JP5957085B2 - Drugs fused with ATF for improved bioavailability - Google Patents

Description

  The present invention relates to a method for improving the bioavailability of a drug using a drug having improved bioavailability and an amino terminal fragment of urokinase-type plasminogen activator (ATF). More specifically, the present invention relates to the delivery of ATF bound medicinal proteins into cells via binding of ATF moieties to urokinase-type plasminogen activator receptors expressed on the cell surface. About.

  In humans, urokinase-type plasminogen activator (uPA) is a protein composed of two peptide chains linked to each other by disulfide bonds. Their chains, A and B, are formed by enzymatic cleavage between amino acids 178 and 179 of prourokinase (plasmin, kallikrein, cathepsin, etc.), which is a single chain protein called prepro-urokinase (sc-uPA) The N-terminal signal peptide (amino acids 1 to 20) is removed. uPA is then cleaved in vivo between amino acids 155 and 156, thereby producing a low molecular weight urokinase-type plasminogen activator (LMW-uPA) and an amino-terminal fragment (ATF) of the urokinase-type plasminogen activator.

  uPA has proteolytic activity and, in physiological conditions, converts plasminogen to plasmin, triggering a proteolytic cascade that results in thrombus formation or extracellular matrix degradation. uPA contains two domains, ATF and a protease domain. ATF is a region consisting of amino acids 1-135 on the amino terminal side, while the protease domain is a region consisting of amino acids 136-411 on the carboxy terminal side of uPA. Thus, ATF does not have protease activity and has affinity for urokinase-type plasminogen activator receptor (uPAR) (Non-patent Document 1).

  ATF itself is composed of two independent domains. That is, an amino terminal growth factor-like domain (amino acids 1 to 43 from the N terminus) and one kringle domain (amino acids 43 to 135 from the N terminus) by which uPA binds to uPAR. (Non-Patent Documents 2 and 3). Thus, ATF itself can bind to uPAR via the amino-terminal growth factor-like domain.

  uPA is a GPI-anchor membrane protein that consists of 335 amino acids and has three extracellular domains. Of these, the first domain from the N-terminus serves as the binding site for uPA (non- Patent Document 4). The binding of ATF to uPAR is not internalized inside the cell, whereas the binding of uPA to uPAR is internalized when complexed with a plasminogen activator inhibitor (non-patented). Reference 5).

  uPAR binding protein (uPARAP), also known as ENDO180, is a lectin-like membrane protein belonging to the mannose receptor protein family of macrophages (Non-patent Document 6). ENDO180 is an internalization receptor that directs the ligand bound to lysosomes, where lysosomal enzymes act on catalytic activity to degrade the ligands. ENDO180 is also known to form triple complexes with uPA and uPAR.

  Lysosomal disease is a group of rare genetic metabolic diseases that are caused by various genetic disorders in genes encoding various lysosomal enzymes. The lack of enzymatic activity of any of these enzymes results in intracellular accumulation of their substrates, which in turn causes significant damage to the cells.

  Lysosomal diseases include, for example, Pompe disease, Fabry disease, Gaucher disease, Harrah's syndrome (mucopolysaccharidosis type I, MPS I), Hunter syndrome (MPS II), Maroto Lamy syndrome (MPS VI), Niemann-Pick disease, And Morquio syndrome (MPS IV). Of course, some types of lysosomal disease are treated with enzyme replacement therapy (ERT), and the enzyme missing from the patient is supplied to the patient from the outside.

  Pompe disease is caused by deficiency of α-glucosidase and is a lysosomal storage disease characterized clinically by muscle weakness and cardiomyopathy. For this disease, enzyme replacement therapy (ERT) has been established to be effective in relieving and relieving muscle weakness and improving cardiac function, and recombinant acid alpha-glucosidase is being administered to patients . The effectiveness of this therapy has been confirmed by several clinical studies in patients with different onset age and disease severity (Non-patent Document 7).

  Fabry disease is caused by α-galactosidase deficiency and is a lysosomal storage disease that is clinically characterized by pain, renal failure, cardiomyopathy, and stroke, which are responsible for the morbidity and mortality of the disease. It has made a cause. Two different enzyme preparations are available for patients with Fabry disease. It has been established that enzyme replacement therapy (ERT) using one of these formulations is effective in improving the patient's quality of life (QOL). The effectiveness of this therapy has been demonstrated, including pain relief, reduced heart size, improved hearing and sweating, stabilized renal function, and delayed renal failure in patients with end-stage renal disease (Non-patent document 8).

  Gaucher disease is a genetic disorder caused by a genetic deficiency of glucosylceramidase, a lysosomal storage disease characterized by sputum formation, fatigue, anemia, thrombocytopenia, and liver and spleen enlargement. It is well established that enzyme replacement therapy (ERT) is effective in improving a patient's quality of life (QOL) (Non-Patent Document 9).

  Hurler's syndrome, known as mucopolysaccharidosis type IH, is caused by a genetic disorder of α-L-iduronidase. Schaeer syndrome is mucopolysaccharidosis type IS and is a mild case of Hurler syndrome. These diseases, including intermediate forms, are classified as Halar-Scheier syndrome. Patients with Hurler's syndrome accumulate dermatan sulfate and heparan sulfate in lysosomes, resulting in progressive severe physical deformity and delayed wisdom that develops in the infant stage, and die by age 10. In the case of Schaeer's syndrome, little or no delay in wisdom is observed. In both cases, dermatan sulfate and heparan sulfate in urine are typically observed. Enzyme replacement therapy (ERT) with α-L-iduronidase has been well established to significantly improve respiratory function and physical ability and reduce glycose aminoglycan accumulation in patients with Hurler's syndrome (Non-Patent Literature). 10).

  Hunter syndrome is a genetic disease caused by a genetic disorder of α-L-iduronidase and iduronic acid-2-sulfatase (I2S) (Non-patent Document 11). I2S is a lysosomal enzyme having an activity of hydrolyzing sulfate ester bonds in the molecule of glycoseaminoglycan such as heparan sulfate and dermatan sulfate. This enzyme deficiency results in the accumulation of its substrate in various tissues, such as liver and kidney tissues, which in turn is associated with various symptoms seen in patients with Hunter's syndrome, including skeletal deformities and severe wisdom delays. cause. It has been well established that enzyme replacement therapy (ERT) improves the physical ability of patients (Non-patent Document 12).

  Maroto Lamy syndrome is mucopolysaccharidosis type VI, a genetic disease caused by a genetic disorder of N-acetylgalactosamine-4-sulfatase (ASB). ASB is known as arylsulfatase B and is an enzyme that liberates sulfate ions from chondroitin 4-sulfate, dermatan sulfate, and UDP-N-acetylgalactosamine 4-sulfate by hydrolysis. It has been well established that enzyme replacement therapy (ERT) using this enzyme improves the physical ability of patients (Non-patent Document 13).

  Niemann-Pick disease is classified into types A to F according to the etiological difference and symptoms. Of these, types A and B are caused by a genetic deficiency of acid sphingomyelinase (ASM). ASM is a lysosomal enzyme that hydrolyzes sphingomyelin into choline phosphate and ceramide. The enzyme activity of ASM is markedly reduced in type A, while it remains relatively high in type B. Such differences stem from the corresponding mutation pattern in the ASM gene. In the case of type A, a lack of ASM enzyme activity causes accumulation of sphingomyelin in the central nervous system, liver, spleen, lung and bone marrow, thus leading to hepatosplenomegaly and central nervous system disorders. As a result, most type A patients die before they reach 4 years of age. In contrast, most patients with type B do not suffer from central nervous system disorders and can survive and grow. Although the effectiveness of enzyme replacement therapy (ERT) with ASM has been reported, it has not been fully established (Patent Document 1).

  In enzyme replacement therapy, patients receive an intravenous infusion of the missing enzyme. In order for an injected enzyme to show clinical effects, it is essential that the enzyme is taken up into the cell where it should act. Furthermore, in order for the enzyme to show clinical effects, it is also essential that the enzyme is sorted into lysosomes, which are sites where its catalytic activity is exerted.

  The enzyme used for ERT has a glycan linked to a mannose-6-phosphate or mannose residue and is expressed on the cell surface to a mannose-6-phosphate receptor or mannose receptor. Through the binding, the enzyme is taken into cells and then sorted into lysosomes where it exerts enzyme activity (Non-Patent Documents 14 to 16). However, mannose-6-phosphate receptors and mannose receptors are expressed throughout the human body, and the binding of enzymes to these receptors leads to the tissues where the activity should be exerted and the enzymes to specific tissues. It is not enough to be sorted. In addition, since these receptors are expressed in both the liver and spleen, injected enzymes containing mannose or mannose-6-phosphate residues in the sugar chain are quickly removed by these organs. It can happen (Non-Patent Document 14).

