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WO2013118165A1 - Agents pharmaceutiques liés par fusion à l'atf pour une biodisponibilité améliorée - Google Patents

Agents pharmaceutiques liés par fusion à l'atf pour une biodisponibilité améliorée Download PDF

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Publication number
WO2013118165A1
WO2013118165A1 PCT/JP2012/000805 JP2012000805W WO2013118165A1 WO 2013118165 A1 WO2013118165 A1 WO 2013118165A1 JP 2012000805 W JP2012000805 W JP 2012000805W WO 2013118165 A1 WO2013118165 A1 WO 2013118165A1
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peptide
fused
seq
pharmaceutical agent
hi2s
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PCT/JP2012/000805
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English (en)
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Kenichi Takahashi
Eiji YODEN
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Jcr Pharmaceuticals Co., Ltd.
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Priority to PCT/JP2012/000805 priority Critical patent/WO2013118165A1/fr
Priority to JP2014537404A priority patent/JP5957085B2/ja
Publication of WO2013118165A1 publication Critical patent/WO2013118165A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06013—Iduronate-2-sulfatase (3.1.6.13)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21031—Urokinase (3.4.21.31)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the present invention relates to pharmaceutical agents with improved bioavailability and a method for improving bioavailability of drugs using the amino terminal fragment of the urokinase-type plasminogen activator (ATF). More specifically, the present invention relates to delivery of pharmaceutical proteins fused to ATF into cells via binding of the ATF moiety to the urokinase type plasminogen activator receptor expressing itself on the surface of the cells.
  • ATF urokinase-type plasminogen activator
  • the urokinase-type plasminogen activator is a protein consisting of two peptide chains linked to each other by a disulfide bond. These chains, A and B, are formed by enzymatic cleavage (with plasmin, kallikrein, cathepsin, etc.) between amino acids 178 and 179 of prourokinase, which in turn is formed by removal of the N-terminal signal peptide (amino acids 1-20) from a single-chain protein called prepro-urokinase (sc-uPA).
  • sc-uPA prepro-urokinase
  • uPA then is cleaved between amino acids 155 and 156 in vivo, thereby giving rise to the low molecular weight urokinase-type plasminogen activator (LMW-uPA) and the amino terminal fragment of urokinase type plasminogen activator (ATF)
  • LMW-uPA low molecular weight urokinase-type plasminogen activator
  • ATF amino terminal fragment of urokinase type plasminogen activator
  • uPA possesses a proteolytic activity and converts plasminogen to plasmin in physiological conditions to trigger a proteolytic cascade which results in thrombosis or extracellular matrix degradation.
  • uPA comprises two domains, i.e. ATF and the protease domain.
  • ATF is the region consisting of amino acids 1-135 on the amino terminal side, while the protease domain is the region consisting of amino acids 136 to 411 on the carboxy terminal side of uPA.
  • ATF thus does not have a protease activity but an affinity for the urokinase type plasminogen activator receptor (uPAR)(NPL 1).
  • ATF itself is composed of two independent domains, i.e. the amino-terminal growth factor-like domain (1 to 43 amino acids from the N-terminus), with which uPA binds to uPAR, and a single kringle domain (43 to 135 amino acids from N-terminus) (NPL 2, 3). Therefore, ATF is itself able to bind to uPAR via the amino-terminal growth factor-like domain.
  • uPAR is a GPI-anchored membrane protein, consists of 335 amino acids, and has three extracellular domains, of which the first domain from N-terminus acts as the binding site of uPA(NPL 4).
  • ATF binding to uPAR is not internalized into the cell, whereas uPA binding to uPAR is internalized if complexed with plasminogen activator inhibitors (NPL 5).
  • uPAR associated protein known also as ENDO180
  • ENDO180 is a lectin-like membrane protein belonging to the macrophage mannose receptor protein family (NPL 6).
  • NPL 6 macrophage mannose receptor protein family
  • ENDO180 is an internalization receptor, which directs a ligand bound to it to the lysosome, where lysosomal enzymes exert their catalytic activities to degrade the ligand.
  • ENDO180 is also known to form a triple complex with uPA and uPAR.
  • Lysosomal diseases a group of rare inherited metabolic disorders, are caused by various genetic disorders in the genes encoding lysosomal enzymes. Lack of enzymatic activities of some of those enzymes results in accumulation of their substrates in cells, which then inflicts severe damages on the cells.
  • Lysosomal diseases include, for example, Pompe disease, Fabry's disease, Gaucher's disease, Hurler syndrome (mucopolysaccharidosis type I, MPS I), Hunter syndrome (MPS II), Maroteaux-Lamy syndrome (MPS VI), Niemann-Pick disease, and Morquio syndrome (MPS IV).
  • ERT enzyme replacement therapies
  • Pompe disease caused by acid alpha-glucosidase deficiency, is a lysosomal storage disorder clinically characterized by muscle weakness and cardiomyopathy. It has been well established that an enzyme replacement therapy (ERT) is effective in this disease to ameliorate weakening muscle strength and to improve heart functions, where recombinant acid alpha-glucosidase is administered to the patients. Efficacy of this therapy has been confirmed by several clinical trials in patients with different ages of onset and disease severity (NPL 7).
  • ERT enzyme replacement therapy
  • Fabry's disease caused by alpha-galactosidase deficiency, is a lysosomal storage disorder clinically characterized by complication of pain, kidney failure, cardiomyopathy, and cerebrovascular events, which are responsible for morbidity and mortality of this disease.
  • ERT enzyme replacement therapy
  • QOL quality of life
  • Gaucher's disease a genetic disease caused by an inherited disorder of glucosylceramidase, is a lysosomal storage disorder characterized by bruising, fatigue, anemia, low blood platelets, and enlargement of the liver and spleen. It has been well established that enzyme replacement therapy (ERT) is effective for improving patient' quality of life (QOL) (NPL 9).
  • ERT enzyme replacement therapy
  • Hurler syndrome known as mucopolysaccharidosis type IH, is a genetic disease caused by an inherited disorder of alpha-L-iduronidase. Sheie's syndrome, mucopolysaccharidosis type IS, is a mild case of Hurler's syndrome. These diseases, including an intermediate type, are categorized as Hurler-Sheie's syndrome. Patients of Hurler syndrome accumulate dermatan sulphate and heparan sulphate in the lysosomes, and consequently show progressive strong body deformation and mental detergency which set in at the infantile stage, resulting in death by age 10. In the case of Sheie's syndrome, mental detergency is not, or only slightly, observed.
