WO2019124973A1 - Protein conjugate and fusion protein which comprise albumin and lysosomal enzyme - Google Patents

Abstract

The present invention relates to a protein conjugate and a fusion protein which comprise albumin and a lysosomal enzyme. A protein conjugate or a fusion protein, of the present invention, in which albumin and IDS are bound, has long in vivo persistence, thereby enabling the administration period to be extended more than that of a currently used therapeutic agent. In addition, the protein conjugate or the fusion protein is developed in a dosage form, which can be injected subcutaneously, and can effectively reduce accumulated glycosaminoglycan (GAG) in urine and tissues even when administered subcutaneously. Therefore, a protein conjugate or a fusion protein, of the present invention, in which albumin and IDS are bound, can be effectively used as an agent for treating lysosomal storage disorders.

Description

 TECHNICAL FIELD The present invention relates to protein conjugates and fusion proteins including albumin and lysosomal enzymes. BACKGROUND OF THE INVENTION 1. Field of the Invention [0001]

 The present invention relates to protein conjugates and fusion proteins comprising albumin and a lysosomal enzyme. More particularly, the invention relates to a protein gal conjugate or fusion protein comprising albumin and an euduronate-2-sulfatase. Background technology

 Hunter syndrome or type 2 mucosal saccharosis (or type 2 mucosal saccharosis)

(Mucopolysaccharidosi s type II) has been reported to accumulate in lysosomes due to the lack of degradation of mucopolysaccharides such as glycosaminoglycan (GAG) due to the deficiency of iduronate-2-sulfatase (Iduronate-2-sul fatase, IDS) Lysosomal storage diseases (LSD) are among the most common. GAG accumulates in all cells of the body and causes various symptoms. These symptoms include heaviness, swelling of the abdomen due to hypertrophy of the hepatic nervosa, and hearing loss, heart valve disease, obstructive respiratory disease, and sleep apnea. .

 Hunter syndrome is a recessive hereditary disease caused by abnormalities of the sex chromosome (Xq28.1 locus), which is reported to occur at a frequency of approximately 1 in every 200,000 people. In June 2006, Shire's Elaprase (R) was first approved as an alternative therapy (ERT). In 2012, Hunterase® was developed in the world's second-largest Green Cross. Currently, we visit the hospital every week for the substitute therapy of the enzyme, and the IDS protein is diluted in physiological saline at a dose of 0.5 mg / kg and infused for 2 to 3 hours. However, because of the nature of the disease that requires treatment of such treatment, it is costly and time-consuming to patients and caregivers. Therefore, if the number of drug injections is reduced or a hypodermic injection is developed,

On the other hand, albumin has a long half-life characteristic similar to that of an antibody. It is also known to have relatively low immunogenicity (immiinogenicity) 2019/124973 1 (1 ^ {2018/016240). In the current market (CSL's Idelvion®, Eperzan®, etc.) products that integrate with albumin in the development of medicines are being marketed, and albumin is bound to the drug as a delirium vehicle (Cel lgene abraxane ®) is currently in use.

 Accordingly, the present inventors intend to develop a next-generation enzyme-based therapeutic agent capable of subcutaneous administration and having an extended administration period (Biweekly or Monthly). Technical Challenge

 The inventors of the present invention have conducted studies to develop a therapeutic agent for Hunter's syndrome with improved persistence in the body. As a result, the present inventors have found that a protein conjugate (albuminated IDS, ALBIDS) or a fusion protein (albuminated IDS, ALB IDS fusion) Confirming that the persistence of the body is improved, thereby completing the present invention. Task solution

 One aspect of the present invention provides a protein conjugate having the following general formula (1) or general formula (2).

 [Formula 1]

 Ai-LrXi

 [General expression

X _] _Lr ^~ Ai

 In this formula,

 Ai is an albumin or albumin derivative (derivat ives);

 Is a linker;

 ¾ is a lysosomal enzyme.

 Another aspect of the present invention provides a pharmaceutical composition for preventing or treating Hunter's syndrome comprising the protein conjugate as an active ingredient.

 Another aspect of the present invention provides a method for preventing or treating Hunter's syndrome comprising administering the protein conjugate to a subject.

Yet another aspect of the present invention provides a fusion protein comprising an albumin or albumin derivative and a lysosomal enzyme. Another aspect of the present invention provides a polynucleotide encoding said fusion protein.

 Another aspect of the present invention provides an expression vector comprising the polynucleotide.

 Another aspect of the present invention provides a transformant transformant into which the above expression vector has been introduced and transformed.

 Another aspect of the present invention is a method for culturing a transformant, comprising culturing the transformant to obtain a culture; And recovering the fusion protein from the culture.

 Another aspect of the present invention provides a pharmaceutical composition for preventing or treating Hunter's syndrome comprising the fusion protein as an active ingredient.

 Yet another aspect of the present invention provides a method of preventing or treating Hunter's syndrome comprising administering the fusion protein to a subject. Effects of the Invention

The albumin-bound protein conjugate or fusion protein according to the present invention has a long persistence in the body and can prolong the administration period of the therapeutic agent currently used. In addition, the protein conjugate or fusion protein was developed as a subcutaneous injectable formulation, and glycosaminoglycan 1 (: ₀₃₃ ) ₁₁₀ 1 3 ₁₁ accumulated in the urine and tissues can be efficiently reduced through subcutaneous administration . Therefore, the protein conjugate or fusion protein of albumin and first order fusion of the present invention can be usefully used as a treatment agent for lysosomal accumulation diseases. Brief Description of Drawings

 FIG. 1 is a diagram showing a protein conjugate and a fusion protein which are combined with a silver-binding albumin and a first-order fusion,

 FIG. 2 is a schematic diagram showing a process for preparing a protein conjugate having a 1-mer structure with recombinant albumin.

Fig. 3 is a graph showing the activity of a protein conjugate (monovalent) bound to silver-binding albumin. Fig. FIG. 4 is an analysis of the protein conjugate bound to recombinant albumin and monovalent protein. FIG.

 FIG. 5 is a graphical representation of a functional group in which a protein conjugate or fusion protein bound to recombinant albumin is increased in half-life by the receptor. FIG. 6 is a graph showing the concentration of mouse 105 in blood after administration of a protein conjugate conjugated with recombinant albumin in a mouse. FIG.

FIG. 7 is a cross-

And the recombinant albumin

And the molecular weight of the coupled protein conjugate is measured.

 8 is an isoelectric point analysis of the protein conjugate of recombinant albumin and first order fusion protein.

Figure 9 is a graph showing the effect of recombinant human serum albumin or recombinant mouse serum albumin

FIG. 2 is a graph showing the content of 1-amino acid that is absorbed into a cell of a mouse skin fibroblast treated with a conjugated protein conjugate. FIG.

 FIG. 10 is a graph showing the content of intracellularly absorbed 1 after treating a skin fibroblast of a patient suffering from Hunter's syndrome with a protein conjugate conjugated with recombinant human serum albumin or mouse serum albumin.

FIG. 11 shows the results of the administration of 1 well or conjugate of recombinant albumin and 1 order to iduronate-2-sulfatase knockout mouse 0 (0) or normal mouse at week intervals, followed by 25 days Urine glycosaminoglycans (for example, _{0331 110} , 1 3 _{11, and} urine excretion amount

 FIG. 12 shows a graph showing the results of immunohistochemical analysis after one week has elapsed after administering a protein conjugate bound unilaterally to recombinant albumin or ichiyoshi firstly to iduronate-2-sulfatase knockout mouse (1) or normal mouse , And the activity of one function in the liver of each mouse.

 FIG. 13 shows the results of immunohistochemical staining of the mouse kidney of each mouse when one week was elapsed after administration of a protein conjugate in which 1 or recombinant albumin and 1 protein were bound to normal mice or dyrupate-2-sulfatase knockout mouse 1 This is a comparative analysis of the activity.

14 shows the results of administration of a conjugate of protein conjugated with monoclonal antibody or recombinant albumin to iduronate-2-sulfatase knockout mouse or normal mouse This is a comparative analysis of the activity of IDS in the lung tissues of each mouse after 1 week after 2019/124973 1: 1 {2018/016240.

 FIG. 15 is a diagram showing an animal experiment schedule using this duroate-2-sulfatase knockout mouse for confirming the amount of GAG released into the urine when the recombinant albumin-IDS-conjugated protein conjugate is administered every two weeks .

 FIG. 16 is a graph showing changes in creatinine released into the urine after administration of IDU or recombinant albumin and IDS-conjugated protein conjugate to iduronate-2-sulfatase knockout mouse (K0) (G GAG / g creatinine) corrected for the amount of the GAG.

