Pharmaceutical composition for treating mucopolysaccharidosis type i

Abstract

To provide methods for treating patients with mucopolysaccharidosis type I.SOLUTION: A pharmaceutical composition containing, as an active ingredient, a fusion protein of an anti-human transferrin receptor antibody and human α-L-iduronidase is administered to a patient with mucopolysaccharidosis type I by intravenous infusion at doses of 0.1 to 10 mg / kg body weight. The administration is continued for at least 3 months at intervals of 5 to 21 days. This degrades dermatan sulphate and heparan sulphate accumulated in the patient's tissues, especially in the central nervous system.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for the treatment of mucopolysaccharidosis type I, comprising a fusion protein of an anti-human transferrin receptor antibody and human α-L-idulonidase (hIDUA) as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for the treatment of mucopolysaccharidosis type I, characterized in that the pharmaceutical composition is administered parenterally to a patient with mucopolysaccharidosis type I to decompose dermatan sulfate and heparan sulfate accumulated in organs including the brain. [Background technology]

[0002] α-L-iduronosidic enzyme (IDUA) is a lysosomal enzyme that hydrolyzes the non-sulfated α-L-iduronosidic bond present in glycosaminoglycans (GAGs) such as heparan sulfate and dermatan sulfate. Patients with mucopolysaccharidosis type 1 have a genetic deficiency of some or all of α-L-iduronosidic activity. This deficiency in the enzyme leads to metabolic abnormalities of heparan sulfate and dermatan sulfate, which in turn leads to the accumulation of fragments of these molecules in tissues such as the liver and kidneys, and even to the excretion of heparan sulfate and dermatan sulfate in the urine. As a result, these abnormalities cause a variety of symptoms in patients with mucopolysaccharidosis type 1, including skeletal deformities and severe intellectual disability. Mucopolysaccharidosis type I is classified into three types: the severe form, Hurler syndrome (MPS IH); the intermediate form, Hurler-Scheille syndrome (MPS IH-S); and the mild form, Schye syndrome (MPS IS).

[0003] The fact that patients with mucopolysaccharidosis type 1 show only minimal IDUA activity was already known in the 1970s, and it was suspected that an abnormality in the IDUA gene was the cause of this disease. In 1991, the human gene encoding hIDUA was isolated and confirmed to be the causative gene for this disease (Non-Patent Literature 1). The isolation of the gene encoding hIDUA made it possible to mass-produce recombinant hIDUA (rhIDUA) using recombinant technology and to use this enzyme as a therapeutic agent in enzyme replacement therapy for mucopolysaccharidosis type 1 (Patent Literature 1).

[0004] However, since rhIDUA cannot cross the blood-brain barrier (BBB), it has been problematic that it cannot effectively improve central nervous system disorders in patients with mucopolysaccharidosis type 1. To solve this problem regarding enzyme replacement therapy for mucopolysaccharidosis type 1 using rhIDUA, a fusion protein of an anti-human transferrin receptor antibody and human α-L-iduronidase has been developed (Patent Documents 2, 3). This fusion protein can cross the BBB by binding to human transferrin receptors present on capillary endothelial cells in the brain, which constitute the BBB, via the anti-human transferrin receptor antibody. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] US6149909 [Patent Document 2] US20180171012 [Patent Document 3] US20190338043 [Non-patent literature]

[0006] [Non-Patent Document 1] Scott HS. et. al., Proc Natl Acad Sci USA 88:9695-9 (1991) [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a method for treating patients with mucopolysaccharidosis type 1, characterized by administering a pharmaceutical composition containing a fusion protein of an anti-human transferrin receptor antibody and human α-L-iduronidase as an active ingredient to patients with mucopolysaccharidosis type 1 parenterally, thereby degrading at least the glucosaminoglycans accumulated in the brain. [Means for solving the problem]