  • Hunter's syndrome is a genetic disease caused by an inherited disorder of alpha-L-iduronidase, iduronate-2-sulfatase (I2S) (NPL 11).
  • I2S is a lysosomal enzyme having an activity to hydrolyze sulfate ester bonds in the molecules of glycosaminoglycans, such as heparan sulfate and dermatan sulfate. Lack of this enzyme results in the accumulation of its substrates in tissues, such as those of the liver and kidney, which then causes diverse symptoms as seen in patients suffering Hunter's syndrome, including skeletal deformities and severe mental retardation. It has been well established that the enzyme replacement therapy (ERT) improves physical capacity of patients (NPL 12).
  • ERT enzyme replacement therapy
  • Maroteaux-Lamy syndrome mucopolysaccharidosis type VI, is an inherited disease caused by a genetic disorder of N-acetylgalactosamine-4-sulfatase (ASB).
  • ASB N-acetylgalactosamine-4-sulfatase
  • ASB known as arylsulfatase B
  • ERT enzyme replacement therapy
  • ASM acidic sphingomyelinase
  • a patient receives intravenous infusion of the enzyme that he or she lacks.
  • the enzyme For the infused enzyme to exhibit its clinical effects, it is prerequisite that the enzyme be taken up into the cells where it should act. Further, for the enzyme to exhibit its clinical effect, it is also requisite that the enzyme be sorted into lysosomes where the enzyme is to exert its catalytic activity.
  • the enzymes which are used for ERT have sugar chains linked to them containing mannose-6-phosphate or mannose residues, and through binding to the mannose-6-phosphate receptor or the mannose receptor expressing itself on the surface of the cells, the enzymes are taken up by the cells and then sorted into the lysosomes, where they exert their enzymatic activities (NPL 14-16).
  • the mannose-6-phosphate receptor and the mannose receptor are molecules ubiquitously expressed in human body, the binding of the enzymes to these receptors is not enough for the enzymes to be sorted to the specific tissues where they should exert their activities.
  • these receptors are expressed in both of the liver and spleen, it is possible that infused enzymes containing mannose or mannose-6-phosphate residues in their sugar chains are rapidly removed by these organs (NPL 14).
  • the enzymes should be modified with some or other ligand which has affinity for other molecules on the cells than those of mannose-6-phosphate receptor or mannose receptor.
  • ERT mannose-6-phosphate receptor
  • hI2S human iduronate-2-sulfatase fused to human or mouse ATF can be taken up, via its binding to the urokinase-type plasminogen activator receptor (uPAR), into fibroblasts, where hI2S can exert its catalytic activity.
  • uPAR urokinase-type plasminogen activator receptor
  • a pharmaceutical agent comprising a pharmacologically active compound (A) and a peptide (B) fused thereto, wherein the peptide (B) comprises the receptor binding domain of ATF.
  • the pharmacologically active compound (A) is a protein.
  • the protein is an enzyme.
  • the enzyme is a lysosomal enzyme. 5.
  • lysosomal enzyme is selected from the group consisting of acid alpha-glucosidase, alpha-galactosidase, glucosylceramidase, alpha-L-iduronidase, alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase, or acidic sphingomyelinase.
  • the peptide (B) is fused to the pharmacologically active compound (A) by an amide bond or an ester bond. 7.
  • the peptide (B) is fused at the carboxy-terminal amino acid thereof to the pharmacologically active compound (A) through a linker consisting of at least one amino acid.
  • the linker is selected from the group consisting of -Lys-, Lys-Pro-, -Lys-Pro-Ser-, -Lys-Pro-Ser-Ser- (SEQ ID NO:41), and -Lys-Pro-Ser-Ser-Pro- (SEQ ID NO:42).
  • peptide (B) is fused at the carboxy-terminal amino acid thereof to the pharmacologically active compound (A) through a linker consisting of at least one amino acid.
  • linker is selected from the group consisting of -Lys-, Lys-Pro-, -Lys-Pro-Ser-, -Lys-Pro-Ser-Ser- (SEQ ID NO:41), and -Lys-Pro-Ser-Ser-Pro- (SEQ ID NO:42).
  • a pharmaceutical composition for enzyme replacement therapy comprising the pharmaceutical agent according to one of 1 to 17 above. 19.
  • the pharmacologically active protein is an enzyme.
  • the enzyme is a lysosomal enzyme. 22.
  • lysosomal enzyme is selected from the group consisting of acid alpha-glucosidase, alpha-galactosidase, glucosylceramidase , alpha-L-iduronidase, alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase, acidic sphingomyelinase.
  • An expression vector comprising the DNA according to one of 19 to 22 above.
  • 24. A mammalian cell transformed with the vector according to 23 above.
  • the present invention provides a means which serves to improve cellular uptake of pharmacologically active compounds through their binding to the urokinase type plasminogen activator receptor (uPAR) on the cells, and thus also provides pharmaceutical agents with improved uptake by cells expressing uPAR.
  • uPAR urokinase type plasminogen activator receptor
  • this improved cellular uptake is to be independent from any interaction of the pharmaceutical agent with the mannose-6-phosphate receptor or the mannose receptor on the cells
  • the present invention provides such pharmaceutical agents that, despite having no or only a small number of mannose or mannose-6-phosphate residues associated to their molecule, can still be taken up into cells
  • Fig. 1-1 shows a flow diagram illustrating the method for construction of pBSK(IRES-Hygr-mPGKpA) vector.
  • Fig. 1-2 shows a flow diagram illustrating the method for construction of pBSK(NotI-IRES-Hygr-mPGKpA) vector.
  • Fig. 1-3 shows a flow diagram illustrating the method for construction of pE-IRES-Hygr vector.
  • Fig. 1-4 shows a flow diagram illustrating the method for construction of pPGK-neo vector.
  • Fig. 1-5 shows a flow diagram illustrating the method for construction of pPGK-IRES-Hygr vector.
  • Fig. 1-1 shows a flow diagram illustrating the method for construction of pBSK(IRES-Hygr-mPGKpA) vector.
  • Fig. 1-2 shows a flow diagram illustrating the method for construction of pBSK(NotI-IRES-Hygr-mPGKp
  • 1-6 shows a flow diagram illustrating the method for construction of pPGK-IRES-GS-delta-polyA vector.