  Therefore, to achieve more efficient uptake of these enzymes into the cell, something else that has an affinity for other molecules on the cell surface other than mannose-6-phosphate receptor or mannose receptor. The enzyme must be modified with the ligand. None of the enzymes currently available for ERT contain such ligands, but are not effectively sorted into their target tissues and are thought to have limited effects.

  Furthermore, in the case of an enzyme with a relatively low content of mannose-6-phosphate, such as α-glucosidase, the binding to receptors via mannose-6-phosphate is Insufficient to be taken in. In order to overcome this difficulty, an attempt has been made to fuse an enzyme to a receptor-related protein that is one of LDL receptor ligands, and an improvement in cellular uptake of the enzyme has been reported (Non-patent Document 17).

US Pat. No. 5,686,240

Blasi F., et. Al., J Cell Biol. 104, 801-4 (1987) Novokhatny V., et.al., J Biol Chem. 267: 3878-86 (1992). Mazar AP., Et.al., Fibrinolysis. 6: 49-55 (1992). Ploug M., et.al., J Biol Chem. 266: 1926-33 (1991). Cubellis MV., Et.al., EMBO J. 9: 1079-85 (1990). Behrendt N., et. Al., J Biol Chem. 275, 1993-2002 (2000) Nicolino, M., et.al., Genet Med. 11, 210-9 (2009) West, M., et. Al., J Am. Soc Nephrol. 20, 1132-39 (2009) Kishnani PS, et.al., Mol Genet Metab. 96, 164-70 (2009) Wraith JE., J Pediatr. 144, 581-8 (2004) Wilson PJ et al., Proc Natl Acad Sci USA. 87: 8531-5, 1990. Muenzer J. et al., Genet Med. 8, 465-73 (2006). Harmatz P, et al., J Pediatr. 148, 533-539 (2006) Bielicki J. et. Al., Biochem J. 289, 241-246 (1993) Grubb JH. Et al., Rejuvenation Res. 13, 229-36 (2010) Kornfield S., FASEB J. 1, 462-8 (1987) Prince WS, J Biol Chem. 279 35037-46 (2004)

  In the above background, an object of the present invention is to provide a novel method for delivering a drug that exhibits pharmacological activity only after being taken up into the cell.

  The present inventor has shown that human iduronic acid-2-sulfatase (hI2S) fused to human or mouse AFT is taken into fibroblasts via binding to urokinase-type plasminogen activator receptor (uPAR). It was found that hI2S can exert its activity. The present invention has been completed through further research based on this discovery. That is, the present invention provides the following.

1. A drug comprising a pharmacologically active compound (A) and a peptide (B) fused thereto, wherein the peptide (B) comprises an ATF receptor binding domain.
2. The drug according to 1 above, wherein the pharmacologically active compound (A) is a protein.
3. The drug according to 2 above, wherein the protein is an enzyme.
4). 4. The drug according to 3 above, wherein the enzyme is a lysosomal enzyme.
5. The lysosomal enzyme is selected from the group consisting of acid α-glucosidase, α-galactosidase, glucosylceramidase, α-L-iduronidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-4-sulfatase, or acid sphingomyelinase. 4. The drug according to 4 above.
6). The drug according to any one of 1 to 5 above, wherein the peptide (B) is fused to the pharmacologically active compound (A) by an amide bond or an ester bond.
7). 6. The drug according to any one of 1 to 5 above, wherein the peptide (B) is fused to the pharmacologically active compound (A) at the amino terminal amino acid of the peptide (B).
8). 3 to 5 above, wherein the peptide (B) is fused to the protein by a peptide bond formed between the amino terminal amino acid of the protein and the carboxy terminal amino acid of the peptide (B). Drug.
9. The above-mentioned 3-5, wherein the peptide (B) is fused to the protein by a peptide bond formed between the carboxy terminal amino acid of the protein and the amino terminal amino acid of the peptide (B). Drug.
10. Any one of the above 1 to 9, wherein the peptide (B) comprises the amino acid sequence shown in SEQ ID NO: 33, or a derivative thereof in which 1 to 3 amino acids are added, substituted, deleted, or inserted. Drug.
11. 10. The drug according to any one of 1 to 9 above, wherein the peptide (B) comprises the amino acid sequence shown in SEQ ID NO: 32 or a derivative thereof in which 1 to 10 amino acids are added, substituted, deleted, or inserted.
12 The pharmacologically active compound is composed of the peptide (B) comprising the amino acid sequence shown in SEQ ID NO: 32, and the peptide (B) is at its carboxy terminal amino acid via a linker comprising at least one amino acid. 11. The drug according to 11 above, which is fused to (A).
13. The linker is -Lys-, Lys-Pro-, -Lys-Pro-Ser-, -Lys-Pro-Ser-Ser- (SEQ ID NO: 41), and -Lys-Pro-Ser-Ser-Pro- (sequence 12. The drug according to 12 above, which is selected from the group consisting of No. 42).
14 The drug according to any one of 1 to 9 above, wherein the peptide (B) is composed of the amino acid sequence shown in SEQ ID NO: 31 or a derivative thereof in which 1 to 5 amino acids are added, substituted, deleted, or inserted. .
15. The drug according to any one of 1 to 9 above, wherein the peptide (B) comprises the amino acid sequence shown in SEQ ID NO: 30 or a derivative thereof in which 1 to 10 amino acids are added, substituted, deleted, or inserted. .
16. The peptide (B) consists of the amino acid sequence shown in SEQ ID NO: 30, and the peptide (B) is pharmacologically active via a linker consisting of at least one amino acid at its carboxy terminal amino acid. 15. The drug according to 15 above, which is fused to the compound (A).
17. The linker is -Lys-, Lys-Pro-, -Lys-Pro-Ser-, -Lys-Pro-Ser-Ser- (SEQ ID NO: 41), and -Lys-Pro-Ser-Ser-Pro- (sequence 16. The above 16 drugs, which are selected from the group consisting of No. 42).
18. A drug composition for enzyme replacement therapy, comprising the drugs 1 to 17 above.
19. A pharmacologically active protein (A) and a peptide (B) fused thereto, wherein the peptide (B) comprises a receptor binding domain of ATF, and the peptide (B) comprises DNA encoding a fusion protein which is fused to the pharmacologically active protein (A) by a peptide bond at the amino or carboxy terminus of the peptide (B).
20. 19. The DNA according to 19 above, wherein the pharmacologically active protein is an enzyme.
21. 20. The DNA according to 20 above, wherein the enzyme is a lysosomal enzyme.
22. The lysosomal enzyme is selected from the group consisting of acid α-glucosidase, α-galactosidase, glucosylceramidase, α-L-iduronidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-4-sulfatase, acid sphingomyelinase The 21 drugs as described above.
23. An expression vector comprising the DNA of the above 19-22.
24. A mammalian cell transformed with the above 23 vector.

  The present invention provides a means to help improve the cellular uptake of pharmacologically active compounds through binding to cell surface urokinase-type plasminogen activator receptor (uPAR), and thus also to uPAR Also provided are agents with improved uptake by cells expressing. In particular, since this improved intracellular uptake is not dependent on any interaction of the agent with the mannose-6-phosphate receptor or mannose receptor on the cell surface, the present invention relates to mannose bound to a molecule. Alternatively, an agent is provided that has no or only a few mannose-6-phosphate residues and can still be taken up into cells.

