaceutical composition.

[0146] The total effective dose of the composition of the present invention can be applied to the patient in a single dose according to the fractionated treatment scheme or can be applied in multiple doses for a long period of time. 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 1kg body weight of the patient. However, in addition to the route of administration and the frequency of treatment of the pharmaceutical composition, various factors are also considered, including the patient's age, body weight, health status, sex, severity of disease, diet, excretion rate, etc., to determine the effective dose of the fusion protein. 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. The pharmaceutical composition according to the present invention is not specifically limited to preparation, route of administration and method, as long as it shows the effect of the present invention.

[0147] In the present invention, the actual dosage of the enzyme fusion protein can be determined based on the type of therapeutic enzyme used as the active ingredient and 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 significantly excellent in vivo duration and activity, the dosage, quantity and frequency of administration of the pharmaceutical preparation containing the enzyme fusion protein of the present invention can be significantly reduced.

[0148] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. The pharmaceutically acceptable carrier may be non-naturally occurring.

[0149] As used herein, the term "pharmaceutically acceptable" refers to a property of having a sufficient amount to exhibit a therapeutic effect and not causing adverse effects, and it 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, duration of treatment, mixed or simultaneously administered drug (one or more), etc.

[0150] Pharmaceutically acceptable carriers may include: for oral administration, binders, glidants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc.; for injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. that may be used in combination; and for topical administration, bases, excipients, lubricants, preservatives, etc., but pharmaceutically acceptable carriers are not limited thereto.

[0151] The formulation types of the composition of the present invention can be prepared differently by combining with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the composition can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. For injections, 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.

[0152] Meanwhile, examples of suitable carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the composition may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, preservatives, etc.

[0153] In addition, the enzyme fusion protein can be used by mixing it with a variety of pharmaceutically acceptable carriers approved as drugs, such as physiological saline or organic solvents. In order to increase stability or absorbability, carbohydrates such as glucose, sucrose or dextran, and antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as drugs.

[0154] 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.

[0155] Still another aspect of the present invention provides a polynucleotide encoding the enzyme fusion protein according to the present invention.

[0156] The polynucleotide encoding the enzyme fusion protein according to the present invention may be a polynucleotide in the form of a region encoding a therapeutic enzyme and a region encoding a peptide linker-immunoglobulin Fc region, 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: 1 or 3, but the sequence is not limited thereto, as long as the polynucleotide can encode a fusion protein including a therapeutic enzyme and an immunoglobulin Fc region.

[0157] Still another aspect of the present invention provides a recombinant expression vector comprising the polynucleotide.

[0158] As used herein, the term "recombinant vector" refers to a DNA construct in which a target peptide (e.g., an enzyme fusion protein) is operably linked to a suitable control sequence so that the target peptide (e.g., an enzyme fusion protein) is expressed in a suitable host. Recombinant vectors according to the present invention can be constructed as vectors for typical cloning or for expression, and prokaryotic or eukaryotic cells can be used as host cell constructions.

[0159] Control sequences include promoters that can initiate transcription, any operator sequences that are used to control transcription, sequences encoding suitable mRNA ribosome binding domains, and sequences that control transcription and translation termination. After being transformed into suitable host cells, recombinant vectors can replicate or function independently of the host genome, or can be integrated into the host genome itself.

[0160] The recombinant vector used in the present invention may not be specifically limited, as long as the vector can be replicated in a host cell, and any vector known in the art may be used to construct the vector. Examples of vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. The vectors available in the present invention are not specifically limited, but any known expression vector may be used.

[0161] Recombinant vectors are used for transformation of host cells to produce enzyme fusion proteins of the present invention. In addition, as 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.

[0162] As used herein, the term "transformation" refers to a method of introducing a recombinant vector comprising a polynucleotide encoding a target protein 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 does not matter whether it is inserted into the chromosome of the host cell and located in the chromosome or located outside the chromosome, and both cases are included.

[0163] In addition, polynucleotides include DNA and RNA encoding target proteins. Polynucleotides can be inserted in any form, as long as they can be introduced into host cells and expressed therein. For example, polynucleotides can be introduced into host cells in the form of expression cassettes, which are gene constructs comprising all necessary elements required for self-expression. Expression cassettes can routinely include promoters, transcription termination signals, ribosome binding domains, and translation termination signals operably connected to polynucleotides. Expression cassettes can be in the form of self-replicating expression vectors. In addition, polynucleotides can be introduced into host cells as they are and operably connected to sequences necessary for their expression in host cells, but polynucleotides are not limited thereto.

[0164] Furthermore, 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.

[0165] As long as the polynucleotides of the present invention can be expressed, the suitable host for the present invention will not be specifically limited. Examples of suitable 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.

[0166] Still another aspect of the present invention provides a transformant introduced with the expression vector.

[0167] For the purpose of the present invention, there is no limitation on the transformant into which the expression vector of the present invention is introduced, as long as the transformant can express and produce the enzyme fusion protein, and the transformant can be a bacterium of 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; a yeast, such as Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces pombe; an insect cell, such as Spodoptera frugiperda (Sf9); and an animal cell, such as CHO, COS, BSC, etc.

[0168] Still another aspect of the present invention provides a method for preparing the enzyme fusion protein according to the present invention.

[0169] Specifically, the method may include (a) culturing the transformant to obtain a culture; and (b) recovering the enzyme fusion protein from the culture, but the method is not limited thereto.

[0170] In the present invention, the culture medium for culturing the transformant must meet the requirements of the host cell culture in a suitable manner. The carbon source that can be included in the culture medium for host cell growth can be appropriately selected according to the type of transformant prepared by it, by the decision of those skilled in the art, and appropriate culture conditions can be selected to control the period and amount of cultivation.

[0171] 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 materials may be used alone or in combination.

[0172] 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.

[0173] 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 required for growth, such as magnesium sulfate or iron sulfate.

[0174] Finally, necessary growth materials, such as amino acids and vitamins, can be used. In addition, suitable precursors for culture medium can also be used. The above-mentioned sources can be suitably added to the culture during the culture period of batch culture or continuous culture. Basic compounds such as sodium hydroxide, potassium hydroxide and ammonia, or acidic compounds such as phosphoric acid or sulfuric acid can be used to suitably regulate the pH of the culture. In addition, defoamers such as fatty acid polyethylene glycol esters can be added to prevent foam from generating. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas (such as air) can be injected into the culture.

[0175] The transformant of the present invention can be cultured at 20 to 45° C., and specifically 25 to 40° C. Furthermore, the culture is continued until the maximum production of the desired enzyme fusion protein is obtained, and in this regard, the culture can be generally continued for 10 to 160 hours.

[0176] As described above, when suitable culture conditions are provided according to the host cells, the transformant of the present invention can produce enzyme fusion protein, and the produced enzyme fusion protein can be secreted in the cytoplasm or secreted into the periplasmic space of the host cells or secreted extracellularly according to the vector construction and characteristics of the host cells.

[0177] 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 fraction precipitation using acetone or ethanol, etc.), dialysis, gel filtration, ion exchange, or chromatography, such as reverse column chromatography, ultrafiltration, etc., and these methods may be used alone or in combination.

[0178] Still another aspect of the present invention provides a method for preventing or treating LSD in a subject, comprising administering an enzyme fusion protein or a composition comprising the enzyme fusion protein.

[0179] Since the enzyme fusion protein of the present invention contains a therapeutic enzyme that can prevent or treat LSD, a subject suspected of having 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.

[0180] 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, cows, etc., but includes, but is not limited to, any subject that can be treated with the enzyme fusion protein of the present invention or a composition comprising the enzyme fusion protein.

[0181] The method of the present invention may include administering a pharmaceutically effective amount of a pharmaceutical composition containing an enzyme fusion protein. The total daily dose suitable for the composition can be determined within the correct medical judgment of the practitioner, and the composition can be administered once or multiple times in divided doses. However, for the purposes of the present invention, preferably, according to a variety of factors, the specific therapeutically effective dose of the composition for any particular patient is differentially applied, including the type and degree of response to be achieved, the specific composition (including whether other reagents are occasionally used together), the patient's age, weight, health status, sex and diet, administration time, administration route, excretion rate of the composition, treatment duration, other drugs used in combination with a specific composition or simultaneously, and similar factors well-known in the medical field.

[0182] Meanwhile, the method for prevention or treatment of LSD may be a combination therapy further comprising administering a compound or material having a therapeutic effect on at least one LSD, but the method is not limited thereto.

[0183] 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 one that should not lose the beneficial effect of the combination.

[0184] 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 body weight of a patient, but the dose is not particularly limited.

[0185] Still another aspect of the present invention provides use of an enzyme fusion protein or a composition comprising the enzyme fusion protein in the preparation of a medicament (or a pharmaceutical composition) for the prevention or treatment of LSD.

[0186] 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.