[0008] In research toward the above objective, the inventors discovered that intravenous injection of a pharmaceutical composition containing a fusion protein of an anti-human transferrin receptor antibody and human α-L-iduronidase as an active ingredient can degrade glucosaminoglycans accumulated in the brains of patients with mucopolysaccharidosis type 1, thus completing the present invention. That is, the present invention includes the following: 1. A pharmaceutical composition containing a fusion protein of an anti-human transferrin receptor antibody and human α-L-iduronidase as an active ingredient, wherein the fusion protein is administered to patients with mucopolysaccharidosis type 1 by intravenous infusion at a dose of 0.1 to 10 mg / kg body weight. 2. The pharmaceutical composition according to item 1 above, wherein the fusion protein is administered in a dose of 0.1 to 8 mg / kg body weight. 3. The pharmaceutical composition according to item 1 above, wherein the fusion protein is administered in a dose of 1 to 6 mg / kg body weight. 4. The pharmaceutical composition according to item 1 above, wherein the fusion protein is administered in a dose of 2 mg / kg body weight or 4 mg / kg body weight. 5. The pharmaceutical composition according to any one of items 1 to 4 above, wherein the fusion protein is administered at a rate of 0.33 mg / hour to 200 mg / hour. 6. The pharmaceutical composition according to any one of items 1 to 4 above, wherein the fusion protein is administered over a period of at least one hour. 7. The pharmaceutical composition according to any one of items 1 to 6 above, wherein the fusion protein is administered by intravenous infusion. 8. The pharmaceutical composition according to any one of items 1 to 7 above, wherein the administration is carried out at intervals of 5 to 21 days for at least 3 months. 9. The pharmaceutical composition according to any one of items 1 to 7 above, wherein the administration is carried out at intervals of 7 days for at least one month. 10. The pharmaceutical composition according to 8 or 9 above, wherein the fusion protein is administered at a dose of 0.1 to 2 mg / kg body weight for the first administration, and the dose is increased for subsequent administrations. 11. The pharmaceutical composition according to 8 or 9 above, wherein the fusion protein is administered at a dose of 0.1 to 2 mg / kg body weight at the first administration, and then at a maintenance dose of 2 to 6 mg / kg body weight. 12. The pharmaceutical composition according to 8 or 10 above, wherein the maintenance dose is 2 mg / kg body weight or 4 mg / kg body weight. 13. The pharmaceutical composition according to any one of 1 to 12 above, wherein the anti-human transferrin receptor antibody is Fab. 14. The pharmaceutical composition according to any one of 1 to 13 above, wherein the α-L-iduronidase is linked via a linker to the C-terminal side or N-terminal side of the light chain of the anti-human transferrin receptor antibody, or to the C-terminal side or N-terminal side of the heavy chain of the anti-human transferrin receptor antibody. 15. The pharmaceutical composition according to any one of 1 to 13 above, wherein the α-L-iduronidase is linked via a linker to the C-terminal side of the heavy chain of the anti-human transferrin receptor antibody. 16. The pharmaceutical composition according to 14 or 15 above, wherein the linker is a peptide consisting of 1 to 150 amino acid residues. 17. The pharmaceutical composition according to 16 above, wherein the linker is a peptide comprising an amino acid sequence selected from the group consisting of 1 glycine, 1 serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, and an amino acid sequence in which these amino acid sequences are continuous for 1 to 10. 18. The pharmaceutical composition according to 16 above, wherein the linker is a peptide consisting of the amino acid sequence of SEQ ID NO: 3. 19. The anti-human transferrin receptor antibody comprises, in the variable region of the light chain, the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 as CDR1, the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or the amino acid sequence Lys-Val-Ser as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3, respectively, and in the variable region of the heavy chain, the amino acid sequence of SEQ ID NO: 9 or 10 as CDR1, the amino acid sequence of SEQ ID NO: 11 or 2 as CDR2, and the amino acid sequence of SEQ ID NO: 13 or 14 as CDR3, respectively. The pharmaceutical composition according to any one of the above 1 to 18, wherein the α-L-iduronidase is bound to the C-terminal side or N-terminal side of the light chain of the anti-human transferrin receptor antibody, or the C-terminal side or N-terminal side of the heavy chain of the anti-human transferrin receptor antibody. 