 FIG. 17 is a diagram illustrating an animal experiment schedule using the duroate-2-sulfatase knockout mouse to confirm the amount of GAG accumulated in each organ when the protein conjugate of the combination of albumin and IDS is administered every two weeks.

 FIG. 18 is a graph showing the effect of the IDS or recombinant albumin-IDS-conjugated protein conjugate on the euduronate-2-sulfatase knockout mouse (K0) or normal mouse (1 or 2 weeks) And the accumulated amount of GAG and FGF after liver was sacrificed.

 FIG. 19 is a graph showing the results of immunohistochemical staining of IDU-conjugated albumin and IDS-conjugated protein conjugate to iduronate-2-sulfatase knockout mouse (K0) or normal mouse (WT) In which the mouse was sacrificed and kidney was collected and the accumulated amount of GAG total protein was measured.

 FIG. 20 shows the results of immunohistochemical staining of IDU or recombinant albumin and protein conjugate conjugated with IDS to iduronate-2-sulfatase knockout mouse (K0) or normal mouse (WT) FIG. 2 is a graph showing the amount of accumulated GAG and FGF after collecting the lungs at the sacrifice of a mouse.

 FIG. 21 shows an animal experiment schedule using this duroate-2-sulfatase knockout mouse to confirm the amount of GAG released in the small intestine or the amount of GAG accumulated in each organ upon single administration of the protein conjugate conjugated with the recombinant albumin and IDS Fig. 22 is a graph showing the amount of GAG in the urine when a single dose of recombinant albumin and IDS-conjugated protein conjugate is administered at a dose of 1 rag / kg.

Figure 23 shows that when a single dose of IDS is administered at a dose of 1 mg / kg, 2019/124973 1: (1 1 2018/016240).

Is a drawing caused by.

FIG. 26 is a graph showing the results of immunohistochemical staining for durotonate-2-sulfatase knockout mouse)

FIG.

FIG. 27 shows the results of repeated administration of the protein conjugate of the combination of albumin and 1-mer.

-2-sulfatase knockout mouse. Fig. 28 is a _{graph showing} the relationship between recombinant albumin and 1-mer protein binding at 1 /

Is a drawing caused by.

 FIG. 32 shows the results of a protein binding assay in which a recombinant albumin and a first order fusion protein

Is a drawing caused by.

FIG. 36 shows the results of repeated administration of a protein conjugate of 1 or recombinant albumin and 1 < RTI ID = 0.0 > Nuclease < / RTI > conjugated to chimeric-2-sulfatase knockout mouse 2019/124973 1: (: 1 ^ {2018/016240 After four months, it is the measurement of the amount of accumulated GAG total amount of the liver after taking the liver at the sacrifice of the mouse.

 FIG. 37 is a graph showing the presence or absence of ADA (antidrug antibody) production by repeated administration of recombinant albumin and IDS-conjugated protein conjugate.

 FIG. 38 is a diagram showing six kinds of fusion proteins in which recombinant albumin and IDS are combined.

 39 is a diagram showing a process of purifying and separating six types of fusion proteins in which recombinant albumin and IDS are bound.

 40 is an S-PAGE analysis of six kinds of fusion proteins in which IDS and recombinant albumin are bound to IDS.

 FIG. 41 is a view showing Western blotting using six anti-IDS antibodies or anti-human serum recombinant albumin antibodies against six kinds of IDS-conjugated fusion proteins with IDS and recombinant albumin. Best Mode for Carrying Out the Invention

 Hereinafter, the present invention will be described in detail.

 One aspect of the present invention provides a protein conjugate having the following general formula (1) or general formula (2).

 [Formula 1]

 Ai-Li-Xi

 [General expression

 Xi-Li-Ai

 In this formula,

 Ai is an albumin or albumin derivative (derivat ives);

 Is a linker;

 Xi is a lysosomal enzyme.

The lysosomal enzyme may be selected from the group consisting of iduronate-2-sul fatase (IDS), beta-gal actosidase, Galactose-6-sulphatase, Beta-glucuronidase, N-acetylgalactosamine-6 sul fatase, N-acetylgalactosamine- Glucocerebrosidase, alpha-galactosidase A, alpha-L-iduronidase (alpha-L- iduronidase, Alpha-N-acetylglucosaminidase Heparai-alpha-glucosaminide, N-acetyl transferase, N-acetyl N-acetylglucosamine 6- sul fatase, hyaluronidase, and the like. Preferably, the lysosomal enzyme may be the duroate 2-sulfatase.

 The IDS is an enzyme involved in the degradation of heparan sulfate and dermatan sulfate, which can lead to Hunter's syndrome or type 2 mucosal saccharification upon IDS deficiency. The IDS may have the amino acid sequence of SEQ ID NO: 2 and may have an amino acid sequence having 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO: 2.

 The albumin may be serum albumin or recombinant albumin. The serum albumin is albumin in the plasma, accounting for more than 50% of plasma proteins. In addition, serum albumin is produced in the liver and is involved in the transport of water or metabolites in tissues. Specifically, serum albumin plays an important role in controlling blood volume by maintaining colloid osmotic pressure. In addition, serum albumin contains some drugs such as water-soluble hormones, bile salts, unconjugated bilirubin, free fatty acids (apoprotein), calcium, ions (transferrin) and warfarin, . On the other hand, albumin is naturally mutated frequently, and there are 79 albumin mutants found so far [Hum Mutat. 2008 Aug; 29 (8): 1007-16). In addition, competition between drugs for the albumin binding site increases the free fraction of one of the drugs, thereby affecting drug efficacy, leading to drug interactions.

The serum albumin may be human serum albumin (HSA) or mouse serum albumin, but is not limited thereto. In addition, the albumin may include an amino acid represented by SEQ ID NO: 1. In addition, the albumin derivative may have 90% or more homology with the amino acid sequence of SEQ ID NO: 1. Specifically, the albumin derivative may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO: . 2019/124973 1 »(: 1 ^ 1 {2018/016240

Bacterial cells such as Salmonella typhimurium C? / _{/ ¾3} ; ₃ e // a typhimurium); Yeast cells such as Saccharomyces cerevisiae, Schi zosaccharomyces pombe); Fungal cells such as P / c / z / a pastor is; Insect cells such as Drosophila (Z? Rosop / z // a), Spodoptera frugiperidae now USpodoptera frugiperda cel l / Sf9 cel l); CHO, COS, NSO,

And albumin produced through gene recombination from plant cells such as Oriza japonica.

The linker may be a polyethylene glycol (PEG) having a size of more than 0 kDa and not more than 5 kDa. Preferably, the linker is more than 0 kDa to less than 5 kDa, more than 0.1 kDa to less than 4.9 kDa, more than 0.2 kDa to less than 4.8 kDa, more than 0.3 kDa to less than 4.7 kDa, more than 0.4 kDa _. From about 0.4 kDa or more to about 4.3 kDa, from about 0.8 kDa to about 4.2 kDa, from about 0.9 kDa or more to about 4. 1 kDa or less, or from 1 kDa or more to about 4.0 kDa or less To 4 kDa or less; Not less than 1 kDa and not more than 3.9 kDa, not less than 1.2 kDa but not more than 3.8 kDa, not less than 1.3 kDa and not more than 3.7 kDa, not less than 1.4 kDa,

3.6 kDa or less, and 1.5 kDa or more to 3.5 kDa or less. Preferably 3.4

 Specifically, the protein conjugate may be albumin and IDS bound through a linker.

One end of the linker can form an amide bond with the IDS. At this time, the amide bond may be bonded through an NHS ester amine reaction. The NHS ester amine reaction is carried out by reacting N-hydroxysuccinic de-ester with primary amines under physiological conditions or weakly alkaline conditions (pH 7.2 to 9). Specifically, the amide bond may be a primary amine, which is a residue in a lysine among the amino acids constituting the IDS, via a NHS ester amine reaction. At this time, the amide bond constitutes IDS and can be formed from any one of 19 lysines in amino acids. Specifically, 2019/124973 1 (1: 1) 2018/016240 The combination is selected from the group consisting of 57th, 124th, 164th, 169th, 199th, 211th, 213th, 236th, 240th, 295th, 376th, 436th, 440th, 483th or 486th position of the lysine residue.

 In addition, the other end of the linker may form a thioether bond with albumin. At this time, the thioether bond may be bonded through maleimide thiol reacting. Specifically, the maleimide thiol reaction is carried out by reacting with a SH group (sul fhydryl group) under a maleimide group weighted pH condition (pH 6.5 to 7).