[0187] Example 1: Preparation of fusion protein expression vector

[0188] To produce enzyme fusion proteins, expression vectors (IDS cDNA, catalog number EX-C0003-M02, Gencopoeia; ARSB cDNA, catalog number EX-C0073-M02, Genecopoeia) in which naturally occurring iduronate-2-sulfatase (IDS, SEQ ID NO: 1) and arylsulfatase B (ARSB, SEQ ID NO: 3) were inserted, respectively, a synthetic linker (SEQ ID NO: 5) and an IgG4 Fc region (SEQ ID NO: 7) were used to prepare expression vectors of fusion proteins by overlapping PCR. Since overlapping PCR technology includes sequences overlapping with primers when amplifying each of the enzyme and linker-Fc, the resulting PCR products will include overlapping sequences. To amplify the fusion protein, the following PCRs were performed: 1) primary PCR (25 cycles consisting of 95°C for 1 min; 57°C for 30 sec; 68°C for 3 min) and 2) secondary PCR (25 cycles consisting of 95°C for 1 min; 57°C for 30 sec; and 68°C for 4 min).

[0189] Specifically, for IDS, PCR was performed using primers of SEQ ID NOS: 10 and 11, and for Linker-Fc, PCR was performed using primers of SEQ ID NOS: 12 and 13. As a result, the IDS PCR product included the Linker-Fc sequence at the 3' end, and the Linker-Fc PCR included the IDS sequence at the 5' end.

[0190] Secondary PCR was performed using the two PCR products obtained in the primary PCR as templates together with primers (SEQ ID NOS: 10 and 13), and then a PCR product having an IDS-Fc sequence was obtained. The overlapping sequences in the product having the IDS-Fc sequence were digested with restriction enzymes (KpnI and XhoI), and the resulting PCR product was inserted into the XOGC vector to prepare an expression vector (pXOGC-enzyme-Fc).

[0191] In the same manner, a PCR product having an ARSB-Fc sequence was obtained using primers (SEQ ID NOs: 14, 15, 16, and 17). The resulting PCR product was digested with restriction enzymes (KpnI and XhoI) and inserted into the XOGC vector that had been digested with the same restriction enzymes (KpnI and XhoI) to prepare an expression vector for the fusion protein.

[0192] [Table 1] Overlapping PCR primers

[0193]

[0194] The chain exchange and N-glycosylation sites in the Fc region of the prepared fusion protein sequence were removed by site-directed mutagenesis PCR technology.

[0195] Specifically, primers (SEQ ID NOS: 18 and 19) were used to replace the 2nd amino acid (i.e., serine) of the Fc region involved in chain exchange with proline, and the 71st amino acid (i.e., asparagine) of the Fc region involved in N-glycosylation with glutamine. In the protein sequences shown in Table 3 below, each bold letter indicates that the target amino acid is substituted, and italic letters indicate linkers.

[0196] [Table 2] Mutagenic primers

[0197]

[0198] The expression vectors for the enzyme fusion proteins prepared in the above examples are named IDS-Fc vector and ARSB-Fc vector, respectively. Alternatively, these vectors can be used interchangeably with pX0GC-enzyme-Fc.

[0199] [Table 3] DNA sequence and protein sequence of enzyme fusion protein

[0200]

[0201]

[0202]

[0203]

[0204] Example 2: Transformation of CHO cell lines using expression vectors of fusion proteins

[0205] The recombinant expression vector pX0GC-enzyme-Fc prepared in Example 1 was introduced into the DG44 / CHO cell line (CHO / dhfr-) (Urlaubet et al., Somat. Cell. Mol. Genet., 12, 555 to 566, 1986) in which the DHFR gene was disrupted and thus the biosynthesis process of its nucleic acid was imperfect, to obtain a transformant, and the enzyme fusion protein (enzyme-Fc) was expressed in the transformant.

[0206] Specifically, DG44 / CHO cell lines were cultured to confluence such that the cells covered about 80% to about 90% of the bottom of the container, and the cells were washed three times with Opti-MEM (Gibco, catalog number 51985034).

[0207] Meanwhile, a mixture of Opti-MEM (3 mL) and pX0GC-enzyme-Fc (expression vector, 5 μg) and a mixture of Opti-MEM (3 mL) and lipofectamine 2000 (Gibco, catalog number 11668-019, 20 μL) were left at room temperature for 30 minutes. Then, the two mixtures were mixed together, and the cultured DG44 / CHO cell line was added thereto and cultured at 37° C. and 5% CO 2 for about 18 hours to introduce the pX0GC-enzyme-Fc expression vector into the DG44 / CHO cell line.

[0208] Then, the cultured cells were washed 3 times with DMEM-F12 medium containing 10% FBS (Gibco, catalog number 11330), and the culture medium was added thereto and cultured for another 48 hours. Trypsin was added to the cultured cells to separate the cultured cells, and these separated cells were inoculated into a selection medium (α-MEM medium (WELGENE, catalog number LM008-02)) that does not contain HT supplements (hypoxanthine-thymidine) but contains 10% FBS and 1mg / mL G418 (Cellgro, catalog number 61-234-RG). By culturing the cells, while replacing the culture medium at intervals of 2 or 3 days, until only the transformed cells survive and form colonies, the transformed cells are selected from the selection medium. Specifically, in order to improve the expression level of the enzyme fusion protein in the selected transformed cells, 10nM MTX (Sigma, catalog number M8407) is added to the selection medium, and the concentration is gradually increased, and thereby the MTX amount of these transformed cells is increased to 20nM in one to two weeks thereafter.

[0209] Example 3: Confirmation of the expression of IDS-Fc and ARSB-Fc fusion proteins by ELISA

[0210] A portion of the transformed cells prepared in Example 2 was concentrated to 1×10 7 The concentration of 10 cells was transferred to each 175-T cell culture flask and cultured until the cells almost covered the bottom of the culture vessel, and then 15 mL of serum-free Ex-cell medium (purchased from Sigma by custom order, catalog number 14360C) containing 1 mM sodium butyrate (Sigma, catalog number B5887) was added to each flask and cultured in an incubator (33°C, 5% CO2) for 48 hours. Each cell culture was transferred to a 50 mL tube, centrifuged and the supernatant was collected again, and the expression level of the fusion protein (IDS-Fc and ARSB-Fc) was measured.

[0211] First, the expression level of IDS-Fc was measured by applying the indirect ELISA method. Human α-IDS antibody (R&D Systems, catalog number AF2449) diluted in PBS at a concentration of 1 μg / mL was added to a 96-well ELISA plate (Nunk, catalog number 44-2404-21) at an amount of 100 μL / well and reacted overnight in a refrigerator (4°C). On the second day, the resultant was washed 5 times with PBS-T buffer, and the culture sample and IDS standard product (Shire Pharmaceuticals Group, Lot No. TEPE09A17) - which was diluted in various concentrations - each was dispensed in an amount of 100 μL / well and reacted at room temperature for 1 hour. One hour later, the plate was washed, and a biotin-labeled human α-IDS antibody (R&D Systems, catalog number BAF2449) was added thereto, and the mixture was reacted at room temperature for one hour. Finally, streptavidin-HRP (GE Healthcare, catalog number RPN440IV) was diluted at a ratio of 1:30,000 and the diluted mixture was added in an amount of 100 μL / well and reacted for 1 hour. The resultant was washed, and a substrate solution was added thereto and reacted for about 10 minutes. After terminating the reaction with a reaction stop solution, the absorbance of the resultant was measured at 450 nm. After obtaining a standard curve and function using the concentration and absorbance values ​​of the human IDS standard product, the amount of human IDS-Fc fusion protein was quantified. As a result, it was confirmed that the human IDS-Fc fusion protein was expressed in a certain amount ( Figure 1 ).

[0212] In addition, the expression level of ARSB-Fc fusion protein was measured by an enzyme immunoassay (Bethyl, catalog number E80-104) that can quantify human IgG. Human IgG-Fc antibody (Bethyl, catalog number A80-104A-9) diluted in carbonate buffer (0.05M carbonate-bicarbonate, pH 9.6) at a concentration of 10 μg / mL was added to a 96-well ELISA plate (Nunk, catalog number 44-2404-21) in an amount of 100 μL / well and reacted at room temperature for 1 hour. After one hour, the ELISA plate was washed 5 times with a washing solution, and each culture sample and the human IgG standard product contained in the human IgG quantitative kit (Bethyl, catalog number RS10-110-4) were diluted at various concentrations, each of which was distributed in an amount of 100 μL / well, and reacted at room temperature for 1 hour. After one hour, the plate was washed, and HRP-labeled human IgG-Fc antibody (Bethyl, catalog number A80-104P-87) diluted at a ratio of 1:150,000 was added thereto, and reacted at room temperature for one hour. Finally, streptavidin-HRP (GE Healthcare, catalog number RPN440IV) was diluted at a ratio of 1:30,000, added in an amount of 100 μL / well, and reacted for 1 hour. The resultant was washed, and a substrate solution was added thereto and reacted for about 15 minutes. After the reaction was terminated with a reaction termination solution, the absorbance of the resultant was measured at 450 nm.