FIG. 1-1 shows a flow diagram illustrating a method for construction of a pBSK (IRES-Hygr-mPGKpA) vector. FIG. 1-2 shows a flow diagram illustrating the method for construction of the pBSK (NotI-IRES-Hygr-mPGKpA) vector. FIG. 1-3 shows a flow diagram illustrating the method for construction of the pE-IRES-Hygr vector. 1-4 shows a flow diagram illustrating the method for construction of the pPGK-neo vector. FIG. 1-5 shows a flow diagram illustrating the method for construction of the pPGK-IRES-Hygr vector. FIG. 1-6 shows a flow diagram illustrating the method for construction of the pPGK-IRES-GS-ΔpolyA vector. FIG. 1-7 shows a flow diagram illustrating the method for construction of the pE-puro vector. Figure 1-8 shows a flow diagram illustrating the method for construction of the pE-puro (XhoI) vector. FIG. 1-9 shows a flow diagram illustrating the method for construction of the pE-IRES-GS-puro vector. FIG. 2 shows a flow diagram illustrating the method for construction of the pE-mIRES-GS-puro vector. FIG. 3 shows a partial schematic diagram of the pE-mIRES-GS-puro (mATF-hI2S) vector. FIG. 4 shows a partial schematic diagram of the pE-mIRES-GS-puro (hATF-hI2S) vector. FIG. 5 shows the results of SDS PAGE analysis of purified mATF-fused hI2S. The fraction containing hI2S activity from HiTrap Q-Sepharose FF was loaded. FIG. 6 is a graph showing the measurement results of cell uptake of hATF-fused hI2S into human fibroblasts. The vertical axis represents the amount (ng) of hATF-fused hI2S or hIS2 incorporated per unit mass (mg) of total cell protein (ng / mg total cell protein). The horizontal axis indicates the concentration of hATF-fused hI2S or hI2S added to the medium. Black circles indicate the cellular uptake value of hATF-fused hI2S, white circles indicate the cellular uptake value of hATF-fused hI2S in the presence of 10 mM M6P, white triangles in the presence of 10 mM M6P and 4.02 μg / mL hATF , Cell uptake values of hATF-fused hI2S are shown. Black squares indicate hI2S cell uptake values, and white squares indicate hI2S cell uptake values in the presence of 10 mM M6P. FIG. 7 shows the measurement results of inhibition by M6P of the uptake of hATF-fused hI2S into human fibroblasts. The vertical axis represents the cell uptake ratio (%) of hATF-fused hI2S or hI2S. The horizontal axis indicates the concentration (mM) of M6P added to the medium. The black circle indicates the value of the cell uptake ratio of hATF-fused hI2S, and the black square indicates the value of the cell uptake ratio of hI2S. FIG. 8 shows the measurement results of cellular uptake of mATF-fused hI2S into mouse fibroblasts. The vertical axis shows the amount (ng) of mATF-fused hI2S or I2S incorporated per unit mass (mg) of total cell protein (ng / mg total cell protein). The horizontal axis indicates the concentration of mATF-fused hI2S or hI2S added to the medium. Black circles indicate cellular uptake values of mATF-fused hI2S, and white circles represent cellular uptake values of mATF-fused hI2S in the presence of 10 mM M6P. Black squares indicate hI2S cell uptake values, and white squares indicate hI2S cell uptake values in the presence of 10 mM M6P. FIG. 9 shows the measurement results of inhibition by M6P on the cellular uptake of mATF-fused hI2S into mouse fibroblasts. The vertical axis represents the cell uptake ratio (%) of mATF-fused hI2S or hI2S. The horizontal axis indicates the concentration (mM) of M6P added to the medium. The black circle indicates the value of mATF-fused hI2S uptake ratio, and the black square indicates the value of hI2S uptake ratio. FIG. 10 shows an alignment of amino acid sequences of human ATF (upper) and mouse ATF (lower). Asterisks indicate amino acids that differ between human ATF and mouse ATF.

  In the present invention, the amino terminal fragment (ATF) of urokinase type plasminogen activator is preferably mammalian ATF, more preferably human or mouse ATF, and particularly preferably human ATF. The amino acid sequences of wild type mouse and human ATF are shown in SEQ ID NO: 30 and SEQ ID NO: 32, respectively. In the present invention, ATF is preferably a wild type, but mutant ATF, that is, one that has undergone addition, substitution, deletion, and / or insertion of one or more amino acids can also be preferably used. . In such mutant ATF, the number of amino acids added, substituted, deleted and inserted is preferably 1-10, more preferably 1-8, still more preferably 1-5, and Even more preferably, it is one.

  In the present invention, the peptide comprising the ATF receptor binding domain is particularly preferably a mammalian peptide, more preferably a human or mouse peptide, and particularly preferably a human peptide. As used herein, the term “peptide comprising a receptor binding domain of ATF” does not include compounds containing uPA. The amino acid sequences of the receptor binding domains of wild type mouse and human ATF are shown in SEQ ID NO: 31 and SEQ ID NO: 33, respectively. In the present invention, the receptor binding domain of ATF is preferably wild-type, but ATF mutant receptor binding that has undergone addition, substitution, deletion, and / or insertion of one or more amino acids. Domains can also be preferably used. The number of amino acids that have undergone addition, substitution, deletion, and insertion in the mutant receptor binding domain of ATF is preferably 1-5, more preferably 1-3, and even more preferably 1. It is a piece.

  The drug of the present invention is a drug administered to mammals, preferably domestic animals such as humans, horses, pigs, cows and goats, and pet animals such as dogs and cats. The medicament is preferably administered intravenously. In the present invention, the term “drug” does not include compounds containing uPA.

  In the present invention, it is the ATF receptor binding domain that plays a major role in leading pharmacologically active compounds to binding to and then into cell surface uPARs. There is no particular limitation on the range of pharmacologically active compounds to which peptides containing the receptor binding domains of the above will be fused. Preferred examples of pharmacologically active compounds include, but are not limited to, proteins, polysaccharides, lipids, and other types of chemicals including antibiotics, anticancer agents and anti-inflammatory agents. Of these, proteins are particularly preferred. The peptide comprising the receptor binding domain of ATF may be fused to a pharmacologically active compound using any kind of chemical bond, if desired, of which amide bonds and ester bonds are preferred. This peptide comprising the receptor binding domain of ATF may be fused to a pharmacologically active compound by an amide bond or an ester bond formed at the carboxy or amino terminal amino acid of the peptide. The number of peptides containing the receptor binding domain of ATF to be fused per molecule of a pharmacologically active compound is not particularly limited, but the number is preferably 1 to 10, more preferably 1 to 5 , Still preferably 1-3, and even more preferably 1.

  When the pharmacologically active compound to which the peptide containing the receptor binding domain of ATF is to be fused is a protein, the peptide is preferably fused to the protein by peptide bonds. More preferably, the peptide comprising the receptor binding domain of ATF may be fused to the protein at either the carboxy or amino terminal amino acid of the protein. Furthermore, the peptide containing the receptor binding domain of ATF may be bound to the protein via a linker. The linker for this purpose may be a single amino acid or two or more amino acids between the protein and the peptide containing the receptor binding domain of ATF. The linker may also consist of 1-20 amino acids, more preferably 1-10 amino acids, and even more preferably 1-5 amino acids. The amino acid sequence of such a linker is preferably -Lys-, Lys-Pro-, -Lys-Pro-Ser-, -Lys-Pro-Ser-Ser- (SEQ ID NO: 41), and -Lys-Pro- You may select from the group which consists of Ser-Ser-Pro- (sequence number 42).

  A fusion protein as a drug made by fusing a peptide containing the receptor binding domain of ATF and a pharmacologically active protein between the former carboxy-terminal amino acid and the latter amino-terminal amino acid is an ATF receptor. A fusion gene in which a gene that encodes a peptide containing a body-binding domain and a gene that encodes a pharmacologically active protein are fused in-frame in this order in the 5 'to 3' direction. Can be produced. Examples of such fusion genes are SEQ ID NO: 26 (ie, the fusion gene of the mouse ATF gene and the human I2S gene) and SEQ ID NO: 28 (ie, the fusion gene of the human ATF gene and the human I2S gene). ).

  A fusion protein as a drug made by fusing a peptide containing an ATF receptor binding domain and a pharmacologically active protein between the former amino terminal amino acid and the latter carboxy terminal amino acid is a pharmacological agent. A fusion gene consisting of a gene encoding an active protein and a gene encoding a peptide containing the receptor binding domain of ATF, which are fused in-frame in this order in the 5 'to 3' direction. It can be produced by expressing a gene.

  In order to express any of the above fusion genes, the selected gene is inserted into an expression vector, and the expression vector thus obtained is then introduced into a host cell. The species to which the host cell used belongs is not particularly limited, but is preferably E. coli, yeast, insect cells, or mammalian cells, of which mammalian cells are more preferred, and CHO cells are particularly preferred. The host cell thus obtained has been transformed with an expression vector and then incubated to express the fusion protein.

  In the present invention, there is no particular limitation regarding the range of pharmacologically active proteins to which a peptide containing an ATF receptor binding domain is bound. Preferred examples of the pharmacologically active protein include enzymes, and among them, lysosomal enzymes, particularly acid α-glucosidase, α-galactosidase, α-L-iduronidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-4-sulfatase or acid sphingomyelinase. The pharmacologically active protein used in the present invention is preferably a mammalian protein, more preferably a human protein.

  When a lysosomal enzyme is used, a wild-type enzyme is preferable, but a mutant enzyme is also preferably used as long as it retains enzyme activity. A lysosomal enzyme and a peptide containing the receptor binding domain of ATF can be fused via a linker consisting of at least one amino acid that may form a peptide. Such a linker is inserted covalently between the amino and carboxy terminal amino acids of those two parts to be bound, ie the peptide used and the peptide containing the receptor binding domain of ATF. The linker preferably comprises 1 to 20 amino acids, more preferably 1 to 10 amino acids, still more preferably 1 to 5 amino acids.