20. The pharmaceutical composition according to 19 above, wherein the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 16. 21. The pharmaceutical composition according to 20 above, wherein the heavy chain is a Fab heavy chain, and the Fab heavy chain comprises the amino acid sequence of SEQ ID NO: 19. 22. The pharmaceutical composition according to any one of 19 to 21 above, wherein the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 17. 23. The pharmaceutical composition according to 22 above, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 18. 24. The pharmaceutical composition according to any one of 1 to 23 above, wherein the human α-L-iduronidase comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21. 25. The pharmaceutical composition according to any one of 1 to 23 above, wherein the human α-L-iduronidase comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21. 26. The pharmaceutical composition according to paragraph 1, wherein the light chain of the anti-human transferrin receptor antibody comprises the amino acid sequence of SEQ ID NO: 18, and the heavy chain of the anti-human transferrin receptor antibody comprises the amino acid sequence of SEQ ID NO: 19, and the heavy chain is conjugated at its C-terminus to a human α-L-iduronidase having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21 via a linker of the amino acid sequence of SEQ ID NO: 3. 27. The pharmaceutical composition according to 19, wherein the light chain of the anti-human transferrin receptor antibody comprises the amino acid sequence of SEQ ID NO: 18, and the heavy chain of the anti-human transferrin receptor antibody comprises the amino acid sequence of SEQ ID NO: 19, and the heavy chain, at its C-terminus, is bound to a human α-L-idulonidase having the amino acid sequence of SEQ ID NO: 20 via a linker of the amino acid sequence of SEQ ID NO: 3, thereby forming the amino acid sequence of SEQ ID NO: 24. 28. A pharmaceutical composition according to any one of items 1 to 27 above, which is a freeze-drying agent or an aqueous liquid. 29. The pharmaceutical composition according to 28 above, further comprising at least one of a neutral salt, a disaccharide, a nonionic surfactant, and a buffering agent. 30. The pharmaceutical composition according to 28 or 29 above, wherein the nonionic surfactant comprises polysorbate and / or poloxamer. 31. The polysorbate is polysorbate 20 or polysorbate 80, The pharmaceutical composition according to 30 above, wherein the poloxamer is selected from the group consisting of polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, polyoxyethylene (42) polyoxypropylene (67) glycol, polyoxyethylene (3) polyoxypropylene (17) glycol, polyoxyethylene (20) polyoxypropylene (20) glycol, and polyoxyethylene (120) polyoxypropylene (40) glycol. 32. The pharmaceutical composition according to 30, wherein the polysorbate is polysorbate 80 and the poloxamer is polyoxyethylene (160) polyoxypropylene (30) glycol. 33. A pharmaceutical composition which is an aqueous solution according to any one of the above 30 to 32, wherein the concentration of the polysorbate is 0.005 to 1.5 mg / mL and the concentration of the poloxamer is 0.1 to 0.6 mg / mL. 34. A pharmaceutical composition which is an aqueous solution according to any one of 30 to 32 above, wherein the concentration of the polysorbate is 0.025 to 1.0 mg / mL and the concentration of the poloxamer is 0.2 to 0.5 mg / mL. 35. A pharmaceutical composition which is an aqueous solution according to any one of 30 to 32 above, wherein the concentration of the polysorbate is 0.05 to 0.15 mg / mL and the concentration of the poloxamer is 0.25 to 0.45 mg / mL. 36. The pharmaceutical composition according to any one of the above 29 to 35, wherein the neutral salt is sodium chloride. 37. The pharmaceutical composition according to any one of the above 29 to 36, wherein the disaccharide is selected from the group consisting of trehalose, sucrose, maltose, lactose, and combinations of two or more of these. 38. The pharmaceutical composition according to any one of the above 29 to 37, wherein the buffering agent is selected from the group consisting of citrate buffering agents, phosphate buffering agents, glycine buffering agents, histidine buffering agents, carbonate buffering agents, acetate buffering agents, and combinations of two or more of these. 