  • Fig. 1-7 shows a flow diagram illustrating the method for construction of pE-puro vector.
  • Fig. 1-8 shows a flow diagram illustrating the method for construction of pE-puro(XhoI) vector.
  • Fig. 1-9 shows a flow diagram illustrating the method for construction of pE-IRES-GS-puro vector.
  • Fig. 2 shows a flow diagram illustrating the method for construction of pE-mIRES-GS-puro vector.
  • Fig. 3 shows a partial schematic diagram of pE-mIRES-GS-puro(mATF-hI2S) vector.
  • Fig. 1-7 shows a flow diagram illustrating the method for construction of pE-puro vector.
  • Fig. 1-8 shows a flow diagram illustrating the method for construction of pE-puro(XhoI) vector.
  • FIG. 4 shows a partial schematic diagram of pE-mIRES-GS-puro(hATF-hI2S) vector.
  • Fig. 5 shows a result of SDS PAGE analysis of purified mATF-fused hI2S. Fractions from HiTrap Q-Sepharose FF which contained hI2S activities were loaded.
  • Fig. 6 shows a graph showing the result of measurement of cellular uptake of hATF-fused hI2S into human fibroblasts. The vertical axis shows the amount of hATF-fused hI2S or hI2S (ng) taken up per unit mass (mg) of the total cellular protein (ng/mg total cellular protein).
  • the horizontal axis shows the concentration of hATF-fused hI2S or hI2S added to the medium.
  • Filled circles indicate the values of cellular uptake of hATF-fused hI2S
  • an open circle indicates the value of cellular uptake of hATF-fused hI2S in the presence of 10 mM M6P
  • an open triangle indicates the values of cellular uptake of hATF-fused hI2S in the presence of 10 mM M6P and 4.02 micrograms/mL hATF.
  • Filled boxes indicate the values of cellular uptake of hI2S
  • an open box indicates the value of cellular uptake of hI2S in the presence of 10 mM M6P.
  • Fig. 7 shows the result of measurement of blockage by M6P of cellular uptake of hATF-fused hI2S into human fibroblasts.
  • the vertical axis shows the cellular uptake ratio of hATF-fused hI2S or hI2S (%).
  • the horizontal axis shows the concentration of M6P added to the medium (mM). Filled circles indicate the values of cellular uptake ratio of hATF-fused hI2S, and filled boxes indicate the values of cellular uptake ratio of hI2S.
  • Fig. 8 shows the result of measurement of cellular uptake of mATF-fused hI2S into mouse fibroblast.
  • the vertical axis shows the amount of mATF-fused hI2S or hI2S (ng) taken up per unit mass (mg) of the total cellular protein (ng/mg total cellular protein).
  • the horizontal axis shows the concentration of mATF-fused hI2S or hI2S added to the medium. Filled circles indicate the values of cellular uptake of mATF-fused hI2S, and an open circle indicates the value of cellular uptake of mATF-fused hI2S in the presence of 10mM M6P.
  • Filled boxes indicate the values of cellular uptake of hI2S, and an open box indicates the value of cellular uptake of hI2S in the presence of 10mM M6P.
  • Fig. 9 shows the result of measurement of blockage by M6P of cellular uptake of mATF-fused hI2S into mouse fibroblasts.
  • the vertical axis shows the cellular uptake ratio of mATF-fused hI2S or hI2S (%).
  • the horizontal axis shows the concentration of M6P added to the medium (mM).
  • Filled circles indicate the value of uptake ratio of mATF-fused hI2S, and filled boxes indicate the value of uptake ratio of hI2S.
  • Fig. 10 shows the alignment of amino acid sequences of human ATF (top row) and mouse ATF (bottom row). Asterisks indicate the different amino acids between human ATF and mouse ATF.
  • the amino terminal fragment of urokinase type plasminogen activator is preferably the mammalian ATF, more preferably the human or mouse ATF, and particularly preferably the human ATF.
  • the amino acid sequences of the wild type mouse and human ATFs are shown as SEQ ID NO:30 and SEQ ID NO:32, respectively.
  • ATF is preferably of the wild type, but mutated ATF, which has undergone addition, substitution, deletion, and/or insertion of one or more amino acids, is also preferably used.
  • the number of amino acids added, substituted, deleted, and inserted in such a mutated ATF is preferably 1 to 10, more preferably 1 to 8, still more preferably 1 to 5, and further more preferably 1.
  • a peptide comprising the receptor binding domain of ATF is preferably a mammalian peptide, more preferably the human or mouse peptide, and particularly preferably the human peptide.
  • the term "peptide comprising the receptor binding domain of ATF" herein does not include a compound comprising uPA.
  • the amino acid sequences of the receptor binding domains of the wild type mouse and human ATFs are shown as SEQ ID NO:31 and SEQ ID NO:33, respectively.
  • the receptor binding domain of ATF is preferably of the wild type, but a mutated receptor binding domain of ATF which has undergone addition, substitution, deletion, and/or insertion of one or more amino acids is also preferably used.
  • the number of amino acids added, substituted, deleted, and inserted in such a mutated receptor binding domain of ATF is preferably 1 to 5, more preferably 1 to 3, and still more preferably 1.
  • the pharmaceutical agent according to the present invention is an agent to be administered to mammals, preferably human, livestock including horse, pig, cattle, and sheep, and pet animals including dog and cat.
  • the agent is preferably administered intravenously.
  • the term "pharmaceutical agent" does not include a compound comprising uPA.
  • pharmacologically active compounds since it is the receptor binding domain of ATF that plays the key role in leading a pharmacologically active compound to the binding to the uPAR on the surface of cells and then into them, there is no particular limitation as to the scope of pharmacologically active compounds to which the peptide comprising the receptor binding domain of ATF is to be fused.
  • Preferred examples of pharmacologically active compounds include, but are not limited to, proteins, polysaccharides, lipids, and other kind of chemicals including antibiotics, anticancer agents, and anti-inflammatory agents. Among them, proteins are particularly preferred.
  • the peptide comprising the receptor binding domain of ATF may be fused to a pharmacologically active compound by any kind of chemical bond as desired, of which amide bond and ester bond are preferred.
  • the 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 on its carboxy- or amino-terminal amino acid of the peptide. While there is no particular limitation on the number of the peptide comprising the receptor binding domain of ATF to be fused per molecule of a pharmacologically active compound, the number is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3, and further more preferably 1.