  In the present invention, a lysosomal enzyme conjugated to a peptide containing the ATF receptor binding domain can be used as a drug in enzyme replacement therapy for lysosomal disease. For example, α-glucosidase, α-galactosidase, α-L-iduronidase, glucosylceramidase, α-L-iduronidase, iduronic acid-2-sulfatase, N-acetylgalactosamine fused to a peptide containing the receptor binding domain of ATF -4 sulfatase or acid sphingomyelinase is an enzyme replacement therapy for Pompe disease, Fabry disease, Gaucher disease, Harler syndrome (mucopolysaccharidosis type I, MPS I), Hunter syndrome (MPS II), and Niemann-Pick disease In, each is used as a drug.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, it is not intended that the present invention be limited to these examples.

(Construction of pE-IRES-GS-puro vector)
pPGKIH expression vector (Miyahara M. et al., J. Biol. Chem. 275,613-618 (2000)) was digested with XhoI and BamHI, and the internal ribosome entry site (IRES) derived from murine encephalomyelitis virus (EMCV) , A hygromycin resistance gene (Hygr gene), and a DNA fragment containing a mouse phosphoglycerate kinase (mPGK) polyadenylation signal (mPGKpA) were excised. This fragment was named IRES-Hygr-mPGKpA. Its nucleotide sequence is shown in SEQ ID NO: 1, wherein (i) nucleotides 1-6 are XhoI sites, (ii) nucleotides 7-119 are intermediate sequences, and (iii) nucleotides 120-718 are 3 of the region. 'Contains a sequence containing IRES from the 5' untranslated region of murine encephalomyelitis virus, including the terminal 'atg', (iv) nucleotides 716 to 1741 represent the hygromycin resistance gene (Hygr gene), (v Nucleotides 1742 to 1746 are intermediate sequences, (vi) nucleotides 1747 to 2210 are sequences containing the polyadenylation signal sequence of the mouse phosphoglycerate kinase gene (mPGKpA) (mPGKpA), and (vii) nucleotides 2211 to 2216 I.e., the 6 nucleotides at the 3 'end represent the BamHI site. This DNA fragment was inserted between the XhoI and BamHI sites of the pBSK vector (STRATAGENE), and the vector thus obtained was named pBSK (IRES-Hygr-mPGKpA) (FIG. 1-1).

  A DNA fragment containing the EMCV-IRES part was amplified by PCR using a pair of primers IRES5 '(SEQ ID NO: 2) and IRES3' (SEQ ID NO: 3), and pBSK (IRES-Hygr-mPGKpA) as a template. . The DNA fragment amplified by PCR was digested with XhoI and HindIII, then inserted between the XhoI and HindIII sites of the pBSK vector, and the vector thus obtained was named pBSK (NotI-IRES-Hygr-mPGKpA) (FIGS. 1-2). pBSK (NotI-IRES-Hygr-mPGKpA) was digested with NotI and BamHI, and the obtained DNA fragment containing the EMCV-IRES part was inserted between the NotI and BamHI sites of the pE-hygr vector. The pE-hygr vector was constructed according to the procedure described in the patent document (JP2011-172602). The obtained vector was named pE-IRES-Hygr (FIGS. 1-3).

  PCR was performed using a set of primers mPGKP5 ′ (SEQ ID NO: 4) and mPGKP3 ′ (SEQ ID NO: 5) and pPGKIH as a template to amplify a DNA fragment containing the mPGK promoter region (mPGKp). Its nucleotide sequence is shown in SEQ ID NO: 6. Here, (i) nucleotides 1 to 3 are intermediate sequences, (ii) nucleotides 4 to 9 are BamHI sites, (iii) nucleotides 10 to 516 are sequences containing mPGKp, and (iv) nucleotides 517 to 523 are intermediates. Sequence (v) Nucleotides 524-529 represent the EcoRI site, and (vi) Nucleotides 530-523 represent the last three nucleotides but represent an intermediate sequence. This amplified DNA fragment was digested with BglII and EcoRI, and inserted between the BglII and EcoRI sites of pCl-neo (Promega). The vector thus obtained was named pPGK-neo (FIGS. 1-4).

  pE-IRES-Hygr was digested with NotI and BamHI, and the resulting fragment (IRES-Hygr) was inserted between the NotI and BamHI sites of pPGK-neo. The vector thus obtained was named pPGK-IRES-Hygr (FIG. 1-5).

  A DNA fragment containing the glutamine synthetase gene (GS gene) was made with a set of primers GS5 '(SEQ ID NO: 7) and GS3' (SEQ ID NO: 8), and cDNA live made from mRNA of CHO-K1 cells as a template. Amplification was performed by PCR using a rally. The obtained DNA fragment was digested with BalI and BamHI and inserted between the BalI and BamHI sites of pPGK-IRES-Hygr. The obtained vector was named pPGK-IRES-GS-ΔpolyA (FIG. 1-6).

  A set of primers puro5 ′ (SEQ ID NO: 9) and puro3 ′ (SEQ ID NO: 10), using pCAGIPuro (Miyahara M. et al., J. Biol. Chem. 275,613-618 (2000)) as a template, PCR was performed to amplify a DNA fragment containing the puromycin resistance gene (Puro gene). This nucleotide sequence is shown in SEQ ID NO: 11. Here, (i) nucleotides 2 to 7 represent the AflII site, (ii) nucleotides 8 to 607 represent the sequence containing the Puro gene, and (iii) nucleotides 608 to 619 represent the BstXI site. The obtained DNA fragment was digested with AflII and BstXI and inserted between the AflII and BstXI sites of the pE-neo vector. The pE-neo vector was constructed according to the procedure disclosed in the patent document (JP2011-172602). The vector thus obtained was named pE-puro (FIGS. 1-7).

  A DNA fragment containing the SV40 late polyadenylation region was amplified by PCR using a set of primers SV40polyA5 '(SEQ ID NO: 12) and SV40polyA3' (SEQ ID NO: 13) and pE-puro as a template. This amplified DNA fragment was digested with NotI and HpaI and inserted between the NotI and HpaI sites of pE-puro. The obtained vector was named pE-puro (XhoI) (FIGS. 1-8).

  pPGK-IRES-GS-ΔpolyA was digested with NotI and XhoI and inserted between NotI and XhoI sites of pE-puro (XhoI). The obtained vector was named pE-IRES-GS-puro (FIG. 1-9).

(Construction of pE-mIRES-GS-puro vector)
Perform PCR with a set of primers mIRES-GS5 '(SEQ ID NO: 14) and mIRES-GS3' (SEQ ID NO: 15) using pE-IRES-GS-puro as a template to destroy the second start codon. A DNA fragment containing EMCV-IRES was amplified. Next, PCR was performed to amplify the region from EMCV-IRES to GS gene using the DNA fragment thus amplified and the aforementioned IRES5 ′ as a primer and pE-IRES-GS-puro as a template. The amplified DNA fragment containing the region from EMCV-IRES to GS gene was digested with NotI and PstI and inserted between the NotI and PstI sites of pE-IRES-GS-puro. The obtained vector was named pE-mIRES-GS-puro (FIG. 2).

(Cloning of human cDNA encoding human ATF (amino terminal fragment of human uPa: hATF) gene)
With a set of primers hATF-f (SEQ ID NO: 16, where nucleotides 4-9 represent the MluI site) and hATF-r (SEQ ID NO: 17, where nucleotides 4-9 represent the XhoI site), the template The human kidney Quick Clone cDNA (Clontech) was used to amplify the hATF gene. In the nucleotide sequence of hATF-f or hATF-r, nucleotides 4 to 9 represent an MluI site or an XhoI site, respectively. The fragment thus amplified was digested with MluI and XhoI, and then inserted between the MluI and XhoI sites of the pCl-neo vector (Promega) to obtain the pCl-neo (hATF) vector.

(Cloning of cDNA encoding mouse ATF (mouse uPA amino-terminal fragment; mATF) gene)
The mATF gene was amplified by PCR using a set of primers mATF-f (SEQ ID NO: 18) and mATF-r (SEQ ID NO: 19), using mouse kidney Quick Clone cDNA (Clontech) as a template. In the mATF-f or mATF-r nucleotide sequence, nucleotides 4-9 represent the MluI or XhoI site, respectively. The thus amplified fragment was digested with MluI and XhoI and inserted between the MluI and XhoI sites of pCI-neo (Promega) to obtain a pCI-neo (mATF) vector.