 Specifically, the thioether bond may be a bond in an amino acid-rich cysteine constituting albumin through a maleimide thiol reaction. At this time, the thioether bond may be formed in one of the 36 cysteines in the amino acid constituting albumin. Specifically, the thioether linkage can be made in one free cysteine (C34) naturally present in the albumin domain I.

 In one embodiment, the protein conjugate of the invention comprises: i) reacting with a primary amine group in the lysine of IDS and an NHS ester at the PEG terminus to form a stable amide bond; And i) adding serum albumin to prepare a stable thioether bond by reacting the maleimide group at the other PEG end with the anticipation of free cysteine of albumin.

 The protein conjugate may be one to fifteen albumin combined with IDS. Specifically, the protein conjugate may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, It may be that albumin is bound to IDS. Preferably, the protein conjugate may be one or two albumin conjugated to IDS.

In one embodiment, the protein conjugate may be one in which one IDS and one albumin are bound (IDS-PEG-albumin). In addition, the protein conjugate may be one in which one IDS and two albumin are bound (IDS-PEG- (albumin) ₂₎ . In one embodiment, the protein conjugate is prepared in the form of IDS-PEG-albumin and IDS-PEG- (albumin) ₂ at a ratio of 3: 1 when prepared via steps i) and ii) . The present inventors have studied to develop a therapeutic agent for type 2 mucosal saccharosis or Hunter's syndrome disease with increased persistence and stability. As a result, Protein conjugate was prepared (Fig. 7). In addition, 1 activity of the protein conjugate prepared above was measured, and it was confirmed that the 1 potential activity was maintained (FIG. 3). Further, the protein conjugate prepared above was administered to the euduronate-2-sulfatase knockout mouse at various intervals, and as a result,

And the amount decreased. In particular, it was confirmed that the administration frequency was increased and the number of administration was decreased, but it was effectively reduced in the body (Figs. 11 to 36).

 The protein conjugate of the present invention is a recombinant protein that is monovalently linked to albumin, and it has been confirmed that the protein conjugate is superior to the conventional one in terms of persistence and 1 activity in the body. Therefore, the protein conjugate can be used as an active ingredient of a pharmaceutical composition for preventing or treating Hunter's syndrome.

 Another aspect of the present invention provides a pharmaceutical composition for preventing or treating Hunter's syndrome comprising the protein conjugate as an active ingredient.

At this time, the protein conjugate

(Albumin) _2, or a combination thereof. The protein conjugate according to the present invention, which is an active ingredient, may be contained in an amount of 10 to 95% by weight based on the total weight of the pharmaceutical composition. The pharmaceutical composition of the present invention may be formulated to contain at least one pharmaceutically acceptable carrier in addition to the active ingredient described above for administration.

 The dosage of the pharmaceutical composition may vary depending on factors such as the type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the form and the kind, and the patient's age, weight, general health status, sex and diet, The route and fraction of the composition, the duration of the treatment, the drug being co-administered, and the like. In addition, the pharmaceutical composition may be administered to a subject by various methods known in the art. The administration route can be appropriately selected by a person skilled in the art in consideration of the administration method, the volume of the body fluid, the viscosity, and the like. The pharmaceutical composition may be administered to a patient suffering from Hunter's syndrome according to known methods.

 Yet another aspect of the present invention provides a method for preventing or treating Hunter's syndrome comprising administering the protein conjugate to a subject.

The protein conjugate is the same as described above. The object The subject may be a mammal, including Hunter syndrome. The term " Hunter "

 Such administration can be by intravenous, subcutaneous, intradermal, intramuscular, intranasal, intracerebral or intrathecal administration. In addition, the administration can be administered once every three months, once every two months, once every month, once every three weeks, once every two weeks, or once a week.

 Yet another aspect of the present invention provides a fusion protein comprising an albumin or albumin derivative and a lysosomal enzyme.

 The fusion protein may have any structure selected from the following structures:

_{A 2 -X 2, X 2 ~} A 2, A 2- L 2 -X 2, X 2 ~ L 2 _ A 2, X 2 _ A 2 _ X2, k fd 2 _1 theta父 _2, 父₂ - Shows ₂ _ 7 ₂ -

X3 ₂ , Father ₂ ^_ 1 ₂ _ Public ₂ - X ₂ , ₂ -1 - ₂ ᄀ ¾3, Show ₂ - Father ₂ ^_ 1 - Show 3, ᅀ ₂ -1 ᅥ ⁽ ₂ ^_ 1 Crave 3;

A ₂ or A ₃ is an independent albumin or albumin derivative, respectively;

L ₂ or L ₃ are each independently a linker;

 Is a lysosomal enzyme.

 The albumin, the albumin derivative and the lysosomal enzyme are the same as described above. Specifically, the lysosomal enzyme may be an iduronate-2-sulfatase (IDS). The IDS is the same as described above.

 The fusion protein may be one in which albumin and IDS are directly linked. In addition, the linker may be a peptide linker. Specifically, the peptide linker may be composed of 1 to 100, 3 to 50 or 5 to 40 amino acids.

 The peptide linker may be a non-flexible linker or a flexible linker. The peptide linker may be a non-flexible linker, a flexible linker, a cleavable linker, or a dipeptide, either singly or in combination. Specifically, the peptide linker may have any one of the amino acid sequences of SEQ ID NOS: 4-6.

Specifically, the non-compliant linker may have an amino acid sequence of A (EMAK) nA, wherein n may be an integer of 1 to 5. The non-compliant linker may be one having an amino acid sequence of PAPAP or (XP) n. At this time, X is May be alanine (Ala), lysine (Lys) or glutamic acid (Glu), and the above may be an integer of 5 to 17. In one embodiment, the non-compliant linker may comprise the amino acid sequence of SEQ ID NO: 4 or 5.

 The flexible linker may have an amino acid sequence of (GGGGS) n, wherein n may be an integer of 1 to 5. In addition, the flexible linker may have an amino acid sequence of (G) n, wherein n may be an integer of 6 to 8. In one embodiment, the flexible linker may comprise the amino acid sequence of SEQ ID NO: 6.

 The peptide linker may be one comprising a cleavable linker. The cleavable linker may be a cathepsin B cleavage sequence, a Furin cleavage motif sequence or a Thrombin cleavage sequence. The cathepsin B truncation sequence may be one having an amino acid sequence of GFLG (Gly-Phe-Leu-Gly) or FKFL (Phe-Lys-Phe-Leu) specifically degraded to cathepsin B. Furthermore, The purine cleavage sequence may have a sequence of R- (R / K / X) -R comprising a purine cleavage motif, wherein the amino acid may be naturally occurring. The sequence comprising the purine cleavage motif may comprise a sequence of RXRRR, RXKXR or RXR. Further, the thrombin cleavage sequence may be one having the amino acid sequence of AB-Pro-Arg-XY, The A and B may be hydrophobic amino acids, and the X may be a non-acidic amino acid.

 In one embodiment, the cleavable linker may comprise any of the amino acid sequences of SEQ ID NOS: 7 to 12. In addition, the peptide linker may include any one of the amino acid sequences of SEQ ID NOS: 7-12.

The C-terminus of the peptide linker may be linked to the N-terminus of the IDS, and the N-terminus of the peptide linker may be linked to the C-terminus of the albumin. Terminal of the peptide linker, wherein the C-terminus of the peptide linker may be linked to the N-terminus of the albumin. In one embodiment, the fusion protein is selected from the group consisting of IDS-HSA, IDS- (G4S) ₃ -HSA, IDS-A (EMAK) 4A-HSA, HSA- IDS, HSA- (G4S) ) ≪ / RTI > 4A-IDS, 2019/124973 1 (1 ^ {2018/016240 Not limited.

The fusion protein is? A gene the art prepared by a chemical peptide synthesis method known in the art, or encoding the fusion protein 111 _{(1) 0} 1 ₅ sheets _{6 36} ^ ₈₁₁₁

Can be amplified using. An expression vector can be synthesized by a known method using the gene amplified by the above-mentioned method. Alternatively, the synthetic expression vector can be cloned and then expressed in cells.

 Another aspect of the present invention relates to a method for producing the fusion protein using the polynucleotide encoding the fusion protein, the expression vector comprising the polynucleotide, the transformant into which the expression vector is introduced, and the transformant .

Another aspect of the invention provides a polynucleotide encoding said fusion protein. The polynucleotide may comprise a single nucleotide sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 18. Specifically, the polynucleotide is 1 Ah狀I (SEQ ID NO: 13), 1 -½4幻_3-狀show (SEQ ID NO: 14), 1的-此four £始04-狀show (SEQ ID NO: 15), 1 ( SEQ ID NO: _{16),狀½43) 3 -1}此( SEQ ID NO: 17), HSA-a (EAAAK ) 4 a-IDS ( SEQ ID NO: 18) can contain a DNA sequence encoding.