  • a pharmacologically active compound to which the peptide comprising the receptor binding domain of ATF is to be fused is a protein
  • the peptide is preferably fused to the protein by a peptide bond. More preferably, the peptide comprising the receptor binding domain of ATF may be fused to the protein, either at the protein's carboxy- or amino-terminal amino acid. Further the peptide comprising the receptor binding domain of ATF may be fused to the protein via linker.
  • a linker for this purpose may be a single or more amino acids interposed between two adjacent termini of the protein and the peptide comprising the receptor binding domain of ATF.
  • the linker may consist of preferably 1 to 20 amino acids, more preferably 1 to 10 amino acids, and still more preferably 1 to 5 amino acids.
  • the amino acid sequence of such a linker may be preferably selected from the group consisting of -Lys-, Lys-Pro-, -Lys-Pro-Ser-, -Lys-Pro-Ser-Ser- (SEQ ID NO:41), and -Lys-Pro-Ser-Ser-Pro- (SEQ ID NO:42).
  • a fusion protein as a pharmaceutical agent made of the peptide comprising the receptor binding domain of ATF and a pharmacologically active protein fused between the carboxy-terminal amino acid of the former and the amino-terminal amino acid of the latter can be produced by letting a fusion gene express itself which comprises the gene encoding the peptide comprising the receptor binding domain of ATF and the gene encoding the pharmacologically active protein fused in frame in this order tandemly in the 5' to 3' orientation.
  • fusion genes are shown as SEQ ID NO:26, i.e., a fusion gene between the mouse ATF and the human I2S genes, and as SEQ ID NO:28, i.e., a fusion gene between the human ATF and the human I2S genes.
  • a fusion protein as a pharmaceutical agent made of the peptide comprising the receptor binding domain of ATF and a pharmacologically active protein fused between the amino-terminal amino acid of the former and the carboxy-terminal amino acid of the latter can be produced by letting a fusion gene express itself which comprises the gene encoding the pharmacologically active protein and the gene encoding the peptide comprising the receptor binding domain of ATF fused in frame in this order tandemly, in the 5' to 3' orientation.
  • a gene chosen is inserted into an expression vector, and the expression vector thus obtained then is introduced into host cells.
  • the species to which the host cells to be used above belongs are preferably E. coli, yeast, insect cells, or mammalian cells, among which mammalian cells are more preferred, and CHO cells are particularly preferred.
  • pharmacologically active proteins there is no particular limitation as to the scope of pharmacologically active proteins to which the peptide comprising receptor binding domain of ATF is to be fused.
  • Preferred examples of pharmacologically active proteins include, but are not limited to, enzymes, among which particularly preferred are lysosomal enzymes, inter alia acid alpha-glucosidase, alpha-galactosidase, alpha-L-iduronidase, alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase, or acidic sphingomyelinase.
  • Pharmacologically active proteins to be employed in the present invention are preferably mammalian proteins, and more preferably human proteins.
  • a lysosomal enzyme and the peptide comprising the receptor binding domain of ATF can be fused via a linker consisting of at least one amino acid, which may form peptide.
  • a linker is covalently interposed between the amino- and carboxy-terminal amino acids of the two moieties to be linked, i.e., an enzyme employed and the peptide comprising receptor binding domain of ATF.
  • a linker comprises preferably 1 to 20 amino acids, more preferably 1 to 10 amino acids, and still more preferably 1 to 5 amino acids.
  • a lysosomal enzyme fused to the peptide comprising the receptor binding domain of ATF can be used as a pharmaceutical agent in enzyme replacement therapy for lysosomal disease.
  • alpha-glucosidase, alpha-galactosidase, alpha-L-iduronidase, glucosylceramidase, alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase, or acidic sphingomyelinase fused to the peptide comprising the receptor binding domain of ATF is used as pharmaceutical agent in enzyme replacement therapy for Pompe disease, Fabry's disease, Gaucher's disease, Hurler syndrome (mucopolysaccharidosis type I, MPS I), Hunter syndrome (MPS II), and Niemann-Pick disease, respectively.
  • pPGKIH expression vector (Construction of pE-IRES-GS-puro vector) was digested with XhoI and BamHI to cut out a DNA fragment containing Internal Ribosome Entry Site (IRES) derived from murine encephalomyelitis virus (EMCV), hygromycin resistance gene (Hygr gene), and polyadenilation signal (mPGKpA) of mouse phosphoglycerate kinase (mPGK).
  • IRES Internal Ribosome Entry Site
  • EMCV murine encephalomyelitis virus
  • Hygr gene hygromycin resistance gene
  • mPGKpA polyadenilation signal of mouse phosphoglycerate kinase
  • nucleotide sequence is set forth as SEQ ID NO:1, in which (i) nucleotides 1-6 represent an XhoI site, (ii) nucleotides 7-119 an intermediate sequence (iii) nucleotides 120-718, including the "atg” at the 3' end of this region, represent a sequence containing IRES derived from 5' untranslated region of murine encephalomyelitis virus, (iv) nucleotides 716-1741 a hygromycin resistance gene (Hygr gene), (v) nucleotides 1742-1746 an intermediate sequence (vi) nucleotides 1747-2210 the sequence containing polyadenilation signal sequence of mouse phosphoglycerate kinase gene(mPGKpA), and (vii) nucleotides 2211-2216, i.e., the six nucleotides at the 3'-terminus, a
  • a DNA fragment containing part of EMCV-IRES was amplified by PCR performed with a set of primers, IRES5' (SEQ ID NO:2) and IRES3' (SEQ ID NO:3), and using pBSK (IRES-Hygr-mPGKpA) as a template.
  • the PCR-amplified DNA fragment was digested with XhoI and HindIII, and subsequently inserted between XhoI and HindIII sites of pBSK vector, and the vector thus obtained was designated pBSK (NotI-IRES-Hygr-mPGKpA)(Fig. 1-2).
  • pBSK NotI-IRES-Hygro-mPGKpA
  • pE-hygr vector was constructed as per the procedure disclosed in a patent literature (JP2011-172602). The resulting vector was designated pE-IRES-Hygr (Fig. 1-3).