(Cloning of DNA encoding human iduronic acid-2-sulfatase (hI2S) gene)
Using human placenta cDNA library (Takara Bio) as a template, two sets of primers, ie, one set of outer primers hI2S-f (SEQ ID NO: 20) and hI2S-r (SEQ ID NO: 21) for the first reaction, Then, in the second reaction, nested PCR was performed using a pair of inner primers I2S-f2 (SEQ ID NO: 22) and hI2S-r2 (SEQ ID NO: 23) to amplify a DNA fragment containing hI2S cDNA. The PCR fragment thus amplified was inserted into the EcoRV site (blunt end cloning site) of the pT7Blue vector (Novagen) to obtain the pT7Blue (hI2S) vector. The cDNA encoding hI2S lacking the signal sequence was then PCR-combined with a set of primers hI2S-f3 (SEQ ID NO: 24) and hI2S-r3 (SEQ ID NO: 25) using the pT7Blue (hI2S) vector as a template. Amplified by In the nucleotide sequence of hI2S-f3, nucleotides 4-9 are XhoI sites, and in hI2S-r3, nucleotides 4-11 are NotI sites. The PCR fragment thus amplified was digested with XhoI and NotI and then inserted between the XhoI and NotI sites of pCI-neo (Promega) to obtain a pCI-neo (ΔS-hI2S) vector.

(Construction of expression vector for ATF-fusion hI2S)
The pCI-neo (mATF) vector was digested with MluI and XhoI to excise cDNA encoding mATF. pCI-neo (ΔS-hI2S) was digested with XhoI and NotI to cut out cDNA encoding hI2S lacking the signal sequence. Next, both cDNAs were inserted in series between the MluI and NotI sites of pE-mIRES-GS-puro to obtain pE-mIRES-GS-puro (mATF-ΔS-hI2S) (FIG. 3). . The DNA sequence of the mATF-fused hI2S gene embedded in the above vector is shown in SEQ ID NO: 26. The amino acid sequence of mATF-fused hI2S translated from this gene is shown in SEQ ID NO: 27. Here, the part consisting of the first 160 amino acids corresponds to the part of mouse sc-uPA, of which the first 21 amino acids correspond to the leader sequence, and the following 135 amino acids (amino acids 22 to 156) are Corresponds to mouse ATF (mATF, SEQ ID NO: 30). The first 43 amino acids of mATF contain the receptor binding domain of mATF (SEQ ID NO: 31). When expressed in CHO cells, mATF-fused hI2S is secreted from the cell after removal of the leader sequence.

  The pCI-neo (hATF) vector was digested with MluI and XhoI to excise cDNA encoding hATF. pCI-neo (ΔS-hI2S) was digested with XhoI and NotI, and a cDNA encoding hI2S lacking the signal sequence was excised. Next, both cDNAs were inserted in series between the MluI and NotI sites of pE-mIRES-GS-puro to obtain pE-mIRES-GS-puro (hATF-ΔS-hI2S) (FIG. 4). The DNA sequence of the hATF-fused hI2S gene embedded in the above vector is shown as SEQ ID NO: 28. The amino acid sequence of the hATF-fused hI2S gene translated from this gene is shown as SEQ ID NO: 29. Here, the part consisting of the first 159 amino acids corresponds to the human sc-uPA part, of which the first 20 amino acids correspond to the leader sequence, followed by 135 amino acids (amino acids 21 to 155) Corresponds to mouse ATF (hATF, SEQ ID NO: 32). The sequence consisting of the first 43 amino acids of hATF comprises the receptor binding domain of hATF (SEQ ID NO: 33). When expressed in CHO cells, hATF-fused hI2S is secreted from the cell after removal of the leader sequence.

(Manufacture of recombinant cells for expression of hATF-fused hI2S)
CHO-K1 cells (sourced from the American Type Culture Collection) with one of the above expression vectors, namely pE-mIRES-GS-puro (mATF-hI2S) or pE-mIRES-GS-puro (hATF-hI2S) Using Lipofectamine2000 (Invitrogen), transfection was carried out by the following method. That is, the day before transfection, 1 × 10 ⁶ CHO-K1 cells were added to a 3.5 cm culture dish containing 3 mL of D-MEM / F12 medium (D-MEM / F12 / 5% FCS) containing 5% FCS. And cultured overnight at 37 ° C in a humidified atmosphere of 5% CO2, 95% air. The next day, 300 μL of a 1: 1 mixture of Lipofectamine 2000 solution diluted 25-fold in Opti-MEM I medium (Invitrogen) and plasmid DNA solution diluted to 13.2 μg / mL in Opti-MEM I medium at 37 ° C. Then, the cells were transfected overnight in a humidified atmosphere consisting of 5% CO ₂ and 95% air.

After transfection, CD Opti CHO culture medium supplemented with selective medium (10 μg / mL insulin, 100 μmole / L hypoxanthine, 16 μmole / L thymidine and 30 μmole / L methionine sulfoximine (MSX, Sigma)) (Invitrogen)) and selective culture was performed at 37 ° C. in a humidified atmosphere consisting of 5% CO ₂ and 95% air. Cells grown in the selective medium were subjected to several successive subcultures in the medium to obtain recombinant cells. During the selective culture, the concentrations of MXS and puromycin were increased to 30-100 μmole / L and 0-10 μg / mL, respectively. Cells that survived the above selection process were used as recombinant cells for expression of ATF-fused hI2S.

(Culture of recombinant cells for expressing ATF-fusion hI2S)
Dilute the above recombinant cells to a density of 2 × 10 ⁵ cells / mL, 4 mM L-glutamine, 10 μg / mL insulin, 100 μmole / L hypoxanthine, 16 μmole / L thymidine, and 10 μg / mL puro. The cells were cultured in a CD Opti CHO culture medium supplemented with mycin at 37 ° C. for 7 days in a humidified atmosphere of 5% CO ₂ and 95% air. The culture supernatant was then collected by centrifugation.

(Purification of ATF-fusion hI2S)
Acetic acid was added to the culture supernatant collected above to adjust the pH of the culture supernatant to 5.0. The resulting precipitate was removed by filtration through a 0.22 μm membrane (Millipore). The collected culture supernatant was equilibrated with 100 mM acetate buffer (pH 5.0) in three times the column volume. The cation has selectivity based on both hydrophobic interaction and hydrogen bond formation. The sample was loaded onto a Capto MMC column (GE Healthcare) as an exchange column and adsorbed. The column was then washed with 4 column volumes of the same buffer. Subsequently, the adsorbed ATF-fused hI2S was columned from 100% 100 mM acetate buffer (pH 5.0) to 100% 100 mM HEPES buffer (pH 8.0) containing 1.5 M sodium chloride. Elution and fractionation was performed using a linear gradient with 20 volumes of buffer. After measuring I2S activity by the method described below, the fraction having hI2S activity was collected as the target eluate. This eluate from the Capto MMC column above was diluted 10-fold with 25 mM sodium phosphate buffer (pH 7.4) and equilibrated with the same buffer. HiTrap Q-Sepharose FF 5 mL column (GE health care company). The column was then washed with the same buffer volume 4 times the column volume. Subsequently, the adsorbed ATF-fused hI2S is transferred from 100% 25 mM sodium phosphate buffer (pH 7.4) to 100% 25 mM sodium phosphate buffer (pH 7.4) containing 1.5 M sodium chloride. The fraction was eluted and fractionated using a linear gradient of 20 times the column volume. After measuring I2S activity by the method described below, fractions with hI2S activity are collected and analyzed by SDS-PAGE, thereby having a single molecular weight corresponding to the molecular weight of ATF-fused hI2S (approximately 92 kD). One band was shown (Figure 5). The collected fractions were used for further analysis as purified ATF-fused hI2S.

(Measurement of rhI2S activity)
The sample was desalted by membrane filtration using a vertical polyethersulfone membrane (VIVASPIN 2 5,000 MWCO PES; Sartorius) as the ultrafiltration membrane, and then the desalted sample was reduced to about 1001 ng / mL with the reaction buffer (5 It was diluted with mM sodium acetate, 0.5 mg / L BSA, 0.1% Triton X-100, pH 4.45). 10 μL of each rhI2S sample was added to each well of a 96-well microtiter plate (FluoroNunc Plate, Nunc), and preincubation was performed at 37 ° C. for 15 minutes. A substrate solution was prepared by dissolving 4-methylumbelliferyl sulfate (SIGMA) in substrate buffer (5 mM sodium acetate, 0.5 mg / mL BSA, pH 4.45) to a final concentration of 1.5 mg / mL. 100 μL of the substrate solution was added to each well containing the ATF-fused hI2S sample, and the plate was left in the dark at 37 ° C. for 1 hour. After this incubation, 190 μL of stop buffer (0.33 M glycine, 0.21 M sodium carbonate, pH 10.7) was added to each well containing the sample. 150 μL of 0.4 μmole / L 4-methylumbelliferyl phosphate (4-MUF, Sigma) solution and 150 μL of stop buffer are added to the wells as a standard, and then the plate in a 96-well plate reader at excitation wavelength 330 nm and fluorescence wavelength 440 nm.