 Also, in the fusion protein, when 1 is preceded by a terminal, 1 may comprise a signal peptide. In this case, the amino acid sequence of one amino acid including the signal peptide is shown in SEQ ID NO: 2. On the other hand, in the fusion protein, when 1 is followed to the 0 terminal, 1 may not include a signal peptide. In this case, the first amino acid sequence that does not include the signal peptide may be the amino acid sequence of SEQ ID NO: 2 in which 25 amino acids have been deleted from the terminal. Specifically, one amino acid sequence that does not contain a signal peptide is shown in SEQ ID NO: 3.

The polynucleotide may be mutated by substitution, deletion, insertion, or a combination thereof, of one or more bases. When prepared by chemical synthesis with a polynucleotide, a synthetic method well known in the art, e.g., literature (此₂₆ 1 ₃ ^^ _81111, is _{0 ½111 1 £ (1 £ 1} 塔1. , 37: 73-127, 1988) can be used, and the triester, phosphite, phosphoramidite and table-phosphate method, And other auto primer methods, oligonucleotide synthesis methods on solid supports, and the like.

 Yet another aspect of the present invention provides an expression vector comprising the polynucleotide.

As used herein, the term " expression vector "refers to a recombinant vector capable of expressing a desired protein in a desired host cell, and includes a necessary regulatory element operatively linked to the expression of the gene insert. The vector may include expression control elements such as initiation codon, termination codon, promoter, operator, etc. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide, It must exhibit an action,

. The promoter of the vector may be constitutive or inducible.

The term "operably "

The nucleic acid expression control sequence and the nucleic acid sequence encoding the desired protein or protein are functionally

11 and ₃ Ding ₆₎ is meant a state in which. For example, a nucleic acid sequence encoding a promoter and a protein or 1/4 peach may be operatively linked to affect the expression of the coding sequence. Operational linkage with an expression vector can be made using recombinant techniques well known in the art, and site-specific cleavage and linkage can be achieved using enzymes generally known in the art.

In addition, the expression vector may include a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture medium _. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include four initiation codons and contiguous sequences. In some cases, an exogenous translational control signal, which may include the initiation codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of appropriate transcriptional or translational enhancers.

Wherein the expression vector comprises a polynucleotide encoding the fusion protein 2019/124973 1 (1 ^ {2018/016240). Here, the vector used is not particularly limited as long as it can produce the fusion protein of the present invention. Preferably, the expression vector may be plasmid 0, phage 0, or the like. More preferably, the expression vector comprises the commercially developed plasmid ( ₁ ) X18,

_??? 3.4, etc.), within one derived from E. coli plasmid 060 322 8 John _{325, 1) 1018, p \} ] Cll9 etc.), Bacillus subtilis-derived plasmids _(> 1] 6110, 5, etc.), yeast-derived plasmids ₁ , 13, YEp24, ¥ 0 _{? 50,} etc.), phage (幻 3 !, ¾3】 "0112:

入· 10,入1: 11 ,入· etc.), animal virus vectors (retrovirus _{10 \} ^ _1113), adenovirus (greater seonhae times), vaccinia virus 0 _\ (; (: ₁₁₁ times), etc.) Insect virus vector (baculovirus 0 _{330110 1113} ), etc.) and the like. Since the amount of expression of the protein and the expression of the expression vector are different depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose _. For example _{, in} an embodiment of the present invention, 0 3.4 vectors with show ₃ (: 1 and Pacl restriction sites were used.

 Yet another aspect of the present invention provides a transformant into which the expression vector is introduced. The transformant can be produced by introducing an expression vector into a host and transforming the vector. In addition, the fusion protein of the present invention can be produced by expressing the polynucleotide contained in the expression vector.

The transformation can be carried out by various methods. Not limited to one that can produce a fusion protein of the present invention, particularly to, particularly, the transformation method ₀₃ 01 ₂ precipitation method, a precipitation method ₀₃ 01 ₂ »(111 11

₃₁₁ 1 £ ₀ (1 ₆ ) 11 _811311311 method by using a reducing material, electric drilling method ( _{616 101} ) _{0 0} 1 ₀₁₁ ), calcium phosphate precipitation method, protoplast fusion method, stirring using silicon carbide fiber Methods, Agrobacterium-mediated transformation,

Transgenic transformation methods, dextran sulfate, lipofectamine and dry / inhibition-mediated transformation methods can be used.

Also, the host cell used for the production of the transformant is not particularly limited as long as it can produce the fusion protein of the present invention. Specifically, the host cell is E. coli. ₀₀ 10, streptomyces, Salmonella typhimurium; Sakaromasse Serebijia, ski survey Karamoisse Pombe, etc. 2019/124973 1 ^»(1 ^ 1 {2018/016240 yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NS0, 293, Bowmanella cells; Or plant cells.

 Yet another aspect of the present invention provides a method for producing the fusion protein. The fusion protein production method comprises the steps of: i) culturing the transformant to obtain a culture; And i) recovering the fusion protein of the present invention from the culture.

 The term "cultivation " of the present invention means a method of growing a microorganism under an appropriately artificially controlled environmental condition.

 The method for culturing the transformant may be carried out using a method well known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing the fusion protein of the present invention. Specifically, the culturing can be continuously performed in a batch process, an injection batch, or a repeated batch or batch fed batch process.

 The medium used for culturing can meet the requirements of a specific strain in an appropriate manner while controlling the temperature, pH and the like under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like. As the carbon source that can be used, a mixed sugar of glucose and xylose can be used as a main carbon source. In addition, carbon sources include sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil and coconut oil, palmitic acid, stearic acid and linoleic acid Alcohols such as fatty acids, glycerol, ethanol, and organic acids such as acetic acid. In addition, the carbon sources may be used individually or as a mixture.

 Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, carcaen hydrolyzate, fish or their degradation products, defatted soybean cake, The nitrogen sources may be used alone or in combination.

The culture medium is supplemented with potassium phosphate, potassium phosphate, 2019/124973 1 (1) {2018/016240 Sodum-containing salts may be included. The number of people that can be used may include potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts. Examples of the inorganic compound include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate. Finally, essential growth substances such as amino acids and vitamins can be added to the medium.

 In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in the culture process in a batch manner, in an oil-feeding manner or in a continuous manner by an appropriate method, but it is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to control the egg of the culture.

In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. An oxygen or oxygen-containing gas (e.g., air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is

To < RTI ID = 0.0 > 370, < / RTI >

 In addition, the step of recovering the fusion protein from the culture can be carried out by a method known in the art. Specifically, the recovering method is not particularly limited as long as it can recover the produced fusion protein of the present invention. Preferably, the recovery method is carried out by centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (eg ammonium sulfate precipitation), chromatography (eg ion exchange, affinity , Hydrophobicity and size exclusion), and the like.

 Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating Hunter's syndrome comprising the fusion protein as an active ingredient.

 The fusion protein is the same as described above.

 At this time, the protein conjugate or fusion protein according to the present invention, which is an active ingredient, may be contained in an amount of 10 to 95% by weight based on the total weight of the pharmaceutical composition. The pharmaceutical composition may be formulated to contain at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration.

The dosage of the pharmaceutical composition may vary depending on the kind of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of the formulation and the age, It can be adjusted according to various factors including weight, general health status, sex and diet, time of administration, route of administration and fraction of composition, duration of treatment, concurrent medication, etc. have. In addition, the pharmaceutical composition may be administered to a subject by various methods known in the art. The administration route can be appropriately selected by a person skilled in the art in consideration of the administration method, the volume of the body fluid, the viscosity, and the like. The pharmaceutical composition may be administered to a patient suffering from Hunter's syndrome according to known methods.

 Yet another aspect of the present invention provides a method of preventing or treating Hunter's syndrome comprising administering the fusion protein to a subject.

 The pharmaceutical composition is the same as described above. The subject may be a mammal, including a human. Specifically, the subject may be a human suffering from Hunter's syndrome.

 Such administration can be by intravenous, subcutaneous, intradermal, intramuscular, intranasal, intracerebral or intrathecal administration. In addition, the administration can be administered once every three months, once every two months, once every month, once every three weeks, once every two weeks, or once a week.

 Another aspect of the present invention provides the use of a protein conjugate of the invention for treating Hunter's syndrome.

 Another aspect of the present invention provides the use of a protein conjugate of the invention for the manufacture of a medicament for the treatment of Hunter's syndrome.

 Another aspect of the invention provides the use of a fusion protein of the invention to treat Hunter's syndrome.