39. A pharmaceutical composition according to any one of the above 30-32, which is an aqueous liquid preparation selected from the group consisting of (1) to (3) below: (1) The fusion protein concentration is 1 to 10 mg / mL, the neutral salt concentration is 0.3 to 1.2 mg / mL, the disaccharide concentration is 50 to 100 mg / mL, the buffer concentration is 10 to 30 mM, the polysorbate concentration is 0.005 to 1.5 mg / mL, and the poloxamer concentration is 0.1 to 0.6 mg / mL; (2) The fusion protein concentration is 2-8 mg / mL, the neutral salt concentration is 0.5-1.0 mg / mL, the disaccharide concentration is 55-95 mg / mL, the buffer concentration is 15-25 mM, the polysorbate concentration is 0.05-1.0 mg / mL, and the poloxamer concentration is 0.25-0.45 mg / mL; and (3) The fusion protein concentration is 4-6 mg / mL, the neutral salt concentration is 0.7-0.9 mg / mL, the disaccharide concentration is 60-90 mg / mL, the buffer concentration is 15-25 mM, the polysorbate concentration is 0.05-0.15 mg / mL, and the poloxamer concentration is 0.25-0.45 mg / mL. 40. A pharmaceutical composition according to any one of the above 28 to 41, which is an aqueous solution with a pH of 4.5 to 6.5, 5.0 to 6.0, or 5.2 to 5.8. 41. A lyophilizing agent selected from the group consisting of (1) to (3) below, the pharmaceutical composition described in any of 30 to 32 above: (1) When dissolved in pure water, the concentration of the fusion protein is 1 to 10 mg / mL, the concentration of the neutral salt is 0.3 to 1.2 mg / mL, the concentration of the disaccharide is 50 to 100 mg / mL, the concentration of the buffer is 10 to 30 mM, the concentration of the polysorbate is 0.005 to 1.5 mg / mL, and the concentration of the poloxamer is 0.1 to 0.6 mg / mL; (2) When dissolved in pure water, the concentration of the fusion protein is 2-8 mg / mL, the concentration of the neutral salt is 0.5-1.0 mg / mL, the concentration of the disaccharide is 55-95 mg / mL, the concentration of the buffer is 15-25 mM, the concentration of the polysorbate is 0.05-1.0 mg / mL, and the concentration of the poloxamer is 0.25-0.45 mg / mL; and (3) When dissolved in pure water, the concentration of the fusion protein is 4-6 mg / mL, the concentration of the neutral salt is 0.7-0.9 mg / mL, the concentration of the disaccharide is 60-90 mg / mL, the concentration of the buffer is 15-25 mM, the concentration of the polysorbate is 0.05-0.15 mg / mL, and the concentration of the poloxamer is 0.25-0.45 mg / mL. 42. A lyophilized agent having a pH of 4.5 to 6.5, 5.0 to 6.0, or 5.2 to 5.8 when dissolved in pure water, the pharmaceutical composition according to any of the above 28 to 32, 36 to 38, and 41. 43. The pharmaceutical composition according to any one of items 1 to 42 above, wherein the patient has a disorder of the central nervous system. 44. A pharmaceutical composition according to any one of items 1 to 43 above, which has the effect of reducing the concentration of dermatan sulfate and heparan sulfate contained in cerebrospinal fluid, serum, and urine. 45. A pharmaceutical composition according to any one of items 1 to 44 above, used for enzyme replacement therapy in patients with mucopolysaccharidosis type 1. 46. ​​Pharmaceutical compositions according to items 1 to 45 above, which are used in combination with immunosuppressants. 47. Enzyme replacement therapy for patients with mucopolysaccharidosis type 1 using any of the pharmaceutical compositions described in items 1 to 46 above. 48. The enzyme replacement therapy described in 47 above, wherein the patient has a central nervous system disorder. 49. A pharmaceutical composition according to any of items 1 to 46 above, wherein when the pharmaceutical composition is administered to a patient with mucopolysaccharidosis type 1, the concentration of heparan sulfate contained in cerebrospinal fluid collected from the patient after administration is 2 / 3 or less, 1 / 2 or less, or 1 / 3 or less compared to the concentration of heparan sulfate contained in cerebrospinal fluid collected from the patient before the pharmaceutical composition was first administered. 50. A pharmaceutical composition according to any of items 1 to 46 above, wherein when the pharmaceutical composition is administered to a patient with mucopolysaccharidosis type 1, the concentration of heparan sulfate in the cerebrospinal fluid collected from the patient after administration is 2000 ng / mL or less, 1800 ng / mL or less, 1600 ng / mL or less, 1500 ng / mL or less, or 1400 ng / mL or less. 50. A pharmaceutical composition according to any of items 1 to 46 above, wherein when the pharmaceutical composition is administered to a patient with mucopolysaccharidosis type 1, the patient exhibits at least one of the following functional improvements compared to before the initial administration of the pharmaceutical composition: (1) Allows for longer conversations; (2) It is possible to write more characters; (3) Joint pain in the lower back, knees, etc. is reduced; (4) Pain and muscle and joint stiffness associated with walking are reduced; (5) It becomes easier to perform fine motor skills such as opening cans and full-body exercises such as playing basketball; (6) Language ability improves. [Effects of the Invention]