  A standard curve was generated by measuring fluorescence intensity at various concentrations of 4-MUF in solution. The fluorescence intensity of each sample was extrapolated to a standard curve. Results were calculated as activity in units / mL. One unit of activity corresponds to the production of 1 μmole of 4-MUF per minute at 37 ° C. To make this measurement, reference was made to a published US patent application (publication number 2004-0229250).

(Measurement of mannose-6-phosphate (M6P) content)
A desalted sample containing about 0.1 mg of protein was dried under reduced pressure, dissolved in 50% trifluoroacetic acid and heated at 100 ° C. for 2 hours. The sample was then dissolved in a 100 mM boric acid solution adjusted to pH 9.0 with NaOH. 20 mg sodium mannose-6-phosphate was dissolved in water to make 100 mL, and this was used as an M6P standard solution. This M6P standard stock solution was frozen in 1 mL portions and stored at −20 ° C., thawed before use, and diluted twice with pure water to obtain an M6P standard solution.

  6.2 g boric acid is added to the purified water and dissolved, and the pH of this solution is adjusted to 9.0 with 2N sodium hydroxide. Purified water is added to make a total volume of 1000 mL, which is then added to a 0.22 μm membrane filter (Millipore ) Through suction. The solution thus obtained was named Solution A (100 mM boric acid solution (pH 9.0)). 6.2 g boric acid and 11.7 g sodium chloride are added to purified water and dissolved, and the pH of the solution is adjusted to 9.0 with 2N sodium hydroxide, and then purified water is added to make a total volume of 1000 mL. This is a 0.22 μm filter. Suction filtered through. The solution thus obtained was named Solution B (100 mM boric acid-200 mM sodium chloride solution (pH 9.0)).

  10 g of L-arginine and 30 g of boric acid are dissolved in purified water, adjusted to a total volume of 1000 mL, suction filtered through a membrane filter with a pore size of 0.22 μM or less, and the solution thus obtained is used as a reaction reagent. Used as a solution.

  An anion exchange column, Shimpack ISA-07 / S2504 (4.0 mm ID x 250 mm) (base: polystyrene gel, stationary phase: quaternary ammonium group), Shimazu HPLC System LC-10Avp (reducing sugar analysis system) ) And Shim-pack guard column ISA (4.0 mm ID × 50 mm) was set as a column oven used to heat the column. A heat block (ALB-221, manufactured by Asahi Techno Glass) for heating reaction was set downstream of the column outlet. The column was heated to 65 ° C in a column oven and the heat block was set to 150 ° C. A fluorescence detector system was installed downstream of the heat block and adjusted to detect fluorescence at 430 nm by irradiating with 320 nm excitation light.

  Solutions A and B were set in the autosampler of the reducing sugar analysis system, which was then set so that the reagent solution for the reaction was supplied downstream of the column outlet (upstream of the heat block). After equilibrating the column with a mobile phase (Solution A) that would initiate chromatography, the column was loaded with M6P standard solution or M6P sample solution.

  A first mobile phase (thus 100 mM boric acid and 20 mM sodium chloride) prepared by loading a standard solution or sample solution of M6P onto the column and then mixing solution A and solution B in a volume ratio of 90:10. Was applied to the column at a flow rate of 0.3 mL / min for 35 minutes. The volume ratio of solution A and solution B is then linearly changed to 25:75 at the same flow rate over 25 minutes (thus containing 100 mM boric acid, whereas sodium chloride increases to 150 mM. ), And then set the volume ratio of solution B to 100% (thus containing 100 mM boric acid and 200 mM sodium chloride) for 10 minutes at the same flow rate, then the same as the first mobile phase Thus, solution A and solution B were run at a volume ratio of 90:10 (thus containing 100 mM boric acid and 20 mM sodium chloride). The reagent solution for reaction was supplied to the flow path downstream of the column outlet at a flow rate of 0.2 mL / min.

  The peak area corresponding to M6P was compared between the standard solution and the sample, and the average number of M6P per protein molecule was calculated in mol / mol.

  The average number of M6P contained in hATF-fused hI2S and mATF-fused hI2S was calculated as 2.6 mol / mol and 2.2 mol / mol, respectively. On the other hand, the average number of M6P contained in recombinant human I2S provided as a pharmaceutical (hereinafter referred to as rhI2S) was calculated to be 4.4 mol / mol.

(Manufacture of His-tag-hMPR9)
To measure the affinity of ATF-fused hI2S for human mannose-6-phosphate receptor (hM6PR), His-tag-hMPR9, which contains domain 9 of human M6P receptor and to which ATF binds, was used. , Produced as described below.

  A plasmid containing a cDNA encoding human mannose-6-phosphate receptor (human M6P receptor) essential for binding of M6P to cells was obtained from ATCC (ATCC No. 95660). A DNA fragment (hMPR9) encoding domain 9 of the human M6P receptor was amplified from the plasmid by PCR using a set of primers hMPR9-f (SEQ ID NO: 35) and hMPR9-r (SEQ ID NO: 36).

  The amplified DNA fragment was digested with NcoI and NotI and inserted between the NcoI and NotI sites of the pET26 vector (Novagen). The resulting plasmid was named pET26-MPR9. In order to amplify a DNA fragment encoding hMPR9 having an additional sequence at both the 5 ′ end and the 3 ′ end and whose sequence is shown in SEQ ID NO: 34, a two-step PCR was performed. This amplified DNA fragment encoding hMPR9 with a Bip signal at the N-terminus and a His tag at the C-terminus was designated Bip tag-hMPR9.

  In the above, the first reaction of the two-step PCR was performed using pET26-MPR9 as a template and a set of primers, MPR9-f2 (SEQ ID NO: 37) and MPR9-r2 (SEQ ID NO: 38). Subsequently, using this amplified DNA fragment as a template, a second PCR was performed using a set of primers, MPR9-f3 (SEQ ID NO: 39) and MPR9-r3 (SEQ ID NO: 40).

  The resulting DNA fragment was then digested with EcoRV and NotI and ligated into the Eco47III-NotI site of the pIB / V5-His-DEST vector (Invitrogen). The resulting plasmid was named pXBi-MPR9 and was used to transfect High Five cells to obtain cells expressing His-tag-hMPR9 derived from Bip-tag hMPR9 by removal of the Bip signal sequence. It was.

High Five cells (Invitrogen) are grown to 50% confluency in 24-well plates using Express Five medium (Invitrogen) and pXBi-MPR9 using Hily Max transfection reagent (Dojin chemical, Japan). Transfected. Cells were cultured in the presence of 30 μg / mL blasticidin to select stable transfectants. Stably transfected cells were then expanded into Erlenmeyer flasks (100 mL) and cultured for 4 days. The culture was then harvested and centrifuged at 3000 rpm for 30 minutes and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter (Millipore) and then diluted 5-fold with equilibration buffer (10 mM phosphate buffer (pH 7.2) containing 300 mM sodium chloride). The diluted supernatant is applied to a chromatography column packed with Profinity IMAC Ni-charged resin (bed volume: 1 mL, Bio-Rad) that has been equilibrated with equilibration buffer, and the column is then placed in a 5 × bed. Wash with volume of equilibration buffer. The resin-bound His-tag hMPR9 is then applied to 5 × bed volume of 10 mM NaPO ₄ , 300 mM NaCl and 10 mM imidazole (pH 7.2) followed by 5 × bed volume of 10 mM NaPO ₄ , 300 mM NaCl and Elution was performed by applying 300 mM imidazole (pH 7.2). Fractions containing His-tag-hMPR9 were collected and concentrated with Amicon 3K (Millipore) while exchanging the buffer into 20 mM Tris buffer (pH 7.4) containing 150 mM NaCl. The concentration of His-tag-hMPR9 was determined by measuring the absorbance at 280 nm.

(Measurement of affinity of ATF-fused hI2S for hM6PR and huPAR)
The binding affinity of ATF-fused hI2S to hM6PR (human mannose-6-phosphate receptor) and huPAR (human urokinase-type plasminogen activator) was measured using a nitrilotriacetic acid immobilized sensor chip (Series S Sensor Chip NTA "BR-1005 -32 ") with a Biacore T100 (GE Healthcare). The Biacore T100 is a measurement device based on surface plasmon resonance (SPR), in which a sample containing a ligand is sent at a constant flow rate to the surface of a sensor chip on which a receptor is immobilized. If the ligand binds to the receptor surface, the mass of the sensor chip increases due to the ligand bound to the receptor, and the SPR signal shifts as a change in resonance units (RU) proportional to the amount of bound ligand. Can be detected. For proteins, 1RU generally corresponds to about 1 pg / mm ² .