  • PCR was performed with a set of primers, mPGKP5' (SEQ ID NO:4) and mPGKP3' (SEQ ID NO:5), and using pPGKIH as a template to amplify a DNA fragment containing mPGK promoter region (mPGKp), whose nucleotide sequence is set forth as SEQ ID NO:6, in which (i) nucleotides 1-3 represent an intermediate sequence (ii) nucleotides 4-9 a BamHI site, (iii) nucleotides 10-516 a sequence containing mPGKp, (iv) nucleotides 517-523 an intermediate sequence , (v) nucleotide 524-529 an EcoRI site, and (vi) nucleotides 530-532, the last three nucleotides, represent an intermediate sequence.
  • the amplified DNA fragment then was digested with BglII and EcoRI, and inserted between BglII and EcoRI sites of pCI-neo (Promega).
  • the vector thus obtained was designated pPGK-neo (Fig. 1-4).
  • pE-IRES-Hygr was digested with NotI and BamHI, and the DNA fragment thus obtained (IRES-Hygr) was inserted between NotI and BamHI sites of pPGK-neo.
  • the vector thus obtained was designated pPGK-IRES-Hygr (Fig. 1-5).
  • a DNA fragment containing a glutamine synthetase gene was amplified by PCR performed with a set of primers, GS5' (SEQ ID NO:7) and GS3' (SEQ ID NO:8), and using a cDNA library prepared from mRNA of CHO-K1 cells as a template.
  • the DNA fragment thus obtained was digested with BalI and BamHI, and inserted between BalI and BamHI sites of pPGK-IRES-Hygr.
  • the vector obtained was designated pPGK-IRES-GS-delta-polyA (Fig. 1-6).
  • PCR was performed with a set of primers, puro5' (SEQ ID NO:9) and puro3' (SEQ ID NO:10), and using pCAGIPuro (Miyahara M. et.al., J. Biol. Chem. 275,613-618(2000)) as a template to amplify a DNA fragment containing puromycin resistance gene (Puro gene), whose nucleotide sequence is set forth as SEQ ID NO:11, in which (i) nucleotides 2-7 represent an AflII site, (ii) nucleotides 8-607 the sequence containing Puro gene, and (iii) nucleotides 608-619 a BstXI site.
  • pE-neo vector was constructed as per the procedure disclosed in a patent literature (JP2011-172602). The vector thus obtained was designated pE-puro (Fig. 1-7).
  • PCR was performed with a set of primers, SV40polyA5' (SEQ ID NO:12) and SV40polyA3' (SEQ ID NO:13), and using pE-puro as a template to amplify a DNA fragment containing the SV40 late polyadenilation region.
  • the DNA fragment thus amplified was digested with NotI and HpaI, and inserted between NotI and HpaI sites of pE-puro.
  • the vector obtained was designated pE-puro(XhoI) (Fig. 1-8).
  • pPGK-IRES-GS-delta-polyA was digested with NotI and XhoI, and inserted between NotI and XhoI sites of pE-puro(XhoI).
  • the vector obtained was designated pE-IRES-GS-puro (Fig. 1-9).
  • PCR reaction was performed with a set of primers, mIRES-GS5' (SEQ ID NO:14) and mIRES-GS3' (SEQ ID NO:15), and using pE-IRES-GS-puro as a template to amplify a DNA fragment containing EMCV-IRES of which the second start codon was disrupted. Then, using the DNA fragment thus amplified and aforementioned IRES5' as primers and, as a template, pE-IRES-GS-puro, PCR reaction was performed to amplify the region from EMCV-IRES to GS gene.
  • the DNA fragment thus amplified containing the region from EMCV-IRES to GS gene, was digested with NotI and PstI and inserted between NotI and PstI sites of pE-IRES-GS-puro.
  • the vector obtained was designated pE-mIRES-GS-puro (Fig. 2).
  • hATF gene was amplified by PCR performed with a set of primers, hATF-f (SEQ ID NO:16, in which nucleotides 4-9 represent an MluI site) and hATF-r (SEQ ID NO:17, in which nucleotides 4-9 represent an XhoI site), and using human kidney Quick Clone cDNA (Clontech) as a template.
  • hATF-f SEQ ID NO:16, in which nucleotides 4-9 represent an MluI site
  • hATF-r SEQ ID NO:17, in which nucleotides 4-9 represent an XhoI site
  • nucleotides 4-9 represent MluI or XhoI site, respectively.
  • the fragment thus amplified was digested with MluI and XhoI, and then inserted between MluI and XhoI sites of pCI-neo vector (Promega) to give pCI-neo (hATF) vector.
  • mATF gene was amplified by PCR performed with a set of primers, mATF-f (SEQ ID NO:18) and mATF-r (SEQ ID NO:19), and using mouse kidney Quick Clone cDNA (Clontech) as a template.
  • mATF-f SEQ ID NO:18
  • mATF-r SEQ ID NO:19
  • nucleotides 4-9 represent MluI or XhoI site, respectively.
  • the fragment thus amplified was digested with the MluI and XhoI, and then inserted between MluI and XhoI sites of pCI-neo (Promega) to give pCI-neo (mATF) vector.
  • hI2S human placenta cDNA library
  • hI2S-f a set of outer primers
  • hI2S-r a set of inner primers
  • hI2S-f2 SEQ ID NO:22
  • hI2S-r2 SEQ ID NO:23
  • telomere sequence was amplified by PCR performed with a set of primers, hI2S-f3 (SEQ ID NO:24) and hI2S-r3 (SEQ ID NO:25), and using pT7Blue(hI2S) vector as a template.
  • nucleotides 4-9 represent a XhoI site
  • nucleotides 4-11 represent a NotI site.
  • the PCR fragment thus amplified was digested with the XhoI and NotI, and then inserted between XhoI and NotI sites of pCI-neo (Promega) to give pCI-neo(delta-S-hI2S) vector.
  • pCI-neo (mATF) vector was digested with MluI and XhoI to cut out the cDNA encoding mATF.
  • pCI-neo (delta-S-hI2S) was digested with XhoI and NotI to cut out the cDNA encoding hI2S lacking the signal sequence. Then both cDNAs were tandemly inserted between MluI and NotI sites of pE-mIRES-GS-puro to give pE-mIRES-GS-puro (mATF-delta-S-hI2S) (Fig. 3).
  • the DNA sequence of mATF-fused hI2S gene embedded in the above vector is set forth as SEQ ID NO:26.