[0009] According to the present invention, for example, dermatan sulfate and heparan sulfate accumulated in organs, including the brain, of patients with mucopolysaccharidosis type 1, and especially heparan sulfate accumulated in the central nervous system, including the brain, can be broken down, thereby suppressing the progression of organ dysfunction in such patients, particularly in the central nervous system, including the brain. [Brief explanation of the drawing]

[0010] [Figure 1] This graph shows the change in heparan sulfate concentration in the cerebrospinal fluid of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in a clinical trial (Phase I). The left side shows the value before administration of the investigational drug, and the right side shows the value after administration. The vertical axis represents the concentration of heparan sulfate (ng / mL), and the vertical lines on the graph represent the SD bars. [Figure 2] This graph shows the change in dermatan sulfate concentration in the cerebrospinal fluid of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in a clinical trial (Phase I). The left side shows the value before administration of the investigational drug, and the right side shows the value after administration. The vertical axis represents the dermatan sulfate concentration (ng / mL), and the vertical lines on the graph represent the standard deviation (SD) bars. [Figure 3] This graph shows the change in heparan sulfate concentration in the serum of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in a clinical trial (Phase I). The left side shows the value before administration of the investigational drug, and the right side shows the value after administration. The vertical axis represents the heparan sulfate concentration (ng / mL), and the vertical lines on the graph represent the SD bars. [Figure 4] This graph shows the change in dermatan sulfate concentration in the serum of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in a clinical trial (Phase I). The left side shows the value before administration of the investigational drug, and the right side shows the value after administration. The vertical axis represents the dermatan sulfate concentration (ng / mL), and the vertical lines on the graph represent the standard deviation (SD) bars. [Figure 5]This graph shows the change in heparan sulfate concentration in the urine of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in a clinical trial (Phase I). The left side shows the value before administration of the investigational drug, and the right side shows the value after administration. The vertical axis represents the amount of heparan sulfate (μg / mg creatinine), and the vertical lines on the graph represent the standard deviation (SD) bars. [Figure 6] This graph shows the change in dermatan sulfate concentration in the urine of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in a clinical trial (Phase I). The graph shows the value before administration of the investigational drug, and the graph on the right shows the value after administration. The vertical axis represents the amount of dermatan sulfate (μg / mg creatinine), and the vertical lines on the graph represent the standard deviation bars. [Figure 7] This figure shows the change in heparan sulfate concentration in the cerebrospinal fluid of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in the first trial of Phase II of the clinical trial. × and ◇ indicate the heparan sulfate concentration in each patient. The vertical axis represents the heparan sulfate concentration (ng / mL), and the dotted line represents the average heparan sulfate concentration in the cerebrospinal fluid of patients without mucopolysaccharidosis type 1. [Figure 8] This figure shows the change in dermatan sulfate concentration in the cerebrospinal fluid of patients with mucopolysaccharidosis type I following administration of the investigational drug in the first trial of Phase II clinical trials. × and ◇ indicate the dermatan sulfate concentration in each patient. The vertical axis shows the dermatan sulfate concentration (ng / mL). [Figure 9] This figure shows the change in heparan sulfate concentration in the cerebrospinal fluid of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in the second trial of Phase II of the clinical trial. The white circles, triangles, and squares indicate the heparan sulfate concentration in each patient in the 2.0 mg / kg body weight dose group, while the black circles, triangles, and squares indicate the heparan sulfate concentration in each patient in the 4.0 mg / kg body weight dose group. The vertical axis represents the heparan sulfate concentration (ng / mL), and the dotted line represents the average heparan sulfate concentration in the cerebrospinal fluid of patients without mucopolysaccharidosis type 1. [Figure 10]This figure shows the change in dermatan sulfate concentration in cerebrospinal fluid of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in the second trial of Phase II of the clinical trial. The white circles, triangles, and squares indicate the dermatan sulfate concentration in each patient in the 2.0 mg / kg body weight dose group, while the black circles, triangles, and squares indicate the dermatan sulfate concentration in each patient in the 4.0 mg / kg body weight dose group. The vertical axis shows the dermatan sulfate concentration (ng / mL). [Figure 11] This figure shows the change in heparan sulfate concentration in the serum of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in the first trial of the Phase II clinical trial. × and ◇ indicate the heparan sulfate concentration in each patient. The vertical axis shows the heparan sulfate concentration (ng / mL). [Figure 12] This figure shows the change in dermatan sulfate concentration in the serum of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in the first trial of the Phase II clinical trial. × and ◇ indicate the dermatan sulfate concentration in each patient. The vertical axis shows the dermatan sulfate concentration (ng / mL). [Figure 13] This figure shows the change in heparan sulfate concentration in the serum of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in the second trial of Phase II of the clinical trial. The white circles, triangles, and squares indicate the heparan sulfate concentration in each patient in the 2.0 mg / kg body weight dose group, while the black circles, triangles, and squares indicate the heparan sulfate concentration in each patient in the 4.0 mg / kg body weight dose group. The vertical axis shows the heparan sulfate concentration (ng / mL). [Figure 14] This figure shows the changes in dermatan sulfate concentration in the serum of patients with mucopolysaccharidosis type 1 following administration of the investigational drug in the second trial of Phase II of the clinical trial. The white circles, triangles, and squares indicate the dermatan sulfate concentration in each patient in the 2.0 mg / kg body weight dose group, while the black circles, triangles, and squares indicate the dermatan sulfate concentration in each patient in the 4.0 mg / kg body weight dose group. The vertical axis shows the dermatan sulfate concentration (ng / mL). [Modes for carrying out the invention]