[0213] After obtaining a standard curve and function using the concentration and absorbance value of the human IgG standard product, the amount of human ARSB-Fc fusion protein was quantified. As a result, it was confirmed that human ARSB-Fc fusion protein was expressed in a certain amount from the selected transformed cells ( Figure 2 ).

[0214] Example 4: Confirmation of the pharmacokinetics of long-acting enzyme fusion protein

[0215] By examining the pharmacokinetics of the long-acting enzyme fusion protein prepared above and the enzyme not fused to the Fc region, the effects of the fusion protein preparations were compared.

[0216] Example 4-1: Pharmacokinetics of long-acting iduronate-2-sulfatase fusion protein

[0217] The present inventors attempted to confirm the therapeutic duration of the fusion protein of the present invention by examining the pharmacokinetics of the long-acting fusion protein of iduronate-2-sulfatase prepared in the above examples.

[0218] For this purpose, iduronate-2-sulfatase (IDS-sulfatase, control group) and a long-acting fusion protein of iduronate-2-sulfatase (IDS-Fc fusion protein, experimental group) were administered to three ICR mice, respectively, and the pharmacokinetic coefficients based on the blood stability and each blood sample collection of each group were compared.

[0219] Specifically, based on the concentration of iduronate-2-sulfatase, the protein was administered to the ICR mice of the control and experimental groups by intravenous and subcutaneous injection at concentrations of 0.5 mg / kg and 1.0 mg / kg, respectively. Blood samples were collected from the group administered by intravenous injection at 0, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 144 and 168 hours after injection, and blood samples were collected 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 the serum was measured by ELISA method using human-specific anti-iduronate-2-sulfatase antibodies. The results of the analysis are presented in Figure 3 and shown in Table 4.

[0220] [Table 4]

[0221]

[0222] As can be seen in the above results, the long-acting fusion protein of iduronate-2-sulfatase according to the present invention shows significantly excellent pharmacokinetic properties compared to those of the control group. These results show that, compared with the enzyme of the non-long-acting fusion protein, the long-acting fusion protein of iduronate-2-sulfatase of the present invention has the advantage of reducing the interval of drug administration in the actual administration of the drug through the long-acting effect.

[0223] like Figure 3 As can be seen from the pharmacokinetic results in Table 4, in the case of the long-acting fusion protein of iduronate-2-sulfatase, the half-life (T 1 / 2 ), the maximum drug concentration in blood (C max ) and in vivo bioavailability (AUC). Specifically, the in vivo bioavailability of the long-acting fusion protein of iduronate-2-sulfatase was 64.9%, thus showing excellent in vivo bioavailability compared to the enzyme of the non-long-acting fusion protein.

[0224] Example 4-2: Pharmacokinetics of long-acting arylsulfatase B fusion protein

[0225] The present inventors attempted to examine the pharmacokinetics of the long-acting fusion protein of arylsulfatase B (ARSB-Fc fusion protein) prepared in the above example, and thus measured the pharmacokinetics of the long-acting fusion protein of arylsulfatase B and compared them with those results of arylsulfatase B.

[0226] Specifically, based on the concentration of arylsulfatase B, a control group (naturally occurring arylsulfatase B: BioMarin) and experimental groups (long-acting fusion proteins of arylsulfatase B). Regardless of the method of administration, blood samples were collected from the control group at 0, 0.25, 0.5, 0.75, 1, 1.5, 4, 8 and 24 hours after injection. In the experimental group, blood samples were collected from ICR mice administered by intravenous injection at 0, 0.25, 0.5, 1, 1.5, 2, 4, 8, 24, 48, 96 and 168 hours after injection, and blood samples were collected from those mice administered by subcutaneous injection at 0, 0.5, 1, 2, 4, 8, 24, 48, 96 and 168 hours after injection.

[0227] The blood samples collected in each group were centrifuged and separated into serum, and the amounts of long-acting fusion protein of arylsulfatase B and naturally occurring arylsulfatase B in the blood were quantified by enzyme immunoassay, and the analysis results were analyzed on Figure 4 and shown in Table 5.

[0228] [Table 5]

[0229]

[0230] As can be seen from the above results, Compared with the naturally occurring aromatic sulfatase B, the long-acting fusion protein of aromatic sulfatase B according to the present invention shows significantly excellent pharmacokinetic properties. These results show that compared with the enzyme, the long-acting fusion protein of aromatic sulfatase B of the present invention has the advantage of reducing the interval of drug administration in the actual administration of the drug through the long-acting effect.

[0231] like Figure 4 As can be seen from the pharmacokinetic results in Table 5, in the case of the long-acting fusion protein of arylsulfatase B, the half-life (T 1 / 2 ), maximum drug concentration in blood (C max ) and all items of in vivo bioavailability (AUC). Specifically, the in vivo bioavailability of the long-acting fusion protein of arylsulfatase B was 65.8%, thus showing excellent in vivo bioavailability compared to the enzyme of the non-long-acting fusion protein.

[0232] As a result of examining the pharmacokinetics of the enzyme fusion proteins in Examples 4-1 and 4-2, it was confirmed that these enzyme fusion proteins showed significantly increased half-life, in vivo bioavailability, etc. compared to those enzymes not fused with the Fc region, and thus the long-lasting effects of these enzyme fusion proteins can be expected.

[0233] Example 5: Confirmation of the enzyme activity of the long-acting enzyme fusion protein

[0234] The activity of the enzyme contained in the enzyme fusion protein prepared above was compared with that of the enzyme to which the Fc region was not fused.

[0235] Example 5-1: In vitro enzyme activity of long-acting iduronate-2-sulfatase fusion protein

[0236] The present inventors attempted to measure changes in the enzyme activity of the long-acting fusion protein preparation of iduronate-2-sulfatase prepared according to the above examples, and thus measured the in vitro enzyme activity.

[0237] Specifically, 4-methylumbelliferyl α-L-idopyranosiduronic acid-2-sulfate sodium salt (4MU-α-IdopyraA-2), known as an enzyme substrate, was reacted with iduronate-2-sulfatase and a long-acting fusion protein of iduronate-2-sulfatase at 37° C. for 4 hours, and then reacted with α-iduronidase (i.e., secondary reaction enzyme) at 37° C. for 24 hours. Then, the fluorescence of the final product 4-methylumbelliferyl (4MU) was measured to measure the enzyme activity of the corresponding substance.

[0238] As a result, it was confirmed that the long-acting fusion protein of iduronate-2-sulfatase and iduronate-2-sulfatase had an enzyme activity (specific activity) of 32.0±1.58nmol / min / mM and 87.3±6.49nmol / min / mM, respectively. Since the long-acting fusion protein of iduronate-2-sulfatase has a structure in which two iduronate-2-sulfatases are connected to one Fc molecule-it is a dimerization form of two Fc chains, the measurement results show that the long-acting fusion protein of iduronate-2-sulfatase has an in vitro enzyme activity about 2.7 times higher than that of the iduronate-2-sulfatase of the non-fusion protein. These results indicate that the structural features of the long-acting fusion protein of iduronate-2-sulfatase with two iduronate-2-sulfatases have advantages in enzyme activity relative to the iduronate-2-sulfatase that does not form a fusion protein ( Figure 5 ).

[0239] Example 5-2: In vitro enzyme activity of long-acting arylsulfatase B fusion protein

[0240] The present inventors compared and measured the enzyme activity of the long-acting fusion protein of arylsulfatase B prepared in the above examples with that of the naturally occurring enzyme, arylsulfatase B ( BioMarin)——enzyme activity.

[0241] Specifically, the long-acting fusion protein of arylsulfatase B and arylsulfatase B were reacted with 4-methylcoumarinyl sulfate at 37°C for 20 minutes, and the in vitro enzymatic activity of the long-acting fusion protein of arylsulfatase B was measured by measuring the fluorescence of the 4-methylcoumarin group formed after cleavage of the sulfate group.

[0242] As a result, it was confirmed that the long-acting fusion protein of aromatic sulfatase B and aromatic sulfatase B had an enzyme activity (specific activity) of 438.5 ± 29.4 nmol / min / mM and 823.8 ± 37.0 nmol / min / μM, respectively. Since the long-acting fusion protein of aromatic sulfatase B has a structure in which two aromatic sulfatase Bs are connected to one Fc molecule-it is a dimerization form of two Fc chains, the measurement results show that the long-acting fusion protein of aromatic sulfatase B has an in vitro enzyme activity about 1.9 times higher than that of aromatic sulfatase B of non-fusion protein. These results confirm that the distinguishing structural features of the long-acting fusion protein of aromatic sulfatase B at the molecular level relative to aromatic sulfatase B are attributed to excellent enzyme activity ( Figure 6 ).