  • the amino acid sequence of mATF-fused hI2S translated from the gene is set forth as SEQ ID NO:27, in which the portion consisting of the first 160 amino acids correspond to part of mouse sc-uPA, of which the first 21 amino acids correspond to the leader sequence, with the 135 amino acids that follow (amino acids 22 to 156) corresponding to mouse ATF (mATF, SEQ ID NO:30).
  • the first 43-amino acid sequence of mATF comprises the receptor binding domain of mATF (SEQ ID NO:31).
  • mATF-fused hI2S after removal of the leader sequence, is secreted from the cells.
  • pCI-neo (hATF) vector was digested with MluI and XhoI to cut out the cDNA encoding hATF.
  • pCI-neo (delta-S-hI2S) was digested with XhoI and NotI to cut out the cDNA encoding hI2S lacking the signal sequence. Then both cDNAs were tandemly inserted between MluI and NotI sites of pE-mIRES-GS-puro to give pE-mIRES-GS-puro (hATF-delta-S-hI2S) (Fig. 4).
  • the DNA sequence of hATF-fused hI2S gene embedded in the above vector is set forth as SEQ ID NO:28.
  • the amino acid sequence of hATF-fused hI2S translated from the gene is set forth as SEQ ID NO:29, in which the portion consisting of the first 159 amino acids corresponds to part of human sc-uPA, of which the first 20 amino acids correspond to the leader sequence, with the 135 amino acids that follow (amino acids 21 to 155) corresponding to mouse ATF (hATF, SEQ ID NO:32).
  • the first 43-amino acid sequence of hATF comprises the receptor binding domain of hATF (SEQ ID NO:33).
  • CHO-K1 cells purchased from American Type Culture Collection
  • pE-mIRES-GS-puro(mATF-hI2S) or pE-mIRES-GS-puro(hATF-hI2S) were transfected with one of the above-mentioned expression vectors, pE-mIRES-GS-puro(mATF-hI2S) or pE-mIRES-GS-puro(hATF-hI2S), using Lipofectamine2000 (Invitrogen) according to the following method.
  • CHO-K1 cells were seeded in a 3.5-cm culture dish containing 3 mL of D-MEM/F12 medium containing 5% FCS (D-MEM/F12/5%FCS), and the cells were cultured overnight at 37 deg C in a humidified atmosphere of 5% CO 2 and 95% air.
  • the cells were transfected with 300 microliters of a 1:1 mixture solution consisting of Lipofectamine 2000 solution diluted 25 times with Opti-MEM I medium (Invitrogen) and a plasmid DNA solution diluted with Opti-MEM I medium to 13.2 micrograms/mL, at 37 deg C in a humidified atmosphere of 5% CO 2 and 95% air over night.
  • Opti-MEM I medium Invitrogen
  • the medium was replaced with a selection medium (the CD Opti CHO culture medium (Invitrogen) supplemented with 10 micrograms/mL insulin, 100 micromole/L hypoxanthine, 16 micromole/L thymidine and 30 micromole/L methionine sulfoximine (MSX, Sigma)), and a selective culture was carried out at 37 deg C in a humidified atmosphere of 5% CO 2 and 95% air. Cells that had grown in the selection medium were subjected to several successive rounds of subculture in the medium to give recombinant cells.
  • the CD Opti CHO culture medium the CD Opti CHO culture medium (Invitrogen) supplemented with 10 micrograms/mL insulin, 100 micromole/L hypoxanthine, 16 micromole/L thymidine and 30 micromole/L methionine sulfoximine (MSX, Sigma)
  • the concentration of MSX and puromycin were escalated from 30 to 100 micromole/L and from 0 to 10 micrograms/ml, respectively, in the course of the selective culture.
  • the cells survived in the course of the selective culture above were used as recombinant cells for expression of ATF-fused hI2S.
  • the adsorbed ATF-fused hI2S was eluted and fractionated with a linear gradient of 20 column volumes of a buffer, from 100 % of 100 mM acetate buffer (pH 5.0) to 100% of 100mM HEPES buffer (pH 8.0) containing 1.5 M sodium chloride. After measuring the I2S activity by a method as described below, fractions having hI2S activity were collected as the eluate of interest.
  • This eluate from the Capto MMC column above was diluted 10 times with 25 mM sodium phosphate buffer (pH 7.4) and loaded and adsorbed on a HiTrap Q-Sepharose FF 5 mL column (GE health care company), which had been equilibrated with the same buffer. Then the column was washed with a fourfold column volumes of the same buffer. Subsequently the adsorbed ATF-fused hI2S was eluted and fractionated with a linear gradient of 20 column volumes of a buffer, from 100% of 25 mM sodium phosphate buffer (pH7.4) to 100% of 25 mM sodium phosphate buffer (pH7.4) containing 1.5 M sodium chloride.
  • fractions having hI2S activity were collected and analyzed by SDS-PAGE, which revealed a single band having a molecular weight corresponding to that of ATF-fused hI2S (approximately 92kD) (Fig. 5).
  • the collected fractions were used for further analysis as purified ATF-fused hI2S.
  • Substrate solution was prepared by dissolving 4-methyl-umbelliferyl 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 microliters of Substrate solution was added to each well containing ATF-fused hI2S sample and the plate was let stand for 1 hour at 37 deg C in the dark. After the incubation, 190 microliters of Stop Buffer (0.33 M glycine, 0.21 M sodium carbonate, pH 10.7) was added to each well containing the sample.
  • Stop Buffer (0.33 M glycine, 0.21 M sodium carbonate, pH 10.7
  • a standard curve was produced by measuring fluorescence intensity at various concentrations of 4-MUF in solution. The fluorescence intensity of each sample was extrapolated to the standard curve. Results were calculated as activity in Units/mL where one Unit of activity was equal to 1 micromole of 4-MUF produced per minute at 37 deg C.
  • a published US patent application publication No. 2004-0229250 was referred to for conducting this measurement.
  • M6P mannose-6-phosphate
  • solution A 100 mM boric acid solution (pH 9.0)
  • solution B 100 mM boric acid-200 mM sodium chloride solution (pH 9.0)
  • Shimpack ISA-07/S2504 (4.0 mm I.D. x 250 mm)(base material: polystyrene gel, stationary phase: quaternary ammonium group) was attached to Shimazu HPLC System LC-10Avp (reducing sugar analysis system), and further, Shim-pack guard column ISA (4.0 mm I.D. x 50 mm) was set as a column oven used to heat the column.