  Since M6P binds to domain 9 among the domains in hM6PR, hM6PR His-tag domain 9 (His-tag-hMPR9) prepared in the same manner as described above was used as an M6P receptor. As the ATF receptor, recombinant huPAR (rhuPAR) obtained from R & D Systems (Minneapolis, MN) was used in this experiment.

In order to activate the sensor chip, 10 mM HEPES (pH 7.4) containing 500 μmole / L NiCl ₂ , 150 mM NaCl, 50 μmole / L EDTA, and 0.05% Surfactant P20 was flowed at a flow rate of 10 μL / min for 60 seconds. Then (i) about 2.1 μL of 2.8 μg / mL His-tag-hMPR9 dissolved in 10 mM HEPES (pH 7.4) containing 150 mM NaCl, 50 μmole / L EDTA and 0.05% Surfactant P20 or (ii) the same buffer Apply approximately 2.1 μL of 5 μg / mL rhuPAR (R & D Systems) dissolved in the solution, and then flow the same buffer at a flow rate of 10 μL / min for 60 minutes to the His-tag on the activated sensor chip surface. -Fixed hMPR9 or rhuPAR.

  Each sample was 10 mM HEPES (pH 7.4) containing 150 mM NaCl, 50 μmole / L EDTA and 0.05% Surfactant P20 so that the concentrations of ATF-fused hI2S or rhI2S were 12.5, 6.25, 3.125 and 1.5625 nmol / L. Diluted with Each dilution was then applied for 300 seconds at 50 μL / min so that the ligand (ATF-fusion hI2S or rhI2S) was bound to the corresponding receptor (His-tag-hMPR9 or rhuPAR) on the sensor chip surface. Next, 10 mM HEPES (pH 7.4) containing 150 mM NaCl, 50 μmole / L EDTA and 0.05% Surfactant P20 was flowed for 180 seconds at a flow rate of 50 μL / min. At the same time, the ligand (ATF-fusion hI2S or rhI2S) and the receptor (His -Tag-hMPR9 or rhuPAR) was continuously monitored. Subsequently, 10 mM HEPES (pH 8.3) containing 150 mM NaCl, 350 mM EDTA and 0.05% Surfactant P20 was flowed at a flow rate of 50 μL / min for 60 seconds to denature the sensor chip. The deviation constant (Kd) was automatically calculated by the Biacore T100 Evaluation software based on the deviation state monitored above.

  As a result, the Kd between hATF-fused hI2S and rhuPAR was calculated to be 1.16 nM and between mATF-fused hI2S and rhuPAR was 5.59 nM. Thus, the affinity of hATF-fused hI2S for rhuPAR was 4.8 times higher than that of mATF-fused hI2S for rhuPAR. No specific binding was detected between hI2S and rhuPAR. These data indicate that hI2S fused to hATF can efficiently bind to huPAR, and mATF, in which 31% of the amino acids are different from that of hI2S (42 of 135 amino acids), is still efficient for huPAR. (FIG. 10).

  Furthermore, the Kd between hATF-fused hI2S and hM6PR was calculated to be 0.40 nM, and that between mATF-fused hI2S and hM6PR (domain 9 of hM6PR) was calculated to be 0.43 nM. These values are equivalent to Kd values between hI2S and hM6PR (Kd of 0.47 nM). These data show that either murine or human ATF-fused hI2S has binding affinity for hM6PR, which is close to that of hIS2 despite their lower M6P content It is shown that.

(Measurement of cellular uptake of hATF-fused hI2S using human fibroblasts)
Cultured human fibroblasts (CCD-1076SK, obtained from DS Pharma Biomedical Co., Ltd.) expressing both the M6P receptor and urokinase-type plasminogen activator receptor (uPAR) are treated with 10% The cells were suspended in MEM-Eagle medium (Gibco) containing activated FBS and 2 mM L-glutamine, and the cell density was adjusted to 8.0 × 10 ⁴ cells / mL. 100 μL of this cell suspension was seeded in each well of a 98-well microplate and cultured at 37 ° C. for 2 days in a humidified atmosphere consisting of 5% CO ₂ and 95% air. Samples containing hATF-fused hI2S or rhI2S were serially diluted with the above media to make their various concentrations from 4.88 ng / mL to 20 μg / mL. Next, the medium in the 96-well microplate on which the fibroblasts had been seeded was removed with a micropipette, and 100 μL of each sample diluted above was added to the wells in duplicate and incubated for 18 hours. Here, the final concentration of hATF-fused hI2S or rhI2S was 4.88 ng / mL to 20 μg / mL. To confirm the specific binding of hATF-fused hI2S or rhI2S to the mannose-6-phosphate receptor (M6P) receptor in this assay, 10 mmol / L mannose-6-phosphate (M6P) was used as an antagonist. ) Was prepared, added to a 96 well microplate and incubated in the same manner as above. The assay was performed in duplicate.

After washing three times with ice-cold PBS, the cells were lysed with an M-PER Mammalian Protein extraction kit (Thermo Scientific) supplemented with 0.5% protease inhibitor cocktail (Sigma). Subsequently, the amount of total cell protein was measured by Pierce BCA ^™ Protein Assay kit (Pierce, IL, USA), and the amount of hATF-fused hI2S and rhI2S incorporated into the cells was quantified by ELISA as described above. The amounts of hATF-fused hI2S and rhI2S incorporated were calculated per unit mass (mg) of total cell protein and plotted on the graph.

  Compared with rhI2S, the level of cellular uptake of hATF-fused hI2S into normal human fibroblasts was low (FIG. 6). This is because the number of M6P contained in hATF-fused hI2S (2.2 / protein molecule) is lower than that of rhI2S (4.4 / protein molecule), which is higher than that of rhI2S. This may be due to the reduced efficiency of cell uptake through the body. On the other hand, in the presence of 10 mM M6P, which antagonizes ligand binding to the M6P receptor, cellular uptake of hATF-fused hI2S was higher than rhI2S. In the presence of 10 mM M6P, rhI2S uptake was inhibited by over 99%, whereas hATF-fused hI2S was incorporated into fibroblasts at a rate of over 20%. This uptake of hATF-fused hI2S was inhibited by further addition of 4.02 μg / mL ATF.

  Inhibition of cellular uptake of hATF-fused hI2S and hI2S by the addition of M6P was further analyzed as shown in FIG. 7, where 20 μg / mL hATF-fused hI2S or hI2S was used to measure cellular uptake. Added to media containing various concentrations of M6P. In the presence of 3.33 mM M6P, the inhibition efficiency of M6P appeared to be almost saturated and rhIS2 uptake inhibition exceeded 99%, while more than 15% of hATF-fused hI2S still It was taken up by fibroblasts.

  These data indicate that hATF-fused hI2S can be taken up by cells not only through the M6P receptor but also in an M6P receptor-independent manner, possibly through binding of the hATF moiety to uPAR It is a thing.

(Measurement of cellular uptake of mATF-fused hI2S using normal mouse fibroblasts)
Measurement of cellular uptake of mATF-fused hI2S was performed according to the procedure described in Example 13 above, where primary mouse fibroblasts were used instead of human fibroblasts. Primary cultured mouse fibroblasts were obtained from Kitayama Labes (Japan).

  Compared with rhI2S, the level of cellular uptake of mATF-fused hI2S into mouse fibroblasts was low (FIG. 8). This indicates that the number of M6P contained in mATF-fused hI2S (2.2 / protein molecule) is lower than that in rhI2S (4.4 / protein molecule), and therefore M6P acceptance in mATF-fused hI2S compared to rhI2S. This may be due to the reduced efficiency of cellular uptake through the body. On the other hand, in the presence of 10 mM M6P (which antagonizes ligand binding to the M6P receptor), cellular uptake of mATF-fused hI2S was higher than rhI2S. In the presence of 10 mM M6P, rhI2S uptake was inhibited by about 91%, whereas mATF-fused hI2S was taken up by fibroblasts in excess of 20%.

  Inhibition of cellular uptake of mATF-fused hI2S and hI2S by the addition of M6P was further analyzed as shown in FIG. 9, where 20 μg / mL mATF-fused hI2S or hI2S was used for measurement. It was added to the medium containing various concentrations of M6P. In the presence of 3.33 mM M6P, the inhibition efficiency of M6P appears to be almost saturated, with more than 90% inhibition of rhI2S uptake inhibited, whereas hATF-fused hI2S still contains more than 20% fiber It was taken up by blast cells.