[0243] As a result of examining the pharmacokinetics of the enzyme fusion proteins in Examples 5-1 and 5-2, it was confirmed that these enzyme fusion proteins exhibited high in vitro enzyme activities compared to those to which the Fc region was not fused.

[0244] Example 6: Confirmation of the efficacy of administration of long-acting iduronate-2-sulfatase fusion protein

[0245] The administration efficacy of the enzyme fusion protein of the present invention was examined by studying the changes in the amount of glucosaminoglycan (GAG) in tissues and urine after drug administration to iduronate-2-sulfatase (IDS)-knockout mice.

[0246] Specifically, in addition to normal mice as a negative control group, IDS knockout mice aged 7 to 14 weeks were divided into a total of four groups based on the GAG ​​content in urine. Iduronate-2-sulfatase (IDS) was administered to the tail vein at a concentration of 0.5 mg / kg (control group) for a total of 4 times (day 0, day 7, day 14, and day 21). Genzyme). The administration of the long-acting fusion protein of iduronate-2-sulfatase was performed by dividing into two groups: one group was administered with a long-acting fusion protein of iduronate-2-sulfatase once into the tail vein at a concentration of 2.0 mg / kg (day 0), and the other group was administered with a long-acting fusion protein of iduronate-2-sulfatase once subcutaneously at a concentration of 4.0 mg / kg (day 0).

[0247] Urine samples were collected from each group on the 7th, 14th, 21st and 28th day before administration and after drug administration. On the 28th day after drug administration, all tissues were collected from liver, spleen, heart and bone marrow. Then, tissue crushing buffer (PBS containing aprotinin (1 μg / mL), 1mM PMSF and 2mM EDTA) was added to each tissue in an amount of 5 volumes (9 volumes for bone marrow), and the mixture was crushed and centrifuged using a sonicator, and each supernatant was used for analysis of GAG content.

[0248] Then, 50 μL of the supernatant obtained from the collected urine samples and each tissue after pulverization was added to a 96-well plate, a dimethylmethylene blue solution (250 μL) was added and mixed, and the content of GAG was quantified at a wavelength of 525 nm. In the case of GAG content in urine, the value was calculated by adjusting with reference to the amount of creatine. Statistical analysis was performed between the control group and the test group using one-way ANOVA using the calculated values. The GAG ​​content measured in urine and each tissue was Figure 7 and 8 Displayed in.

[0249] like Figure 7 and Figure 8 As shown in Figure 2, it was confirmed that even a single monthly intravenous or subcutaneous administration of the long-acting fusion protein of iduronate-2-sulfatase significantly reduced the GAG ​​values ​​in urine and each tissue to levels similar to those of weekly intravenous administration compared with IDS knockout mice. The level of therapy, It is an enzyme without fused Fc region.

[0250] By this embodiment, it is confirmed that due to the extended blood half-life, even if it is administered intravenously or subcutaneously once a month, the long-acting fusion protein of iduronate-2-sulfatase can show an effect comparable to the existing drug therapy of administering medicine once a week. In addition, in the group of long-acting fusion protein of iduronate-2-sulfatase administered subcutaneously once a month, the long-acting fusion protein of iduronate-2-sulfatase shows the result of the effect of reducing GAG value, confirming that the long-acting fusion protein of iduronate-2-sulfatase has the potential of subcutaneous injection as the administration route of fusion protein of the present invention. Therefore, it is suggested that by administering once a month or subcutaneously, the long-acting fusion protein of iduronate-2-sulfatase according to the present invention can be used to treat Hunter syndrome patients.

[0251] Example 7: Confirmation of tissue distribution of long-acting fusion protein (arylsulfatase B)

[0252] The present inventors attempted to confirm the distribution degree of the enzyme fusion protein of the present invention prepared in the above examples.

[0253] In this regard, the extent of distribution of arylsulfatase B (control group) and long-acting fusion protein of arylsulfatase B (experimental group) in tissues and organs of three ICR mice were collected for each group and compared according to each group.

[0254] Specifically, based on the concentration of arylsulfatase B, the control group and the experimental group were administered at a concentration of 5.0 mg / kg by intravenous injection.

[0255] For the control group, naturally occurring arylsulfatase B and the experimental group of long-acting fusion protein of arylsulfatase B, by administering naturally occurring arylsulfatase B After the long-acting fusion protein of succinimidyl-1,4-dihydrophenylalanine and arylsulfatase B was removed from the mouse organs, the concentration of each material in the tissues (bone marrow, liver, spleen, lung, kidney and heart) was measured and compared by enzyme immunoassay.

[0256] As a result, compared with naturally occurring arylsulfatase B, which was used as a control group and was not fused with an Fc region, the long-acting fusion protein of arylsulfatase B showed results of being distributed in all tissues to a higher degree or for a longer period of time in the same period of time.

[0257] Specifically, it was confirmed that the long-acting fusion protein of arylsulfatase B has a significantly higher distribution degree in the bone marrow and spleen than the naturally occurring arylsulfatase B. In addition, it was confirmed that the long-acting fusion protein of arylsulfatase B is distributed in the lung, kidney and heart, while arylsulfatase B ( Fig. 9 ).

[0258] From these experimental results, it was confirmed that the long-acting fusion protein of arylsulfatase B was (i.e., naturally occurring arylsulfatase B) has excellent pharmacokinetic properties. Specifically, the potential use of a long-acting fusion protein of arylsulfatase B for monthly administration can not only reduce the frequency of administration, but also help improve the quality of life of patients by switching to subcutaneous administration, compared with the existing weekly intravenous administration therapy.