 Another aspect of the present invention provides the use of a fusion protein of the invention for the manufacture of a medicament for the treatment of Hunter's syndrome. DETAILED DESCRIPTION OF THE INVENTION

 Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the present invention is not limited to the following examples. Production Example 1. Production of recombinant albumin and protein conjugate conjugated with iuduronate-2-sulfatase

Recombinant albumin (Sho - 6608, @ 打 !!) and euduronate - 2 - The process of preparing a protein conjugate using PEG (polyethylene glycol) using Iduronate-2-sul fatase (IDS) is shown in FIG. 2 Respectively. At this time, PEG was purchased from Japan's heterofunctional PEGCsunbright MA-034TS).

 First, the primary amine in the lysine of IDS is reacted with the NH-ester (N-hydroxysuccinimide ester) at the end of PEG to form a stable amide bond and then the remaining PEG is removed using anion exchange resin Respectively. After that, a mouse or human serum albumin was added to react the maleimide on one PEG with the SH group of the free cysteine of albumin to form a stable thioether bond. IDS bound to albumin was isolated and purified using anion exchange resin and size exclusion chromatography. Experimental Example 1. Identification of activity of albumin-IDS conjugated protein conjugate

 IDS activity of the protein conjugate prepared in Preparation Example 1 was measured. Specifically, the activity measurement was performed using a synthetic substrate, 4-methylumbelliferyl-L-iduronide-2-sulfate (4-methylumbel liferyl- doA-2S) was reacted with IDS for 4 hours, and the sulfate was firstly liberated from the substrate (first reaction). After the first reaction, aldulazide of Genzyme was added and reacted for 24 hours. Subsequently, the substrate, 4-methylumbelliferyl-L-iduronide (reactant free of sulfate in the first reaction) and the secondary enzyme The reaction was allowed to proceed. As a result, the activity of IDS was evaluated by measuring the intensity of fluorescence of 4-methylumbelliferyl (Ex.355 nm / Em.460 nm) in which L-iduronide was liberated and finally remained.

 As a result, it was confirmed that the activity of the protein conjugate was 96% as compared to the specific activity of IDS, and the activity decrease due to albumin binding was not significant (FIG. 3). Experimental Example 2. Determination of molecular weight of protein conjugate conjugated with albumin and IDS

To determine the molecular weight of protein conjugates conjugated with albumin and IDS, measurements were made using a multi-angle light scattering detector (SEC-MALS). Specifically, 100 samples containing albumin-IDS-conjugated protein conjugate prepared in Preparation Example 1 were subjected to Shimadzu HPLC (manufactured by Shimadzu Corporation) equipped with a TSK-geut G3000SWXL column At a rate of 0.5 M / min. The signals generated by passing through Wyatt DAWN Heleos 11 (18 angles) and Wyatt Optilab T-Rex (RI), a MALS system linked to Shimadzu HPLC, were analyzed using ASTRA 6 software.

The results were very similar to the calculated theoretical molecular weights of the ALBIDS IDS-PEG-albumin protein conjugate. In addition, it was confirmed that the ratio of IDS-PEG-albumin to IDS-PEG- (albumin) ₂ was approximately 3: 1 in a combined form (FIG. 7). Experimental Example 3: Isotope analysis of albumin-IDS conjugated protein conjugate

 The isoelectric point of the albumin-IDS-conjugated protein conjugate prepared in Preparation Example 1 was confirmed. Specifically, the IEF gel forming the pH gradient is called XCel l

Coupled to a SureLock ™ Mini-Cel Electrophoresis System, the upper chamber was filled with 200 m IEF IEF cathode buffer and the lower chamber was filled with 600 M anode buffer (anode buffer) . A mixture of sample 10 containing albumin and the IDS-conjugated protein conjugate and IEF sample buffer 10 was loaded into each well (well) and loaded with 5 IEF marker. Thereafter, the electrophoresis was carried out while varying the voltage in three steps of 100 V / 1 hour, 200 V / 1 hour and 500 V / 30 min, and fixed with 12% TCA solution for 30 minutes. Thereafter, the isoelectric point of the olfactory sample decolored with a destaining solution was determined by staining with coomassie blue staining buffer for 30 minutes or more. At this time, pH 3-10 IEF gel from Invitron was used, and IEF markers 3-10 and SERVA Liquid Mix from SERVA were used as standard markers.

As a result, it was confirmed that the isoelectric point was located between about 4.2 and 4.5 in both the mouse serum albumin (MSA) or the protein conjugate bound to human serum albumin (HSA) and IDS. It was found that it was located between IDS (<3.5) and albumin (~ 5.0) (FIG. 8). Experimental Example 4: In-vitro cel lular of albumin-IDS-conjugated protein conjugate 2019/124973 1 »(: 1 ^ 1 {2018/016240 uptake analysis

 The IDS or albumin and IDS conjugate were diluted by 1 nM to 128 nM concentration, and then treated with cells for a certain period of time to be absorbed into the cells. Then, the content of IDS or albumin and IDS conjugate absorbed into the cells was measured by ELISA. At this time, the saturation curve for the intracellular absorbed content was determined according to the treatment concentration of IDS or albumin and IDS conjugate, and the maximum content (Vmax) of the intracellularly absorbed protein was determined using a saturation curve. (Km, Kuptake) at the rate corresponding to 1/2 of the maximum rate at which a specific substance is absorbed, by converting it into a reciprocal graph of the absorbed content of the IDS or the albumin and the IDS conjugate Respectively.

Specifically, for each ELISA assay, 100 μl each of anti-IDS ant i body (R & D, AF 2449) was added to each well to a final concentration of 1 mg / M in a 96-well plate, . After 16 hours, the solution was removed and washed with IX PBS. A medium for the culture (IMDM, 10 v / v¾ FBS , 1 v / v% Ant i -ant i) normal mouse fibroblasts (NIH / 3T3) or cells of patients with Hunter syndrome (C0RIELL 1 ₁₁₃ 1 to; ₆八Lt; / RTI &gt; 13203). Dissolve 2-M wells to a 12-well-plate at 1.0x10 5 cells / well and incubate at 37 ^° C, 5% CO _{2 .} And cultured in an incubator for 24 hours.

Each IDS or albumin and IDS conjugate was labeled with 128 nM, 64 nM, 32 nM, 16 nM, 8 nM, and 1 nM using dilution media (IMDM, 5 v / 4 nM, 2 nM and 1 nM concentration. After 24 hours, the medium on the plate was removed and IDS or albumin and IDS conjugate at the respective concentrations were added to each well in an amount of 2 _{1 / well} , followed by culturing in an incubator at 37 ^° C and 5% C0 ₂ for 6 hours.

 After 6 hours, the remaining medium was removed and each well was washed once with IX PBS at room temperature. At this time, care was taken so that the cells attached to the well did not fall off. Then, M-PER Mammalian Extract Reagent 200

UJL was added to spread well in the wells and the plates were left in the refrigerator at 2 ^° C to 8 ^° C for 10 minutes to 20 minutes. Thereafter, the cell lysate was collected downward by tilting the plate by about 40 to 50 degrees, and transferred to a new container to conduct ELISA analysis. Specifically, ELISA plates were treated with biotinylated anti-human IDS antibody (R & D, BAF 2449), washed with IX PBS, and then streptoavidin-peroxidase (Sigraa, S-2438 ) And reacted at room temperature for 1 hour. Thereafter, washings (0.05% PBST) were washed four times in 200 wells of each well using a multipipette. After washing, each well was treated with a solution of TMB peroxidase substrate (KPL, 52-00-00), and the concentration was determined by measuring the absorbance at 450 nra wavelength.

 As a result, the amount and activity of IDS were increased in both cells. This confirmed the intracellular absorption capacity of IDS (FIGS. 9 and 10). Experimental Example 5 After administration of albumin and IDS-conjugated protein conjugate,

Confirming the effect of reducing the amount of GAG

 In order to confirm that the amount of GAG in the urine was decreased when the albumin and IDS conjugated protein conjugate were administered once a week, IDU or IDS was added to the iduronate-2-sulfatase knockout mouse (K0) and normal mouse (WT) Albumin and IDS-conjugated protein conjugates (ALBIDS) were administered weekly for one month at a dose of 0.5 mg / kg (based on IDS). Experiments with mice were carried out for 25 days, and urine collection was performed on day 6 after administration of IDS or protein conjugate.