  These data indicate that hATF-fused hI2S can be taken up by cells not only through the M6P receptor but also in an M6P-independent manner, possibly through binding of the hATF moiety to uPAR. It shows that you can do it.

(Analysis of human I2S by ELISA)
To each well of a 96-well microtiter plate (Nunc), add 100 μL of mouse anti-human monoclonal antibody diluted to 4 μg / mL with 0.05 M carbonate-bicarbonate buffer (pH 9.6), and leave the plate at room temperature for 1 hour. Then, the antibody was adsorbed to the well. Next, after washing each well 3 times with phosphate buffered saline (pH 7.4) (PBS-T) containing 0.05% Tween 20, 200 μL of Starting Block (PBS) blocking buffer (Thermo Fisher Scientific) was added. The plate was left at room temperature for at least 30 minutes. Each well was then washed 3 times with PSB-T, and then 100 μL of test sample or human I2S standard diluted as desired with PBS containing 0.5% BSA and 0.05% Tween 20 (PBS-BT) was added to the wells. The plate was allowed to stand for at least 1 hour at room temperature. Next, each well was washed three times with PBS-T, 100 μL of biotin-labeled anti-human I2S monoclonal antibody diluted with PBS-BT was added, and the plate was allowed to stand for at least 1 hour. Next, each well was washed three times with PBS-T, and then 100 μL of streptavidin-HRP (R & D SYSTEMS) diluted with PBS-BT was added, and the plate was allowed to stand for at least 30 minutes. Next, after washing each well 3 times with PBS-T, 0.4 mg / mL o-phenylenediamine phosphate-citrate buffer (pH 5.0) was added to the well, and the plate was allowed to stand at room temperature for 8-20 minutes. Left to stand. Next, 0.1 mL of 1 mol / LH ₂ SO ₄ was added to each well to stop the reaction, and the absorbance at 490 nm was measured per well with a 96-well plate reader.

  The present invention can be used as a means for delivering various pharmacologically active compounds to organs, tissues and cells in which urokinase-type plasminogen activator receptor (uPAR) is expressed. Thus, the present invention can also be used to provide various drugs that are directed to organs, tissues and cells in which the receptor is expressed.

1 LacZ promoter 2 mPGK promoter 3 Wild-type murine encephalomyelitis virus IRES (EMCV-IRES)
3a Mutant murine encephalomyelitis virus IRES (EMCV-mIRES)
4 mPGK polyadenylation signal (mPGKpA)
5 Sequence containing the EF-1 promoter and its first intron 6 SV40 late polyadenylation signal 7 SV40 early promoter 8 Synthetic polyadenylation signal 9 Cytomegalovirus promoter 10 Glutamine synthetase gene 11 Sequence encoding mouse ATF 12 Human I2S gene 13 Sequence encoding human ATF

SEQ ID NO: 1 = DNA sequence encoding IRES-Hygr-mPGKpA SEQ ID NO: 2 = IRES5 '
Sequence number 3 = IRES3 '
Sequence number 4 = mPGKP5 '
Sequence number 5 = mPGKP3 '
SEQ ID NO: 6 = DNA sequence including mPGK promoter region SEQ ID NO: 7 = GS5 ′
Sequence number 8 = GS3 '
Sequence number 9 = puro5 '
Sequence number 10 = puro3 '
SEQ ID NO: 11 = DNA containing puromycin resistance gene SEQ ID NO: 12 = SV40polyA5 ′
Sequence number 13 = SV40polyA3 '
Sequence number 14 = mIRES-GS5 '
Sequence number 15 = mIRES-GS3 '
Sequence number 16 = hATF-f
Sequence number 17 = hATF-r
SEQ ID NO: 18 = mATF-f
SEQ ID NO: 19 = mATF-r
Sequence number 20 = hI2S-f
Sequence number 21 = hI2S-r
Sequence number 22 = hI2S-f2
Sequence number 23 = hI2S-r2
Sequence number 24 = hI2S-f3
Sequence number 25 = hI2S-r3
SEQ ID NO: 26 = DNA sequence of mATF-fused hI2S gene SEQ ID NO: 27 = amino acid sequence of mATF-fused hI2S SEQ ID NO: 28 = DNA sequence of hATF-fused hI2S gene SEQ ID NO: 29 = amino acid sequence of hATF-fused hI2S SEQ ID NO: 30 = mATF amino acid sequence SEQ ID NO: 31 = mATF receptor binding domain amino acid sequence SEQ ID NO: 32 = hATF amino acid sequence SEQ ID NO: 33 = hATF receptor binding domain amino acid sequence SEQ ID NO: 34 = His signal at the N-terminal and His -Synthetic DNA sequence encoding hMPR9 with tag at C-terminus SEQ ID NO: 35 = hMPR9-f
SEQ ID NO: 36 = hMPR9-r
SEQ ID NO: 37 = MPR9-f2
SEQ ID NO: 38 = MPR9-r2
SEQ ID NO: 39 = MPR9-f3
SEQ ID NO: 40 = MPR9-r3

Claims (16)

Lysosomal enzymes (A) and fused thereto Peptide (B) and the unrealized peptide (B) is comprising an agent which is intended to include receptor binding domain of ATF, enzyme replacement therapy agent composition. The lysosomal enzyme is selected from the group consisting of acid α-glucosidase, α-galactosidase, glucosylceramidase, α-L-iduronidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-4-sulfatase, or acid sphingomyelinase. The pharmaceutical composition of claim 1 , wherein The peptide (B) is one in which the peptide bond formed between the carboxy terminal amino acids of the amino-terminal amino acid and the peptide of the protein (B), are fused to the protein, according to claim 1 or 2 Pharmaceutical composition . The peptide (B) is one in which the peptide bond formed between the amino-terminal amino acids of the carboxy terminal amino acid and the peptide of the protein (B), are fused to the protein, according to claim 1 or 2 Pharmaceutical composition . The peptide (B) is the amino acid sequence shown in SEQ ID NO: 33, or 1 to 3 amino acids are added, substituted, deleted, or made of the insertion derivative thereof, any one of claims 1-4 Pharmaceutical composition . The peptide (B) is an amino acid sequence or 1-10 amino acids shown in SEQ ID NO: 32 are added, substituted, deleted, or those consisting inserted derivative thereof, of any one of claims 1-4 Pharmaceutical composition . The peptide (B) consists of the amino acid sequence shown in SEQ ID NO: 32, and the peptide (B) is fused to the lysosomal enzyme (A) via a linker consisting of at least one amino acid at the carboxy terminal amino acid. 7. The pharmaceutical composition of claim 6 , wherein The linker is -Lys-, Lys-Pro-, -Lys-Pro-Ser-, -Lys-Pro-Ser-Ser- (SEQ ID NO: 41), and -Lys-Pro-Ser-Ser-Pro- (sequence The pharmaceutical composition according to claim 7 , which is selected from the group consisting of No. 42). The peptide (B) is an amino acid sequence or 1-5 amino acids shown in SEQ ID NO: 31 are added, substituted, deleted, or those consisting inserted derivative thereof, of any one of claims 1-4 Pharmaceutical composition . The peptide (B) is an amino acid sequence or 1-10 amino acids shown in SEQ ID NO: 31 are added, substituted, deleted, or those consisting inserted derivative thereof, of any one of claims 1-4 Pharmaceutical composition . The peptide (B) comprises the amino acid sequence shown in SEQ ID NO: 31 , and the peptide (B) is linked to the lysosomal enzyme (A) via a linker consisting of at least one amino acid at the carboxy terminal amino acid. The pharmaceutical composition of claim 10 , which is fused. The linker is -Lys-, Lys-Pro-, -Lys-Pro-Ser-, -Lys-Pro-Ser-Ser- (SEQ ID NO: 41), and -Lys-Pro-Ser-Ser-Pro- (sequence The pharmaceutical composition according to claim 11 , which is selected from the group consisting of No. 42). A lysosomal enzyme (A) and a peptide (B) fused thereto, wherein the peptide (B) comprises a receptor binding domain of ATF, and the peptide (B) comprises the peptide (B) DNA encoding a fusion protein which is fused to the lysosomal enzyme (A) by a peptide bond at the amino or carboxy terminus. The lysosomal enzyme is selected from the group consisting of acid α-glucosidase, α-galactosidase, glucosylceramidase, α-L-iduronidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-4-sulfatase, and acid sphingomyelinase. The DNA of claim 13 , wherein An expression vector comprising the DNA of claim 13 or 14 . A mammalian cell transformed with the vector of claim 15 .