[0259] From the foregoing, it will be understood by those skilled in the art that the present invention may be implemented in other specific forms without modifying the technical concept or basic features of the present invention. 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 exemplary embodiments, but also various options, modifications, equivalents and other embodiments that may be included in the spirit and scope of the present invention as defined by the appended claims. <110> Hanmi Pharmaceutical Co., Ltd. <120> Novel therapeutic enzyme fusion protein and its use <130> OPA18228 <150> KR 10-2017-0086594 <151> 2017-07-07 <160> 26 <170> KoPatentIn 3.0 <210> 1 <211> 1650 <212> DNA <213> Homo sapiens <400> 1 atgccgccac cccggaccgg ccgaggcctt ctctggctgg gtctggttct gagctccgtc 60 tgcgtcgccc tcggatccga aacgcaggcc aactcgacca cagatgctct gaacgttctt 120 ctcatcatcg tggatgacct gcgcccctcc ctgggctgtt atggggataa gctggtgagg 180 240. tccccaata ttgaccaact ggcatcccac agcctcctct tccagaatgc ctttgcgcag caagcagtgt gcgccccgag ccgcgtttct ttcctcactg gcaggagacc tgacaccacc 300 cgcctgtacg acttcaactc ctactggagg gtgcacgctg gaaacttctc caccatcccc 360 cagtacttca aggagaatgg ctatgtgacc atgtcggtgg gaaaagtctt tcaccctggg 420 atatcttcta accataccga tgattctccg tatagctggt cttttccacc ttatcatcct 480 tcctctgaga actccatgcc tgtcgagggc actccatgcc aacctgcttt gccctgtgga tgtgctggat gttcccgagg gcaccttgcc tgacaaacag 600. agcactgagc aagccataca gttgttggaa aagatgaaaa cgtcagccag tcctttcttc 660. ctggccgttg ggtatcataa gccacacatc cccttcagat accccaagga atttcagaag 720 ttgtatccct tggagaacat caccctggcc cccgatcccg aggtccctga tggcctaccc 780 cctgtggcct acaacccctg gatggacatc aggcaacggg aagacgtcca agccttaaac atcagtgtgc cgtatggtcc aattcctgtg gactttcagc ggaaaatccg ccagagctac tttgcctctg tgtcatattt ggatacacag gtcggccgcc tcttgagtgc tttggacgat 960 cttcagctgg ccaacagcac catcattgca tttacctcgg atcatgggtg ggctctaggt 1020 gaacatggag aatgggccaa atacagcaat tttgatgttg ctacccatgt tcccctgata 1080 ttctatgttc ctggaaggac ggcttcactt ccggaggcag gcgagaagct tttcccttac 1140 ctcgaccctt ttgattccgc ctcacagttg atggagccag gcaggcaatc catggacctt 1200 gtggaacttg tgtctctttt tcccacgctg gctggacttg caggactgca ggttccacct 1260 cgctgccccg ttccttcatt tcacgttgag ctgtgcagag aaggcaagaa ccttctgaag 1320 cattttcgat tccgtgactt ggaagaggat ccgtacctcc ctggtaatcc ccgtgaactg 1380 attgcctata gccagtatcc ccggccttca gacatccctc agtggaattc tgacaagccg 1440 agtttaaaag atataaagat catgggctat tccatacgca ccatagacta taggtatact 1500 gtgtgggttg gcttcaatcc tgatgaattt ctagctaact tttctgacat ccatgcaggg 1560 gaactgtatt ttgtggattc tgacccattg caggatcaca atatgtataa tgattcccaa 1620 ggtggagatc ttttccagtt gttgatgcct 1650 <210> 2 <211> 550 <212> PRT <213> Homo sapiens <400> 2 Met Pro Pro Pro Arg Thr Gly Arg Gly Leu Leu Trp Leu Gly Leu Val 1 5 10 15 Leu Ser Ser Val Cys Val Ala Leu Gly Ser Glu Thr Gln Ala Asn Ser 20 25 30 Thr Thr Asp Ala Leu Asn Val Leu Leu Ile Ile Val Asp Asp Leu Arg 35 40 45 Pro Ser Leu Gly Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro Asn Ile 50 55 60 Asp Gln Leu Ala Ser His Ser Leu Leu Phe Gln Asn Ala Phe Ala Gln 65 70 75 80 Gln Ala Val Cys Ala Pro Ser Arg Val Ser Phe Leu Thr Gly Arg Arg 85 90 95 Pro Asp Thr Thr Arg Leu Tyr Asp Phe Asn Ser Tyr Trp Arg Val His 100 105 110 Ala Gly Asn Phe Ser Thr Ile Pro Gln Tyr Phe Lys Glu Asn Gly Tyr 115 120 125 Val Thr Met Ser Val Gly Lys Val Phe His Pro Gly Ile Ser Ser Asn 130 135 140 His Thr Asp Asp Ser Pro Tyr Ser Trp Ser Phe Pro Pro Tyr His Pro 145 150 155 160 Ser Ser Glu Lys Tyr Glu Asn Thr Lys Thr Cys Arg Gly Pro Asp Gly 165 170 175 Glu Leu His Ala Asn Leu Leu Cys Pro Val Asp Val Leu Asp Val Pro 180 185 190 Glu Gly Thr Leu Pro Asp Lys Gln Ser Thr Glu Gln Ala Ile Gln Leu 195 200 205 Leu Glu Lys Met Lys Thr Ser Ala Ser Pro Phe Phe Leu Ala Val Gly 210 215 220 Tyr His Lys Pro His Ile Pro Phe Arg Tyr Pro Lys Glu Phe Gln Lys 225 230 235 240 Leu Tyr Pro Leu Glu Asn Ile Thr Leu Ala Pro Asp Pro Glu Val Pro 245 250 255 Asp Gly Leu Pro Pro Val Ala Tyr Asn Pro Trp Met Asp Ile Arg Gln 260 265 270 Arg Glu Asp Val Gln Ala Leu Asn Ile Ser Val Pro Tyr Gly Pro Ile 275 280 285 Pro Val Asp Phe Gln Arg Lys Ile Arg Gln Ser Tyr Phe Ala Ser Val 290 295 300 Ser Tyr Leu Asp Thr Gln Val Gly Arg Leu Leu Ser Ala Leu Asp Asp 305 310 315 320 Leu Gln Leu Ala Asn Ser Thr Ile Ile Ala Phe Thr Ser Asp His Gly 325 330 335 Trp Ala Leu Gly Glu His Gly Glu Trp Ala Lys Tyr Ser Asn Phe Asp 340 345 350 Val Ala Thr His Val Pro Leu Ile Phe Tyr Val Pro Gly Arg Thr Ala 355 360 365 Ser Leu Pro Glu Ala Gly Glu Lys Leu Phe Pro Tyr Leu Asp Pro Phe 370 375 380 Asp Ser Ala Ser Gln Leu Met Glu Pro Gly Arg Gln Ser Met Asp Leu 385 390 395 400 Val Glu Leu Val Ser Leu Phe Pro Thr Leu Ala Gly Leu Ala Gly Leu 405 410 415 Gln Val Pro Pro Arg Cys Pro Val Pro Ser Phe His Val Glu Leu Cys 420 425 430 Arg Glu Gly Lys Asn Leu Leu Lys His Phe Arg Phe Arg Asp Leu Glu 435 440 445 Glu Asp Pro Tyr Leu Pro Gly Asn Pro Arg Glu Leu Ile Ala Tyr Ser 450 455 460 Gln Tyr Pro Arg Pro Ser Asp Ile Pro Gln Trp Asn Ser Asp Lys Pro 465 470 475 480 Ser Leu Lys Asp Ile Lys Ile Met Gly Tyr Ser Ile Arg Thr Ile Asp 485 490 495 Tyr Arg Tyr Thr Val Trp Val Gly Phe Asn Pro Asp Glu Phe Leu Ala 500 505 510 Asn Phe Ser Asp Ile His Ala Gly Glu Leu Tyr Phe Val Asp Ser Asp 515 520 525 Pro Leu Gln Asp His Asn Met Tyr Asn Asp Ser Gln Gly Gly Asp Leu 530 535 540 Phe Gln Leu Leu Met Pro 545 550 <210> 3 <211> 1599 <212> DNA <213> Homo sapiens <400> 3 atgggtccgc gcggcgcggc gagcttgccc cgaggccccg gtcctcggcg gctgcttctc 60 cccgtcgtcc tcccgctgct gctgctgctg ttgttggcgc cgccgggctc gggcgccggg 120 gccagccggc cgccccacct ggctttcttg ctggcagacg acctaggctg gaacgacgtc 180 ggcttccacg gctcccgcat ccgcacgccg cacctggacg cgctggcggc cggcggggtg 240 ctcctggaca actactacac gcagccgctg tgcacgccgt cgcggagcca gctgctcact 300 ggccgctacc agatccgtac aggtttacag caccaaataa tctggccctg tcagcccagc 360 tgtgttcctc tggatgaaaa actcctgccc cagctcctaa aagaagcagg ttatactacc 420 catatggtcg gaaaatggca cctgggaatg taccggaaag aatgccttcc aacccgccga 480 ggatttgata cctactttgg atatctcctg ggtagtgaag attattattc ccatgaacgc 540 tgtacattaa ttgacgctct gaatgtcaca cgatgtgctc ttgatttcg agatggcgaa 600 gaagttgcaa caggatataa aaatatgtat tcaacaaaca tattcaccaa aagggctata 660 gccctcataa ctaaccatcc accagagaag cctctgtttc tctaccttgc tctccagtct 720 gtgcatgagc cccttcaggt ccctgaggaa tacttgaagc catatgactt tatccaagac 780 aagaacaggc atcactatgc aggaatggtg tcccttatgg atgaagcagt aggaaatgtc 840 actgcagctt taaaaagcag tgggctctgg aacaacacgg tgttcatctt ttctacagat 900 aacggagggc agactttggc agggggtaat aactggcccc ttcgaggaag aaaatggagc 960 ctgtgggaag gaggcgtccg aggggtgggc tttgtggcaa gccccttgct gaagcagaag 1020 ggcgtgaaga accgggagct catccacatc tctgactggc tgccaacact cgtgaagctg 1080 gccaggggac acaccaatgg cacaaagcct ctggatggct tcgacgtgtg gaaaaccatc 1140 agtgaaggaa gcccatcccc cagaattgag ctactgcata atattgaccc gaacttcgtg 1200 gactcttcac cgtgtcccag gaacagcatg gctccagcaa aggatgactc ttctcttcca 1260 gaatattcag cctttaacac atctgtccat gctgcaatta gacatggaaa ttggaaactc 1320 ctcacgggct acccaggctg tggttactgg ttccctccac cgtctcaata caatgtttct 1380 gagataccct catcagaccc accaaccaag accctctggc tctttgatat tgatcgggac 1440 cctgaagaaa gacatgacct gtccagagaa tatcctcaca tcgtcacaaa gctcctgtcc 1500 cgcctacagt tctaccataa acactcagtc cccgtgtact tccctgcaca ggacccccgc 1560 tgtgatccca aggccactgg ggtgtggggc ccttggatg 1599 <210> 4 <211> 533 <212> PRT <213> Homo sapiens <400> 4 Met Gly Pro Arg Gly Ala Ala Ser Leu Pro Arg Gly Pro Gly Pro Arg 1 5 10 15 Arg Leu Leu Leu Pro Val Val Leu Pro Leu Leu Leu Leu Leu Leu Leu 20 25 30 Ala Pro Pro Gly Ser Gly Ala Gly Ala Ser Arg Pro Pro His Leu Val 35 40 45 Phe Leu Leu Ala Asp Asp Leu Gly Trp Asn Asp Val Gly Phe His Gly 50 55 60 Ser Arg Ile Arg Thr Pro His Leu Asp Ala Leu Ala Ala Gly Gly Val 65 70 75 80 Leu Leu Asp Asn Tyr Tyr Thr Gln Pro Leu Cys Thr Pro Ser Arg Ser 85 90 95 Gln Leu Leu Thr Gly Arg Tyr Gln Ile Arg Thr Gly Leu Gln His Gln 100 105 110 Ile Ile Trp Pro Cys Gln Pro Ser Cys Val Pro Leu Asp Glu Lys Leu 115 120 125 Leu Pro Gln Leu Leu Lys Glu Ala Gly Tyr Thr Thr His Met Val Gly 130 135 140 Lys Trp His Leu Gly Met Tyr Arg Lys Glu Cys Leu Pro Thr Arg Arg 145 150 155 160 Gly Phe Asp Thr Tyr Phe Gly Tyr Leu Leu Gly Ser Glu Asp Tyr Tyr 165 170 175 Ser His Glu Arg Cys Thr Leu Ile Asp Ala Leu Asn Val Thr Arg Cys 180 185 190 Ala Leu Asp Phe Arg Asp Gly Glu Glu Val Ala Thr Gly Tyr Lys Asn 195 200 205 Met Tyr Ser Thr Asn Ile Phe Thr Lys Arg Ala Ile Ala Leu Ile Thr 210 215 220 Asn His Pro Pro Glu Lys Pro Leu Phe Leu Tyr Leu Ala Leu Gln Ser 225 230 235 240 Val His Glu Pro Leu Gln Val Pro Glu Glu Tyr Leu Lys Pro Tyr Asp 245 250 255 Phe Ile Gln Asp Lys Asn Arg His His Tyr Ala Gly Met Val Ser Leu 260 265 270 Met Asp Glu Ala Val Gly Asn Val Thr Ala Ala Leu Lys Ser Ser Gly 275 280 285 Leu Trp Asn Asn Thr Val Phe Ile Phe Ser Thr Asp Asn Gly Gly Gln 290 295 300 Thr Leu Ala Gly Gly Asn Asn Trp Pro Leu Arg Gly Arg Lys Trp Ser 305 310 315 320 Leu Trp Glu Gly Gly Val Arg Gly Val Gly Phe Val Ala Ser Pro Leu 325 330 335 Leu Lys Gln Lys Gly Val Lys Asn Arg Glu Leu Ile His Ile Ser Asp 340 345 350 Trp Leu Pro Thr Leu Val Lys Leu Ala Arg Gly His Thr Asn Gly Thr 355 360 365 Lys Pro Leu Asp Gly Phe Asp Val Trp Lys Thr Ile Ser Glu Gly Ser 370 375 380 Pro Ser Pro Arg Ile