As a result, urinary GAG content after IDS and protein conjugate administration was significantly decreased compared to the knockout (K0) group _. My GAG content decreased to normal mouse level. Thus, it was confirmed that the intragastric GAG content was lowered to the normal range by administering the IDS or protein conjugate to the knockout mouse (FIG. 11). Experimental Example 6. Confirmation of reduction effect of GAG accumulation in tissues after administering albumin and IDS-conjugated protein conjugate

In order to confirm that the amount of GAG accumulation in the tissues decreased when the albumin-IDS-conjugated protein conjugate was administered once a week, iridonate-2-sulfatase knockout mice (K0) and normal mice (WT) Or albumin and IDS-conjugated protein conjugates (ALBIDS) were administered weekly at a dose of 0.5 mg / kg (by IDS) for one month. Tissue harvesting was performed on the 7th day (one week after the last drug administration). The tissues collected were stored in a freezer at a temperature of -80 ^° C.

 The stored tissues were homogenized with homogenizer (BIOPEC PRODUCTS INC. Model 398) and then disrupted using an ultrasonic disrupter (Fischer Scientific Model 500). Thereafter, the amount of GAG accumulation in tissues was measured using sGAG assay kit (BP-004) from K-ASSAY. In addition, total protein content in tissues was analyzed using Pierce BCA assay kit. The amount of GAG measured was corrected by dividing by the total protein amount measured in the same sample.

[Table 1]

As a result, as shown in Table 1,

In the case of iduronate-2-sulfatase knockout mice versus wild-type mice,

The accumulation amount increased. On the other hand, in the mice administered with the first primer or the protein conjugate,

There was a significant decrease in all tissues except the brain. Particularly, in mice administered with the protein conjugate,

Lower. Experimental Example 7. After administration of the albumin and 1 &lt; nd &gt; conjugated protein conjugate,

When the albumin-1 protein-binding protein conjugate was administered weekly, the activity of ioduronate-2-sulfapatase knockout mouse 0 (0) and normal mouse (1) or albumin And 1-mer protein conjugate (此) were administered weekly for one month at a dosage of 0.5 / 1 _{¾ (} 1 weight). The collection of tissue 2019/124973 1: (1 ^ {2018/016240) was performed on the 7th day (one week after the last drug administration). The tissues were stored at -80 ^° C.

 The stored tissues were homogenized with homogenizer (BIOPEC PRODUCTS INC. Model 398) and then disrupted using an ultrasonic disrupter (Fi sher Scientific Model 500). Thereafter, the activity of IDS was measured in the same manner as in Experimental Example 1.

 As a result, the IDS activity in the crushed tissue was highly measured in the group administered the IDS-to-protein conjugate in liver, kidney, and lung tissues. In particular, in the group administered with IDS, in the kidney and lung tissues, IDS activity was hardly observed at one week after administration, whereas in the group administered protein conjugate, tissue IDS activity persisted to a considerable extent (Figs. 12 to 14). Experimental Example 8. Confirmation of the effect of decreasing the amount of GAG in the urine after two weeks administration of albumin and IDS-conjugated protein conjugate

 2-sulfatase knockout mouse (GAG / g creatinine), which was calibrated to the amount of creatinine excreted in the urine when bovine albumin and IDS conjugated protein conjugate were administered every other day K0) and a protein conjugate (ALBIDS) conjugated with IDS or albumin and IDS to normal mice were administered weekly or biweekly at a dose of 0.5 rag / kg (by weight of IDS). The experiment using the mouse was carried out for 60 days, and the urine was collected on the schedule shown in Fig.

 As a result, the amount of GAG (g GAG / g creatinine) corrected by urinary creatinine amount after administration of IDS and protein conjugate was significantly decreased, and in particular, in the group administered with protein conjugate, The amount of corrected GAG (g GAG / g creatinine) was measured. It was confirmed that when the protein conjugate is administered, the administration interval can be increased or the number of administrations can be reduced (FIG. 16). Experimental Example 9. Determination of the effect of decreasing the amount of GAG accumulation in tissues after bi-weekly administration of albumin-IDS conjugated protein conjugate

When the albumin and IDS-conjugated protein conjugate were administered every other day, IDS or albumin and IDS were transfected with iduronate-2-sulfatase knockout mouse (K0) and normal mouse (WT) in order to confirm the amount of accumulated GAG accumulated. The combined protein conjugate (ALBIDS) was administered weekly or biweekly at a dose of 0.5 rag / kg (by weight of IDS). Experiments with mice were carried out for 60 days, and tissue samples were collected on the 7th day (one week after the last drug administration). The collected tissues were stored at a temperature of -80 ° C (Fig. 17).

 The tissues were homogenized with a homogenizer (BIOPEC PRODUCTS INC. Model 398) and then disrupted using an ultrasonic disrupter (FiSer Scientific Model 500). Thereafter, the amount of GAG accumulation in tissues was measured using sGAG assay kit (BP-004) from K-ASSAY. In addition, total protein content in tissues was analyzed using Pierce BCA assay kit. The amount of GAG measured was also divided by the total amount of protein measured. As a result, in the case of a group administered with protein conjugate at a dose of 1.0 and 2.0 mg / kg in the group administered with biweekly IDS or protein conjugate, the GAG amount of the positive control group (weekly, 0.5 mpk IDS) And were measured at similar levels. On the other hand, when the concentration of IDS was doubled to 1.0 mg / kg, GAG accumulation was observed in some organs (lungs) compared to the positive control. Therefore, it was confirmed that the protein conjugate was more effective in reducing the amount of GAG accumulation in the organs than the conventional IDS when administered at intervals of two weeks (FIG. 18 to FIG. 20). However, in the brain, the GAG reduction effect could not be confirmed under the current administration conditions. Experimental Example 10. Determination of the effect of decreasing the amount of GAG in the urine after a single administration of albumin and IDS-conjugated protein conjugate

 To determine the amount of GAG (g GAG / g creatinine) corrected to the amount of creatinine excreted in the urine when albumin and IDS conjugated protein conjugate were administered intravenously, a single dose of iduronate-2-sulfatase GAG reductions were compared for 4 weeks after intravenous administration of a protein conjugate (ALBIDS) conjugated with IDS or albumin and IDS to out-mice (K0) and normal mice (WT). The urine was collected according to the schedule shown in Fig.

Specifically, the current dose of 0.5 mg / kg of IDS and the single dose of 1.0 mg / kg or 2.0 mg / kg of IDS The effect of gag / creatinine (g / g) reduction in the urine of group 1 or group 2 (ALBIDS) was compared by repeated measurement ANOVA.

 There was no difference in the level of GAG released into the urine between the single-protein-conjugated group and the IDS-administered group (4 times) after 4 weeks of observation. On the other hand, the group receiving 1.0 mg / kg of IDS showed a statistically significant increase in GAG levels (p = 0.0227). However, there was no difference in urinary GAG levels between the groups receiving IDS at 2.0 mg / kg (p = 0.053). Particularly, in the case of mice administered with 0.5 mg / kg of IDS, the GAG excretion was significantly decreased after 2 weeks. In addition, the amount of GAG excretion decreased from the first week in mice administered 1.0 mg / kg protein conjugate, and the amount of GAG in the urine was kept low for one month without additional administration in the middle. Thus, it was confirmed that the intracellular GAG content was reduced to a normal range by administering a protein conjugate with albumin and IDS once a month at an appropriate dose (FIGS. 22 to 25). Experimental Example 11. Determination of the effect of decreasing the amount of GAG accumulation in liver tissues after single administration of albumin and IDS-conjugated protein conjugate

2-sulfatase knockout mouse (K0) and normal mouse (WT) were injected intravenously with IDS to determine the amount of GAG accumulated in the tissue when the albumin-IDS conjugated protein conjugate was intravenously administered once. Or albumin and IDS-conjugated protein conjugates (ALBIDS) were intravenously injected for 4 weeks. Liver tissue was collected on day 28. The tissues were stored at -80 ^° C.

 The stored tissues were homogenized with homogenizer (BIOPEC PRODUCTS INC. Model 398) and then disrupted using an ultrasonic disrupter (FiSer Scientific Model 500). Thereafter, the amount of GAG accumulation in tissues was measured using sGAG assay kit (BP-004) from K-ASSAY. In addition, total protein content in tissues was analyzed using Pierce BCA assay kit. The amount of GAG measured was also divided by the total amount of protein measured.

As a result, in the case of a single administration of the protein conjugate, the level of GAG in the positive control group (weekly, 0.5 mpk IDS) in the liver tissue was measured at a level similar to that of the GAG. On the other hand, in the case of a single-group administration of IDS, a GAG The accumulation amount of 2019/124973 was observed (FIG. 26). Experimental Example 12. Determination of the effect of decreasing the amount of GAG in urine after repeated administration of albumin and IDS-conjugated protein conjugate

 When albumin and IDS conjugated protein conjugate were repeatedly administered at monthly intervals for 4 months, the amount of corrected GAG (g GAG / g creatinine) was confirmed by the amount of urine creatinine released. Specifically, the repeated administration of iduronate-2-sulfatase knockout mouse (K0) and normal mouse (IDS or albumin-IDS conjugated protein conjugate (ALBIDS) The urine was collected 3 days before the first administration and 4 months after the administration.