  • a heat block (ALB-221, mftd. by Asahi Techno Glass) for heat reaction was set downstream of the outlet of the column. The column was heated in the column oven at 65 deg C, and the heat block was set at 150 deg C.
  • a fluorescence detector system was installed downstream of the heat block, and adjusted so that it would irradiate excitation light at the wavelength of 320 nm and detect fluorescent light at the wavelength of 430 nm.
  • solutions A and B were set on the autosampler of the reducing sugar analysis system, which then was set so that the reagent solution for reaction was supplied downstream of the outlet of the column (upstream of the heat block). After the column was equilibrated with the mobile phase (solution A) with which chromatography was to be started, the M6P standard solution or the samples were loaded onto the column.
  • a first mobile phase prepared by mixing solution A and solution B at a volume ratio of 90:10 (thus, containing 100 mM boric acid and 20 mM sodium chloride) was let flow through the column at a flow rate of 0.3 mL/min for 35 min; then the volume ratio between solution A and solution B was changed in a linear fashion to 25:75 over 25 min at the same flow rate (thus, containing 100 mM boric acid, while sodium chloride being increased up to 150 mM), and further the volume ratio of solution B was set at 100% (thus, containing 100 mM boric acid and 200 mM sodium chloride) and the solution was let flow at the same flow rate for 10 min, and then solution A and solution B were let flow at a volume ratio of 90:10 (thus, containing 100 mM boric acid and 20 mM sodium chloride) as was the case of the first mobile phase.
  • the reagent solution for reaction was supplied to the flow path downstream
  • the average number of M6P contained in hATF-fused hI2S and mATF-fused hI2S was calculated to be 2.6 mol/mol and 2.2 mol/mol, respectively.
  • the average number of M6P contained in recombinant human I2S which had been provided as pharmaceutical medicine was calculated as 4.4 mol/mol.
  • His-tagged-hMPR9 For measurement of the affinity of ATF-fused hI2S for the human mannose-6-phasphate receptor (hM6PR), His-tagged-hMPR9, which contained domain 9 of the human M6P receptor, to which ATF binds, was produced as described below.
  • a DNA fragment encoding the domain 9 of the human M6P receptor (hMPR9) 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 NcoI and NotI sites of pET26 vector (Novagen).
  • the resultant plasmid was designated pET26-MPR9.
  • Two-step PCR was conducted for amplification of a DNA fragment whose sequence is set forth as SEQ ID NO:34, which encoded hMPR9 with added sequences at both of its 5'- and 3'-termini.
  • the first reaction of the two-step PCR was conducted using pET26-MPR9, as template, and a set of primers: MPR9-f2 (SEQ ID NO:37) and MPR9-r2 (SEQ ID NO:38). Subsequently, using the amplified DNA fragment as template, the second PCR was conducted using a set of primers: MPR9-f3 (SEQ ID NO:39), and MPR9-r3 (SEQ ID NO:40).
  • the resultant DNA fragment was digested with EcoRV and NotI, and ligated into the Eco47III-NotI site of the pIB/V5-His-DEST vector (Invitrogen).
  • the resultant plasmid was designated pXBi-MPR9, with which High Five cells were transfected to obtain cells expressing His-tagged-hMPR9 which was derived from Bip-tagged hMPR9 through removal of Bip signal sequence.
  • High Five cells (Invitrogen) were grown in a 24-well plate until 50% confluent using Express Five medium (Invitrogen), and transfected with the pXBi-MPR9 using the Hily Max transfection reagent (Dojin chemical, Japan). The cells were cultured in the presence of 30 micrograms/mL blasticidin to select stable transfectant. Stably transfected cells then were expanded and cultured in a Erlenmeyer flask (100 mL) for 4 days. The culture then was harvested and centrifuged at 3000 rpm for 30 min, and the supernatant was collected.
  • the supernatant was filtrated though a 0.22 micrometer filter (Millipore), then diluted 5-fold with an equilibration buffer (10 mM phosphate buffer containing 300 mM NaCl (pH 7.2)).
  • the diluted supernatant was applied to the chromatography column with Profinity IMAC Ni-charged Resin (bed volume: 1 mL, Bio-Rad) which had been equilibrated with the equilibration buffer, and the column then was washed with 5 bed volumes of the equilibration buffer.
  • the His-tagged-hMPR9 bound to the resin was eluted by applying to the column 5 bed volumes of 10 mM NaPO 4 , 300 mM NaCl and 10 mM imidazole (pH 7.2), and subsequently 5 bed volumes of 10 mM NaPO 4 , 300 mM NaCl and 300 mM imidazole (pH 7.2).
  • Fractions containing His-tagged-hMPR9 were collected and concentrated by Amicon 3K (Millipore) with the buffer exchanged to 20mM Tris buffer containing 150 mM NaCl (pH 7.4).
  • the concentration of the His-tagged-hMPR9 was determined by measuring absorbance at 280 nm.
  • Biacore T100 GE Healthcare
  • nitrilotriacetic acid fixed sensor chip Series S Sensor Chip NTA "BR-1005-32"
  • Biacore T100 is measuring apparatus based on surface plasmon resonance (SPR), where a sample containing a ligand is sent at a constant flow rate onto the surface of the sensor chip on which a receptor is fixed.
  • the mass of the sensor chip is increased due to the ligand bound to the receptor, and a shift of the SPR signal can be detected as a change in the resonance unit (RU) in proportion to the amount of the ligand bound.
  • RU resonance unit
  • 1 RU corresponds to approximately 1 pg/mm 2 .
  • hM6PR As the domain in hM6PR to which M6P binds is the domain 9, His-tagged domain 9 of hM6PR (His-tagged-hMPR9) prepared as above was used as the receptor for M6P. And as the receptor for ATF, a recombinant huPAR (rhuPAR) purchased from R&D Systems (Minneapolis, MN) was used in this experiment.
  • rhuPAR recombinant huPAR
  • 10 mM HEPES pH 7.4 containing 500 micromole/L NiCl 2 , 150 mM NaCl, 50 micromole/L EDTA and 0.05% Surfactant P20 was run at the flow rate of 10 microliters/min for 60 sec, and then, (i) approximately 2.1 microliters of 2.8 micrograms/mL His-tagged-hMPR9 dissolved in 10 mM HEPES (pH 7.4) containing 150 mM NaCl, 50 micromole/L EDTA and 0.05% Surfactant P20 or (ii) approximately 2.1 microliters of 5 micrograms/mL rhuPAR (R&D Systems) dissolved in the same buffer was applied, and subsequently the same buffer was run at the flow rate of 10 microliters/min for 60 sec to fix His-tagged-hMPR9 or rhuPAR on the activated sensor chip.