Glu Leu Leu His Asn Ile Asp Pro Asn Phe Val 385 390 395 400 Asp Ser Ser Pro Cys Pro Arg Asn Ser Met Ala Pro Ala Lys Asp Asp 405 410 415 Ser Ser Leu Pro Glu Tyr Ser Ala Phe Asn Thr Ser Val His Ala Ala 420 425 430 Ile Arg His Gly Asn Trp Lys Leu Leu Thr Gly Tyr Pro Gly Cys Gly 435 440 445 Tyr Trp Phe Pro Pro Pro Ser Gln Tyr Asn Val Ser Glu Ile Pro Ser 450 455 460 Ser Asp Pro Pro Thr Lys Thr Leu Trp Leu Phe Asp Ile Asp Arg Asp 465 470 475 480 Pro Glu Glu Arg His Asp Leu Ser Arg Glu Tyr Pro His Ile Val Thr 485 490 495 Lys Leu Leu Ser Arg Leu Gln Phe Tyr His Lys His Ser Val Pro Val 500 505 510 Tyr Phe Pro Ala Gln Asp Pro Arg Cys Asp Pro Lys Ala Thr Gly Val 515 520 525 Trp Gly Pro Trp Met 530 <210> 5 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Peptide linker <400> 5 ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcg 45 <210> 6 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 6 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 7 <211> 666 <212> DNA <213> Artificial sequence <220> <223> Immunoglobulin Fc variant <400> 7 ccatcatgcc cagcacctga gttcctgggg ggaccatcag tcttcctgtt ccccccaaaa 60 cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 120 agccaggaag accctgaggt ccagttcaac tggtacgtgg acggcgtgga ggtgcataat 180 gccaagacaa agccgcggga ggagcagttc aacagcacgt accgtgtggt cagcgtcctc 240 accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 300 ggcctcccat cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 360 caggtgtaca ccctgccccc atcccaggag gagatgacca agaaccaggt cagcctgacc 420 tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 480 ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 540 tacagcaggc taaccgtgga caagagcagg tggcaggagg ggaacgtctt ctcatgctcc 600 gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctctgggt 660 aaatga 666 <210> 8 <211> 221 <212> PRT <213> Artificial sequence <220> <223> IgG4 Fc <400> 8 Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 1 5 10 15 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 20 25 30 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 50 55 60 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 65 70 75 80 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 85 90 95 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115 120 125 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 165 170 175 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 180 185 190 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 220 <210> 9 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> Immunoglobulin Fc Variant <400> 9 Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 1 5 10 15 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 20 25 30 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 50 55 60 Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu 65 70 75 80 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 85 90 95 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115 120 125 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 165 170 175 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 180 185 190 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 220 <210> 10 <211> 26 <212> DNA <213> Artificial sequence <220> <223> IDS-F (KpnI) <400> 10 caggtaccat gccgccaccc cggacc 26 <210> 11 <211> 44 <212> DNA <213> Artificial sequence <220> <223> IDS-R (Overlap) <400> 11 tgaaccgcct ccaccaggca tcaacaactg gaaaagatct ccac 44 <210> 12 <211> 34 <212> DNA <213> Artificial sequence <220> <223> L15Fc(IDS)-F <400> 12 cagttgttga tgcctggtgg aggcggttca ggcg 34 <210> 13 <211> 33 <212> DNA <213> Artificial sequence <220> <223> L15Fc-R (XhoI) <400> 13 gactcgagtc atttacccag agacaggggag agg 33 <210> 14 <211> 26 <212> DNA <213> Artificial sequence <220> <223> ARSB-F (KpnI) <400> 14 caggtaccat gggtccgcgc ggcgcg 26 <210> 15 <211> 35 <212> DNA <213> Artificial sequence <220> <223> ARSB-R (overlapping) <400> 15 tgaaccgcct ccacccatcc aagggcccca caccc 35 <210> 16 <211> 34 <212> DNA <213> Artificial sequence <220> <223> L15Fc(ARSB)-F <400> 16 tggggccctt ggatgggtgg aggcggttca ggcg 34 <210> 17 <211> 33 <212> DNA <213> Artificial sequence <220> <223> L15Fc-R (XhoI) <400> 17 gactcgagtc atttacccag agacaggggag agg 33 <210> 18 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Fc(S2P)_F <400> 18 ctggcggtgg cggatcgcca ccatgcccag cacctgagtt cct 43 <210> 19 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Fc(S2P)_R <400> 19 aggaactcag gtgctgggca tggtggcgat ccgccaccgc cag 43 <210> 20 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Fc(N71Q)_F <400> 20 agccgcggga ggagcagttc caaagcacgt accgtgtggt cag 43 <210> twenty one <211> 43 <212> DNA <213> Artificial sequence <220> <223> Fc(N71Q)_R <400> twenty one ctgaccacac ggtacgtgct ttggaactgc tcctcccgcg gct 43 <210> twenty two <211> 2361 <212> DNA <213> Artificial sequence <220> <223> IDS-Fc <400> twenty two atgccgccac cccggaccgg ccgaggcctt ctctggctgg gtctggttct gagctccgtc tgcgtcgccc tcggatccga aacgcaggcc aactcgacca cagatgctct gaaacgttctt ctcatcatcg tggatgacct gcgcccctcc ctgggctgtt atggggataa gctggtgagg 180 240. tccccaata ttgaccaact ggcatcccac agcctcctct tccagaatgc ctttgcgcag caagcagtgt gcgccccgag ccgcgtttct ttcctcactg gcaggagacc tgacaccacc 300 cgcctgtacg acttcaactc ctactggagg gtgcacgctg gaaacttctc caccatcccc 360 cagtacttca aggagaatgg ctatgtgacc atgtcggtgg gaaaagtctt tcaccctggg 420 atatcttcta accataccga tgattctccg tatagctggt cttttccacc ttatcatcct 480 tcctctgaga actccatgcc tgtcgagggc actccatgcc aacctgcttt gccctgtgga tgtgctggat gttcccgagg gcaccttgcc tgacaaacag 600. agcactgagc aagccataca gttgttggaa aagatgaaaa cgtcagccag tcctttcttc 660. ctggccgttg ggtatcataa gccacacatc cccttcagat accccaagga atttcagaag 720 ttgtatccct tggagaacat caccctggcc cccgatcccg aggtccctga tggcctaccc 780 cctgtggcct acaacccctg gatggacatc aggcaacggg aagacgtcca agccttaaac 840 atcagtgtgc cgtatggtcc aattcctgtg gactttcagc ggaaaatccg ccagagctac 900 tttgcctctg tgtcatattt ggatacacag gtcggccgcc tcttgagtgc tttggacgat 960 cttcagctgg ccaacagcac catcattgca tttacctcgg atcatgggtg ggctctaggt 1020 gaacatggag aatgggccaa atacagcaat tttgatgttg ctacccatgt tcccctgata 1080 ttctatgttc ctggaaggac ggcttcactt ccggaggcag gcgagaagct tttcccttac 1140 ctcgaccctt ttgattccgc ctcacagttg atggagccag gcaggcaatc catggacctt 1200 gtggaacttg tgtctctttt tcccacgctg gctggacttg caggactgca ggttccacct 1260 cgctgccccg ttccttcatt tcacgttgag ctgtgcagag aaggcaagaa ccttctgaag 1320 cattttcgat tccgtgactt ggaagaggat ccgtacctcc ctggtaatcc ccgtgaactg 1380 attgcctata gccagtatcc ccggccttca gacatccctc agtggaattc tgacaagccg 1440 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 gtgtgggttg gcttcaatcc tgatgaattt ctagctaact tttctgacat ccatgcaggg gaactgtatt ttgtggattc tgacccattg caggatcaca atatgtata tgattcccaa ggtggagatc ttttccagtt gttgatgcct ggtggaggcg gttcaggcgg aggtggctct 1680 ggcggtggcg gatcgccatc atgcccagca cctgagttcc tggggggacc atcagtcttc 1740 ctgttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 1800. gtggtggtgg acgtgagcca ggaagaccct gaggtccagt tcaactggta cgtggacggc 1860. gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc aaggtctcca acaaaggcct cccatcctcc atcgagaaaa cccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc aggaggat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 2160 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 2220 ggctccttct tcctctacag caggctaacc gtggacaaga gcaggtggca ggaggggaac 2280 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 2340 tccctgtctc tgggtaaatg a 2361 <210> 23 <211> 786 <212> PRT <213> Artificial sequence <220> <223> IDS-Fc <400> 23 Met Pro Pro Pro Arg Thr Gly Arg Gly Leu Leu Trp Leu Gly Leu Val 1 5 10 15 Leu Ser Ser Val Cys Val Ala Leu Gly Ser Glu Thr Gln Ala Asn Ser 20 25 30 Thr Thr Asp Ala Leu Asn Val Leu Leu Ile Ile Val Asp Asp Leu Arg 35 40 45 Pro Ser Leu Gly Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro Asn Ile 50 55 60 Asp Gln Leu Ala Ser His Ser Leu Leu Phe Gln Asn Ala Phe Ala Gln 65 70 75 80 Gln Ala Val Cys Ala Pro Ser Arg Val Ser Phe Leu Thr Gly Arg Arg 85 90 95 Pro Asp Thr Thr Arg Leu Tyr Asp Phe Asn Ser Tyr Trp Arg Val His 100 105 110 Ala Gly Asn Phe Ser Thr Ile Pro Gln Tyr Phe Lys Glu Asn Gly Tyr 115 120 125 Val Thr Met Ser Val Gly Lys Val Phe His Pro Gly Ile Ser Ser Asn 130 135 140 His Thr Asp Asp Ser Pro Tyr Ser Trp Ser Phe Pro Pro Tyr His Pro 145 150 155 160 Ser Ser Glu Lys Tyr Glu Asn Thr Lys Thr Cys Arg Gly Pro Asp Gly 165 170 175 Glu Leu His Ala Asn Leu Leu Cys Pro Val Asp Val Leu Asp Val Pro 180 185 190 Glu Gly Thr Leu Pro Asp Lys Gln Ser Thr Glu Gln Ala Ile Gln Leu 195 200 205 Leu Glu Lys Met Lys Thr Ser Ala Ser Pro Phe Phe Leu Ala Val Gly 210 215 220 Tyr His Lys Pro His Ile Pro Phe Arg Tyr Pro Lys Glu Phe Gln Lys 225 230 235 240 Leu Tyr Pro Leu Glu Asn Ile Thr Leu Ala Pro Asp Pro Glu Val Pro 245 250 255 Asp Gly Leu Pro Pro Val Ala Tyr Asn Pro Trp Met Asp Ile Arg Gln 260 265 270 Arg Glu Asp Val Gln Ala Leu Asn Ile Ser Val Pro Tyr Gly Pro Ile 275 280 285 Pro Val Asp Phe Gln Arg Lys Ile Arg Gln Ser Tyr Phe Ala Ser Val 290 295 300 Ser Tyr Leu Asp Thr Gln Val Gly Arg Leu Leu Ser Ala Leu Asp Asp 305 310 315 320 Leu Gln Leu Ala Asn Ser Thr Ile Ile Ala Phe Thr Ser Asp His Gly 325 330 335 Trp Ala Leu Gly Glu His Gly Glu Trp Ala Lys Tyr Ser Asn Phe Asp 340 345 350 Val Ala Thr His