 Specifically, a group of 0.5 mg / kg of IDS, which is currently being used as a clinical dose, was used as a positive control and repeated at a dose of 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg and 4.0 mg / (G / g) reduction in intraperitoneal urine administered IDS or protein conjugates administered intravenously (4 times) were compared by repeated measurement ANOVA.

 Regardless of the dose, there was no difference in the amount of GAG released into the urine compared to the positive control group in the case of the protein conjugate administered repeatedly four times at one month intervals. On the other hand, in IDS-administered groups, the GAG levels in urine were significantly higher in the IDS-treated group than in the positive control group (p <0.05). Thus, it was confirmed that the GAG content in the urine can be maintained within the normal range by administering a protein conjugate having an appropriate dose once a month (MontWy) (FIGS. 28 to 35). Experimental Example 13. Determination of the effect of decreasing the amount of GAG accumulation in liver tissues after repeated administration of albumin and IDS-conjugated protein conjugate

2-sulfatase knockout mouse (K0) and normal mice were injected with IDS (2-mercaptoethanol) to determine the amount of GAG accumulated in the tissues when albumin and IDS-conjugated protein conjugate were repeatedly administered at intervals of one month for 4 months. (ALBIDS) conjugated with albumin and IDS were repeatedly administered at intervals of one month for 4 months. The group receiving the IDS every week for the positive control group (2019/124973) and the group receiving the IDS or protein conjugate every month for 28 days after the last administration At the time of the passage, liver tissues were collected by autopsy and stored in a freezer at -80 ^° C.

 The stored tissue was homogenized with a homogenizer (BIOPEC PRODUCTS INC. Model 398) and then disrupted using an ultrasonic disrupter (Fisher Scientific Model 500). Thereafter, the amount of GAG accumulation in tissues was measured using sGAG assay kit (BP-004) from K-ASSAY. In addition, total protein content in tissues was analyzed using Pierce BCA assay kit. The amount of GAG measured was also divided by the total amount of protein measured. As a result, it was found that the amount of GAG accumulation at the current clinical dose of 0.5 mg / kg IDS was similar to that of GAG accumulation in a group of 2.0 mg / kg or more (in terms of IDS weight) protein conjugate repeatedly administered. On the other hand, in the case of the conventional IDS, even when the administration concentration was 4.0 mg / kg, it was confirmed that GAG was accumulated more than the control group administered every week. As a result, it was confirmed that administration of the protein conjugate was superior to the conventional IDS administration in reducing the GAG in the tissues (FIG. 36). Experimental Example 14. After repeated administration of albumin and IDS-conjugated protein conjugate,

Check for the generation of ADA (anti-drug antibody)

 An experiment was conducted to detect an anti-IDS antibody (anti-IDS ant ibody) that can be generated by repeated administration. When a mouse serum was reacted with a plate coated with IDS, the principle of reacting with coated IDS was used in the presence of an antibody against IDS enzyme in blood.

 Mouse blood obtained by administering PBS, IDS or ALBIDS for 4 months in the same manner as in Example 12 was reacted on a plate coated with IDS, and IDS-peroxidase conjugated body fluid was added thereto. After the addition of the substrate solution, the color reaction was confirmed according to the concentration of the antibody against IDS.

After coating at 4 ° C for 18 hours, the plate was washed six times with physiological saline containing 0.1% Tween 20 (0.1% PBST). Thereafter, the cells were blocked with physiological saline (1% BSAPBS) containing 1% BSA for 1 hour at room temperature, And washed six times with 0.11% PBST.

Each mouse serum and IDS-HRP were dispensed into the coated wells in an IDS-coated well to give a total of 100 in each well. Serum was diluted 10-fold with 1% BSAPBS and reacted at 37 ± 1 ^° C for 2 hours. Finally, the substrate was washed six times with 0.1% PBST and incubated for 30 minutes at room temperature (18 ^° C to 25 ° C). The absorbance was measured at a wavelength of 450 nm and a reference wavelength of 620 nm and was performed within 10 minutes after the reaction was stopped.

 As a result, the anti-hIDS ant i body (BAF2449, R & D system) used as a positive control showed color development at a wavelength of 450 nm due to concentration dependency. On the other hand, it was confirmed that even when the IDS or protein conjugate was repeatedly administered for 4 months, no antibody was generated in the mouse body (FIG. 37). Preparation Example 2. Preparation of albumin-iduronate-2-sulfatase-conjugated fusion protein Preparation Example 2.1 Preparation of fusion protein

 In order to prepare an albumin-iduronate-2-sulfatase conjugated fusion protein, an albumin comprising the amino acid sequence of SEQ ID NO: 1 and a vector expressing an euduronate-2-sulfatase comprising the amino acid sequence of SEQ ID NO: Was used to prepare a fusion protein.

 Various fusion proteins were prepared as shown in Figure 38 to maintain optimal expression and activity. Albumin at the C-terminus of the IDS was directly or indirectly linked to a known labelable inker, G4S l inker or r igid l inker, EAAAK. Separately, a fusion protein linked to IDS was designed using the same linker at the C-terminus of albumin (FIG. 38).

 Gene expression vectors were synthesized to contain Ascl and Pad restriction sites. The expression vector containing the nucleotide sequence encoding the fusion protein was pcDNA 3.4 (ThermoFisher Scientific). In addition, the expression vector contained a signal sequence for secretion of the fusion protein to be synthesized.

 Production Example 2.2 Production of fusion protein

The prepared expression vector was transformed into ExpiCHO-STM cells (ThermoFi sher 2019/124973 1 »(: 1 ^ 1 {2018/016240

Scientific) with Exp i Feet Ami nTM CHO Transfection Kit (ThermoFisher

Scientific) to express the fusion protein.

Specifically, expression of the fusion protein was performed at a level of 30 M based on ExpiCHOTM Expression medium (ThermoFisher Scientific) without animal-derived components. ExpiCHO-STM cells were subcultured to the required number one day before transfection. On the day of transfection, cells were prepared to have a cell number of 6x10 ⁶ cells / m 2. While the expression vector DNA containing the gene encoding the fusion protein, _{30 // g (l // g /} me) into the culture medium and cultured in a few days 8% C0 ₂ and 37 ^° C temperature. After completion of the culturing, the cells were centrifuged and the culture liquid was subjected to purification. The recovered culture supernatant was confirmed by SDS-PAGE and Western blotting using IDS antibody and albumin antibody.

 Production Example 2.3 Purification of Fusion Protein

 Various purification methods can be utilized to purify the various types of fusion proteins produced in Production Example 2.2. The expressed fusion protein is secreted into the medium and can be isolated by an albumin affinity chromatography (CaptureSelect ™ Human albumin affinity matrix, Thermo scientific). In this experiment, purification was carried out by three-step column chromatography.

Specifically, the culture solution was loaded through ion exchange (Q) chromatography, which was equilibrated with 20 mM sodium phosphate buffer (pH 7.2), and then eluted with a 20 mM sodium phosphate buffer containing 0.3 M sodium chloride. Sodium chloride was added to the eluate to prepare a sodium phosphate buffer solution containing 2.0 M sodium chloride. The diluted solution was loaded into a hydrophobic chromatography buffer, butyl sepharose, and the sodium chloride concentration was changed from 2.0 M to 0.0, Respectively. The eluted protein was subjected to size exclusion chromatography using Superdex 200 column and PBS buffer to obtain a fusion protein having a degree of purification of 90% or more (FIG. 39). EXPERIMENTAL EXAMPLE 15 Confirmation of Production of a Fusion Protein Bound by Albumin-iduronate-2-sulfatase SDS-PAGE was performed in order to confirm that the fusion protein bound with iduronate-iduronate-2-sulfatase was properly prepared. The culture supernatant 30 obtained in Production Example 2.2 _! d was mixed with 10 samples of sample buffer containing 5% mercaptoethanol, and then reacted at 951 for 10 minutes. Each sample was then loaded with 15-25 fA on a 4% to 12% Bi s-Tr is gel. Gel electrophoresis was performed for 1 hour and 10 minutes with a 150V voltage (EC250-90, EC apparatus corporation) to separate the proteins according to their sizes and stained with a staining solution (Sun-Gel staining solution, LPS solution) . At this time, electrophoresis was performed using a Novex Xcel l SureLock electrophoresis apparatus (Fig. 40).