  • 10 mM HEPES pH 7.4 containing 500 micromole/L NiCl 2 , 150 m
  • Each sample was diluted so as to adjust the concentration of ATF-fused hI2S, or of rhI2S, to 12.5, 6.25, 3.125 and 1.5625 nmol/L, with 10 mM HEPES (pH7.4) containing 150 mM NaCl, 50 micromole/L EDTA and 0.05% Surfactant P20.
  • Each dilution then were applied at 50 microliters/min for 300 sec for the ligand (ATF-fused hI2S or rhI2S) to be bound to the corresponding receptor (His-tagged-hMPR9 or rhuPAR) on the sensor chip.
  • Kd between hATF-fused hI2S and rhuPAR was calculated to be 1.16 nM, and that between mATF-fused hI2S and rhuPAR to be 5.59 nM.
  • affinity of hATF-fused hI2S for rhuPAR was 4.8-fold higher than that of mATF-fused hI2S for rhuPAR. Specific binding between hI2S and rhuPAR was not detected.
  • Kd between hATF-fused hI2S and hM6PR was calculated to be 0.40 nM, and that between mATF-fused hI2S and hM6PR (domain9 of hM6PR) to be 0.43 nM. These values were comparable to the Kd value between hI2S and hM6PR (Kd of 0.47 nM). These data indicate that either of the murine or human ATF-fused hI2S has a binding affinity for hM6PR, which is similar to that of hI2S, despite their lower contents of M6P.
  • the medium in the 96-well microplate where the fibroblasts had been seeded was removed with a micropipette, and 100 microliters of each sample diluted above was added in a duplicate manner to the wells, and incubation was done for 18 hrs, where the final concentrations of hATF-fused hI2S or rhI2S were 4.88 ng/mL to 20 micrograms/mL.
  • the cells were lysed with M-PER Mammalian protein extraction reagent (Thermo Scientific) supplemented with a 0.5% protease inhibitor cocktail (Sigma). Then the amount of total cellular protein was measured by Pierce BCA TM Protein Assay kit (Pierce, IL, USA), and the amount of hATF-fused hI2S and rhI2S taken up in the cells was determined by ELISA as described below. The amount of hATF-fused hI2S and rhI2S taken up was calculated per unit mass (mg) of the amount of total cellular protein, and plotted on a graph.
  • hATF-fused hI2S Compared with rhI2S, the level of cellular uptake of hATF-fused hI2S into normal human fibroblast was low (Fig 6). That may be because the number of M6P contained in hATF-fused hI2S (2.2 per protein molecule) was lower than that in rhI2S (4.4 per protein molecule), which lowered the efficiency of cellular uptake through M6P receptor in hATF-fused hI2S compared with rhI2S. On the other hand, cellular uptake of hATF-fused hI2S was higher than rhI2S in the presence of 10 mM M6P, which antagonized the binding of the ligand to M6P receptor.
  • hATF-fused hI2S is able to be taken up by cells not only through M6P receptor but also in a M6P receptor-independent manner, possibly through binding of the hATF moiety to uPAR.
  • Measurement of cellular uptake of mATF-fused hI2S was conducted following the procedure described in Example 13 above, where primary mouse fibroblasts were substituted for human fibroblasts.
  • the primary mouse fibroblasts were purchased from Kitayama Labes Co., Ltd., Japan.
  • rhI2S Compared with rhI2S, the level of cellular uptake of mATF-fused hI2S into mouse fibroblasts was low (Fig 8). That may be because the number of M6P contained in mATF-fused hI2S (2.2 per protein molecule) was lower than that in rhI2S (4.4 per protein molecule), which lowered the efficiency of cellular uptake through M6P receptor in mATF-fused hI2S compared with rhI2S. On the other hand, cellular uptake of mATF-fused hI2S was higher than rhI2S in the presence of 10 mM M6P, which antagonized the binding of the ligand to M6P receptor. In the presence of 10 mM M6P, the uptake of rhI2S was blocked by about 91%, whereas more than 20% of mATF-fused hI2S was taken up by the fibroblasts.
  • hATF-fused hI2S is able to be taken up by cells not only through M6P receptor but also in a M6P receptor-independent manner, possibly through binding of the hATF moiety to uPAR.
  • the present invention can be used as a means to deliver various pharmacologically active compounds to organs, tissues and cells where the urokinase-type plasminogen activator receptor (uPAR) is expressed.
  • uPAR urokinase-type plasminogen activator receptor
  • the present invention can also be used to provide various pharmaceutical agents which are directed to organs, tissues and cells where the receptor is expressed.
  • LacZ promoter 2 mPGKpromoter 3 Wild type murine encephalomyelitis virus IRES (EMCV-IRES) 3a Mutated type murine encephalomyelitis virus (EMCV-mIRES) 4 mPGK polyadenilation signal (mPGKpA) 5 Sequence containing EF-1 promoter and its first intoron 6 SV40 late polyadenilation signal 7 SV40 early promoter 8 Synthesized polyadenilation 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

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Abstract

Cette invention concerne des agents pharmaceutiques ayant une disponibilité améliorée et un procédé d'utilisation d'un fragment amino-terminal d'un activateur du plasminogène de type urokinase (ATF) pour administrer des médicaments dans des cellules où ces médicaments exerceront leur activité pharmaceutique. Les agents pharmaceutiques selon l'invention comprennent des principes pharmacologiquement actifs auxquels un peptide comprenant le domaine de liaison aux récepteurs de l'ATF est lié.
PCT/JP2012/000805 2012-02-07 2012-02-07 Agents pharmaceutiques liés par fusion à l'atf pour une biodisponibilité améliorée WO2013118165A1 (fr)

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WO2019151539A1 (fr) * 2018-02-05 2019-08-08 Ｊｃｒファーマ株式会社 Procédé d'administration d'un médicament à un muscle

Citations (3)

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JP2019137675A (ja) * 2018-02-05 2019-08-22 Ｊｃｒファーマ株式会社 薬剤を筋肉に送達するための方法
JP7591340B2 (ja) 2018-02-05 2024-11-28 Ｊｃｒファーマ株式会社 薬剤を筋肉に送達するための方法

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