Val Pro Leu Ile Phe Tyr Val Pro Gly Arg Thr Ala 355 360 365 Ser Leu Pro Glu Ala Gly Glu Lys Leu Phe Pro Tyr Leu Asp Pro Phe 370 375 380 Asp Ser Ala Ser Gln Leu Met Glu Pro Gly Arg Gln Ser Met Asp Leu 385 390 395 400 Val Glu Leu Val Ser Leu Phe Pro Thr Leu Ala Gly Leu Ala Gly Leu 405 410 415 Gln Val Pro Pro Arg Cys Pro Val Pro Ser Phe His Val Glu Leu Cys 420 425 430 Arg Glu Gly Lys Asn Leu Leu Lys His Phe Arg Phe Arg Asp Leu Glu 435 440 445 Glu Asp Pro Tyr Leu Pro Gly Asn Pro Arg Glu Leu Ile Ala Tyr Ser 450 455 460 Gln Tyr Pro Arg Pro Ser Asp Ile Pro Gln Trp Asn Ser Asp Lys Pro 465 470 475 480 Ser Leu Lys Asp Ile Lys Ile Met Gly Tyr Ser Ile Arg Thr Ile Asp 485 490 495 Tyr Arg Tyr Thr Val Trp Val Gly Phe Asn Pro Asp Glu Phe Leu Ala 500 505 510 Asn Phe Ser Asp Ile His Ala Gly Glu Leu Tyr Phe Val Asp Ser Asp 515 520 525 Pro Leu Gln Asp His Asn Met Tyr Asn Asp Ser Gln Gly Gly Asp Leu 530 535 540 Phe Gln Leu Leu Met Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 545 550 555 560 Gly Gly Gly Gly Ser Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 565 570 575 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 580 585 590 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 595 600 605 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 610 615 620 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg 625 630 635 640 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 645 650 655 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 660 665 670 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 675 680 685 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 690 695 700 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 705 710 715 720 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 725 730 735 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 740 745 750 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 755 760 765 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 770 775 780 Gly Lys 785 <210> 24 <211> 2310 <212> DNA <213> Artificial Sequence <220> <223> ARSB-Fc <400> 24 atgggtccgc gcggcgcggc gagcttgccc cgaggccccg gtcctcggcg gctgcttctc 60 cccgtcgtcc tcccgctgct gctgctgctg ttgttggcgc cgccgggctc gggcgccggg 120 gccagccggc cgccccacct ggtcttcttg ctggcagacg acctaggctg gaacgacgtc 180 ggcttccacg gctcccgcat ccgcacgccg cacctggacg cgctggcggc cggcggggtg 240 ctcctggaca actactacac gcagccgctg tgcacgccgt cgcggagcca gctgctcact 300 ggccgctacc agatccgtac aggtttacag caccaaataa tctggccctg tcagcccagc 360 tgtgttcctc tggatgaaaa actcctgccc cagctcctaa aagaagcagg ttatactacc 420 catatggtcg gaaaatggca cctgggaatg taccggaaag aatgccttcc aacccgccga 480 ggatttgata cctactttgg atatctcctg ggtagtgaag attattattc ccatgaacgc 540 tgtacattaa ttgacgctct gaatgtcaca cgatgtgctc ttgatttcg agatggcgaa 600 gaagttgcaa caggatataa aaatatgtat tcaacaaaca tattcaccaa aagggctata 660 gccctcataa ctaaccatcc accagagaag cctctgtttc tctaccttgc tctccagtct 720 gtgcatgagc cccttcaggt ccctgaggaa tacttgaagc catatgactt tatccaagac 780 aagaacaggc atcactatgc aggaatggtg tcccttatgg atgaagcagt aggaaatgtc 840 actgcagctt taaaaagcag tgggctctgg aacaacacgg tgttcatctt ttctacagat 900 aacggagggc agactttggc agggggtaat aactggcccc ttcgaggaag aaaatggagc 960 ctgtgggaag gaggcgtccg aggggtgggc tttgtggcaa gccccttgct gaagcagaag ggcgtgaaga accgggagct catccacatc tctgactggc tgccaacact cgtgaagctg gccaggggac acaccaatgg cacaaagcct ctggatggct tcgacgtgtg gaaaaccatc agtgaagga gcccatcccc cagaattgag ctactgcata atattgaccc gaacttcgtg gactcttcac cgtgtcccag gaacagcatg gctccagcaa aggatgactc ttctcttcca gaatattcag cctttaacac atctgtccat gctgcaatta gacatggaa ttggaaactc ctcacgggct acccaggctg tggttactgg ttccctccac cgtctcaata caatgtttct gagataccct catcagaccc accaaccaag accctctggc tctttgatat tgatcgggac cctgaagaaa gacatgacct gtccagagaa tatcctcaca tcgtcacaaa gctcctgtcc cgcctacagt tctaccataa acactcagtc cccgtgtact tccctgcaca ggacccccgc 1560 tgtgatccca aggccactgg ggtgtggggc ccttggatgg gtggaggcgg ttcaggcgga 1620 ggtggctctg gcggtggcgg atcgccatca tgcccagcac ctgagttcct ggggggacca 1680 tcagtcttcc tgttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 1740 tcagtcttcc tgttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 1740 gtcacatgcg tggtggtgga cgtgagccag gaagaccctg aggtccagtt caactggtac 1800 gtcacatgcg tggtggtgga cgtgagccag gaagaccctg aggtccagtt caactggtac 1800 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 1860 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 1860 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1920 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1920 tacaagtgca aggtctccaa caaaggcctc ccatcctcca tcgagaaaac catctccaaa 1980 tacaagtgca aggtctccaa caaaggcctc ccatcctcca tcgagaaaac catctccaaa 1980 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatccca ggaggagatg 2040 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatccca ggaggagatg 2040 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 2100 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 2100 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 2160 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 2160 gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 2220 gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 2220 gaggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 2280 gaggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 2280 aagagcctct ccctgtctct gggtaaatga 2310 aagagcctct ccctgtctct gggtaaatga 2310 <210> 25 <210> 25 <211> 769 <211> 769 <212> PRT <212> PRT <213> 人工序列 <213> Artificial sequence <220> <220> <223> ARSB-Fc <223> ARSB-Fc <400> 25 Met Gly Pro Arg Gly Ala Ala Ser Leu Pro Arg Gly Pro Gly Pro Arg 1 5 10 15 Arg Leu Leu Leu Pro Val Val Leu Pro Leu Leu Leu Leu Leu Leu Leu 20 25 30 Ala Pro Pro Gly Ser Gly Ala Gly Ala Ser Arg Pro Pro His Leu Val 35 40 45 Phe Leu Leu Ala Asp Asp Leu Gly Trp Asn Asp Val Gly Phe His Gly 50 55 60 Ser Arg Ile Arg Thr Pro His Leu Asp Ala Leu Ala Ala Gly Gly Val 65 70 75 80 Leu Leu Asp Asn Tyr Tyr Thr Gln Pro Leu Cys Thr Pro Ser Arg Ser 85 90 95 Gln Leu Leu Thr Gly Arg Tyr Gln Ile Arg Thr Gly Leu Gln His Gln 100 105 110 Ile Ile Trp Pro Cys Gln Pro Ser Cys Val Pro Leu Asp Glu Lys Leu 115 120 125 Leu Pro Gln Leu Leu Lys Glu Ala Gly Tyr Thr Thr His Met Val Gly 130 135 140 Lys Trp His Leu Gly Met Tyr Arg Lys Glu Cys Leu Pro Thr Arg Arg 145 150 155 160 Gly Phe Asp Thr Tyr Phe Gly Tyr Leu Leu Gly Ser Glu Asp Tyr Tyr 165 170 175 Ser His Glu Arg Cys Thr Leu Ile Asp Ala Leu Asn Val Thr Arg Cys 180 185 190 Ala Leu Asp Phe Arg Asp Gly Glu Glu Val Ala Thr Gly Tyr Lys Asn 195 200 205 Met Tyr Ser Thr Asn Ile Phe Thr Lys Arg Ala Ile Ala Leu Ile Thr 210 215 220 Asn His Pro Pro Glu Lys Pro Leu Phe Leu Tyr Leu Ala Leu Gln Ser 225 230 235 240 Val His Glu Pro Leu Gln Val Pro Glu Glu Tyr Leu Lys Pro Tyr Asp 245 250 255 Phe Ile Gln Asp Lys Asn Arg His His Tyr Ala Gly Met Val Ser Leu 260 265 270 Met Asp Glu Ala Val Gly Asn Val Thr Ala Ala Leu Lys Ser Ser Gly 275 280 285 Leu Trp Asn Asn Thr Val Phe Ile Phe Ser Thr Asp Asn Gly Gly Gln 290 295 300 Thr Leu Ala Gly Gly Asn Asn Trp Pro Leu Arg Gly Arg Lys Trp Ser 305 310 315 320 Leu Trp Glu Gly Gly Val Arg Gly Val Gly Phe Val Ala Ser Pro Leu 325 330 335 Leu Lys Gln Lys Gly Val Lys Asn Arg Glu Leu Ile His Ile Ser Asp 340 345 350 Trp Leu Pro Thr Leu Val Lys Leu Ala Arg Gly His Thr Asn Gly Thr 355 360 365 Lys Pro Leu Asp Gly Phe Asp Val Trp Lys Thr Ile Ser Glu Gly Ser 370 375 380 Pro Ser Pro Arg Ile Glu Leu Leu His Asn Ile Asp Pro Asn Phe Val 385 390 395 400 Asp Ser Ser Pro Cys Pro Arg Asn Ser Met Ala Pro Ala Lys Asp Asp 405 410 415 Ser Ser Leu Pro Glu Tyr Ser Ala Phe Asn Thr Ser Val His Ala Ala 420 425 430 Ile Arg His Gly Asn Trp Lys Leu Leu Thr Gly Tyr Pro Gly Cys Gly 435 440 445 Tyr Trp Phe Pro Pro Pro Ser Gln Tyr Asn Val Ser Glu Ile Pro Ser 450 455 460 Ser Asp Pro Pro Thr Lys Thr Leu Trp Leu Phe Asp Ile Asp Arg Asp 465 470 475 480 Pro Glu Glu Arg His Asp Leu Ser Arg Glu Tyr Pro His Ile Val Thr 485 490 495 Lys Leu Leu Ser Arg Leu Gln Phe Tyr His Lys His Ser Val Pro Val 500 505 510 Tyr Phe Pro Ala Gln Asp Pro Arg Cys Asp Pro Lys Ala Thr Gly Val 515 520 525 Trp Gly Pro Trp Met Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 530 535 540 Gly Gly Gly Ser Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 545 550 555 560 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 565 570 575 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 580 585 590 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 595 600 605 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val 610 615 620 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 625 630 635 640 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 645 650 655 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 660 665 670 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 675 680 685 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 690 695 700 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 705 710 715 720 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 725 730 735 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 740 745 750 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 755 760 765 Lys <210> 26 <211> 12 <212> PRT <213> Homo sapiens <400> 26 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10