As shown in FIG. 40, SDS-PAGE was performed on the culture supernatant. As a result, except for IDS-A (EAAAK) ₄ A-HSA of lane 4, albumin fusion protein was expressed normally in culture medium in CHO cell system .

Each sample was also separated by electrophoresis on a 4% to 12% Bis-Trs gel and transferred to iBlot ™ 2 Transfer stack, PVDF, polyvinylidene membrane (ThermoFisher Scientif ic) The protein was transferred by electrophoresis using an iBlotTM 2 Transfer device (ThermoFisher Scientif ic) on the idene fuoride membrane. Protein-transferred fluorovinylidene membrane was blocked for 30 minutes with 5% skim milk and biotin-conjugated anti-IDS antibody or anti-albumin antibody was added and incubated at 4 ° C Lt; / RTI &gt; overnight. The cells were then washed three times for 10 min each with 0.1% PBST (137 mM NaCl, 2.7 mM KCl, Na2HPO4 10 mM, KH2PO4 1.8 mM, 0.1% Tween 20) buffer and incubated with avidin and horseradish peroxidase in ^° C temperature, the reaction was carried out for 2 hours. After washing three times with 0.1% PBST buffer for 10 minutes, the immunoreacted proteins were visualized using a chemiluminescence detection system (GE Amersham) according to the manufacturer's protocol (FIG. 41). In addition, as shown in FIG. 41, the fusion protein was reacted at the same position by the anti-IDS antibody and the anti-albumin antibody, and the presence of the fusion protein for the two substances was confirmed. Lane 2 was a fusion protein directly linked to IDS-HSA without a linker, and it was confirmed that there were fragments in which a considerable amount of the culture solution was cleaved by IDS and albumin. Other IDS- (G4S) _3- HSA in lane ₃ , HSA- 2019/124973 1 »(: 1/10/06 018/016240

Respectively.

Claims

2019/124973 1 »(: 1 ^ 1 {2018/016240 Patent Claims

 1. A protein conjugate having the following general formula 1 or general formula 2:

 [Formula 1]

 Ai-LrXi

 [General expression

 Xi-Li-Ai

In this formula _,

 Ai is an albumin or albumin derivative (der ivat ives);

 Is a linker;

 ¾ is a lysosomal enzyme.

 2. The method of claim 1,

 The lysosomal enzyme may be selected from the group consisting of iduronate-2-sul fatase (IDS), beta-galactosidase, galactose-6-sulphatase, Beta-glucuronidase, N-acetylgalactosamine-6 sul fatase, glucocerebrosidase,

glucuronidase, glucocerebrosidase, alpha-galactosidase A, alpha-L-auronidase, alpha-N-acetylglucosaminidase, N-acetylglucosaminidase) Heparan-alpha- glucosaminide, N-acetyltransferase, N-acetylghicosamine 6-sul fatase ), Hyaluronidase

(hyaluronidase). &lt; / RTI &gt;

 3. The method of claim 1,

 Wherein the lysosomal enzyme is gyuduronate 2-sulfatase.

4. The method of claim 1,

 Wherein the albumin comprises the amino acid sequence of SEQ ID NO: 1.

 5. The method of claim 1,

 Wherein the albumin derivative has 90% or more homology with the amino acid sequence of SEQ ID NO: 1.

6. The method of claim 1, Wherein the linker is a polyethylene glycol having a size of greater than 0 kDa and less than or equal to 5 kDa. Protein conjugate.

 7. The method of claim 1,

 Wherein the linker comprises an amide bond with an euduronate 2-sulfatase.

 8. The method of claim 1,

 Wherein the linker comprises a recombinant albumin with a thioether bond.

 9. The method of claim 1,

 Wherein the protein conjugate is one to fifteen albumin bound to the euduronate 2-sulfatase.

 10. The method of claim 1,

 Wherein the protein conjugate is one in which one or two albumin is bound to the euduronate 2-sulfatase.

 11. A pharmaceutical composition for preventing or treating Hunter syndrome comprising the protein conjugate of claim 1 as an active ingredient.

 12. A method for preventing or treating Hunter's syndrome comprising administering the protein conjugate of claim 1 to a subject.

 13. The method of claim 12,

 Wherein said administration is intravenous, subcutaneous, intradermal, intramuscular, intranasal, intracerebral or intrathecal administration.

 14. The method of claim 12,

 Wherein said administration is administered once every three months, once every two months, once every month, once every three weeks, once every two weeks, or once a week.

 15. A fusion protein comprising an albumin or albumin derivative and a lysosomal enzyme.

 16. The method of claim 15,

 Wherein the fusion protein has any one of the structures selected from the following structures:

^{_{A 2- X 2, X 2 _}} A 2, A 2 -L 2 -X 2, Table ₂ ^_ 1 ^_公 _{2, 2} group _{^{2, A 2 _ X 2 ~}} A 2,,不石~ 2019/124973 1 »(: 1 ^ 1 {2018/016240

_{L 2 -A 2 -L 3 -X3 2} , A 2 -L 2- X 2 - A 3, A 2 -X 2 -L 2 -A 3, A 2 -L 2 -X 2 - L 3 -A 3 _;

In the above _structure,

A ₂ or A ₃ is an independent albumin or albumin derivative, respectively;

L ₂ or are each independently a linker;

Each X ₂ is independently a lysosomal enzyme.

 17. The method of claim 15,

 The lysosomal enzyme may be selected from the group consisting of iduronate-2-sul fatase (IDS), beta-galactosidase, galactose-6-sulphatase, Beta-glucuronidase, N-acetylgalactosamine-6 sul fatase, glucocerebrosidase,

glucuronidase, glucocerebrosidase, alpha-galactosidase A, alpha-L-iduronidase, alpha- N-acetylglucosaminidase, N-acetyl transferase, N-acetylglucosamine 6-sulphate, Hyaluronan, and the like. Azime

(hyaluronidase). &lt; / RTI &gt;

 18. The method of claim 15,

 Wherein the lysosomal enzyme is a diuronate 2-sulfatase.

 19. The method of claim 15,

 Wherein the topoisomerase comprises the amino acid sequence of SEQ ID NO: &lt; RTI ID = 0.0 &gt; 1. &lt; / RTI &gt;

 20. The method of claim 15,

 Wherein the albumin derivative has 90% or more homology with the amino acid sequence of SEQ ID NO: 1.

 21. The method of claim 16,

 Wherein the linker is a peptide linker.

 22. The method of claim 21,

 Wherein said peptide linker is comprised of 5 to 40 amino acids.

23. The method of claim 21, Wherein said peptide linker is a rigid linker, a flexible linker, or a cleavable linker.

 24. The method of claim 23,

 (I) A (EAAAK) nA is an integer from 1 to 5, ii) PAPAP or iii) (XP) n (X = Ala, Lys, Glu, n is an integer of 5 to 17) &Lt; / RTI &gt;

 25. The method of claim 23,

 Wherein the flexible linker has an amino acid sequence of i) (GGGGS) n is an integer of 1 to 5 or ii) (G) n (n is an integer of 6 to 8).

 26. The method of claim 21,

 Wherein said peptide linker comprises a cleavable linker.

 27. The method of claim 26,

 Wherein the cleavable linker is a Cathepsin B cleavage sequence, a Furin cleavage motif sequence, or a Thrombin cleavage sequence.

 28. The method of claim 21,

 Wherein said peptide linker has the amino acid sequence of any of SEQ ID NOS: 4-6.

 29. The method of claim 21,

 Wherein said peptide linker comprises any of the amino acid sequences selected from SEQ ID NOS: 7-12.

 30. A polynucleotide encoding the fusion protein of any one of claims 15 to 29.

 31. The method of claim 30,

 Wherein the polynucleotide comprises a single nucleotide sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 18.

 32. An expression vector comprising the polynucleotide of claim 30.

 33. A transformant transformed by the introduction of the expression vector of claim 32.

 34. A method for culturing a transformant, comprising: i) culturing the transformant of claim 33 to obtain a culture; and

ii) recovering the fusion protein of claim 15 from said culture 2019/124973 DISCLOSURE OF INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION 1. A method for producing a fusion protein.

 35. A pharmaceutical composition for preventing or treating Hunter syndrome comprising the fusion protein of claim 15 as an active ingredient.

 36. A method for preventing or treating Hunter's syndrome comprising administering the fusion protein of claim 15 to a subject.

 37. Use of the protein conjugate of claim 1 to treat Hunter's syndrome.

 38. The use of the fusion protein of claim 15 to treat Hunter's syndrome.

 39. Use of the protein conjugate of claim 1 for the manufacture of a Hunter syndrome therapeutic agent.

40. Use of the fusion protein of claim 15 for the manufacture of a Hunter syndrome therapeutic agent.