Claims

1. An enzyme fusion protein, wherein an immunoglobulin Fc region is fused to a therapeutic enzyme, and the therapeutic enzyme has an increased in vivo duration compared to the therapeutic enzyme not fused to the immunoglobulin Fc region, wherein there is no chain exchange in the immunoglobulin Fc region, Compared with the therapeutic enzyme not fused with the immunoglobulin Fc region, the enzyme fusion protein has increased stability and reduced binding affinity to lysosomal receptors, thereby having a high tissue distribution, wherein the enzyme is iduronate-2-sulfatase (IDS) or arylsulfatase B (ARSB), The enzyme fusion protein is the amino sequence of SEQ ID NO: 23 or 25.

2. The enzyme fusion protein according to claim 1, wherein the enzyme fusion protein is a fusion of one molecule of immunoglobulin Fc region and a dimeric therapeutic enzyme. The enzyme fusion protein according to claim 1 , wherein the immunoglobulin Fc region is non-glycosylated.

4. Use of the enzyme fusion protein according to any one of claims 1 to 3 in the preparation of a medicament for preventing or treating a lysosomal storage disease (LSD), wherein the lysosomal storage disease (LSD) is selected from mucopolysaccharidosis (MPS) and Hunter syndrome, The enzyme is iduronate-2-sulfatase (IDS) or aromatic sulfatase B (ARSB), and when the enzyme is iduronate-2-sulfatase (IDS), the drug is a drug for preventing or treating Hunter syndrome, and when the enzyme is aromatic sulfatase B (ARSB), the drug is a drug for preventing or treating mucopolysaccharidosis (MPS).

5. The use according to claim 4, wherein the composition reduces the binding affinity of the enzyme to the lysosomal receptor. 6 . A polynucleotide encoding the enzyme fusion protein according to claim 1 .

7. An expression vector comprising the polynucleotide of claim 6.

8. A transformant into which the expression vector according to claim 7 is introduced, wherein the transformant is a non-animal or plant species.

9. A method for preparing an enzyme fusion protein, comprising: (a) culturing the transformant according to claim 8 to obtain a culture; and (b) recovering the enzyme fusion protein from the culture.

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