[0011] Transferrin receptors (hTfRs) are present on the surface of brain capillary endothelial cells (cerebral vascular endothelial cells), which form the human blood-brain barrier. Antibodies that can recognize these receptors as antigens (anti-hTfR antibodies) can bind to them. Antibodies bound to hTfRs on the surface of brain capillary endothelial cells can then cross the blood-brain barrier via hTfR-mediated transcytosis and reach the central nervous system. Therefore, by binding human α-L-iduronidase (hIDUA) to these antibodies, hIDUA can reach the central nervous system.

[0012] In the present invention, the anti-hTfR antibody is preferably Fab. Here, Fab is a variable region and C L A single light chain containing a region (the steady region of the light chain), and a variable region and C H This refers to a molecule in which one heavy chain, containing one region (part 1 of the constant region of the heavy chain), is linked to the cysteine ​​residues present in each region by disulfide bonds. In Fab, the heavy chain consists of a variable region and a C H In addition to one region (part 1 of the constant region of the heavy chain), a portion of the hinge region may also be included, but in this case, the hinge region lacks the cysteine ​​residues present in the hinge region that bind the heavy chains of the antibody together. In Fab, the light chain and the heavy chain are defined as the constant region of the light chain (C L Cysteine ​​residues located in the region and the constant region of the heavy chain (C H The bonds are formed by disulfide bonds between cysteine ​​residues located in the 1st region or the hinge region. The heavy chain that forms the Fab is called the Fab heavy chain. Since the Fab lacks cysteine ​​residues located in the hinge region that bind antibody heavy chains together, it consists of one light chain and one heavy chain. The light chain that makes up the Fab consists of a variable region and C L It includes a region. The heavy chains that make up Fab are variable regions and C H It may consist of one region, a variable region, C HIn addition to one region, a portion of the hinge region may also be included. However, in this case, the hinge region is selected so as not to contain cysteine ​​residues that connect the heavy chains, so that a disulfide bond is not formed between the two heavy chains in the hinge region.

[0013] In the present invention, the anti-hTfR antibody is preferably a human antibody or a humanized antibody. A human antibody is an antibody whose entirety is encoded by a human-derived gene. However, an antibody encoded by a gene that has been mutated from the original human gene for purposes such as increasing the gene expression efficiency is also a human antibody. Furthermore, an antibody obtained by combining two or more genes encoding human antibodies and replacing a part of one human antibody with a part of another human antibody is also a human antibody. A human antibody has three complementarity-determining regions (CDRs) in the immunoglobulin light chain and three complementarity-determining regions (CDRs) in the immunoglobulin heavy chain. The three CDRs in the immunoglobulin light chain are called CDR1, CDR2, and CDR3, in order from the N-terminus. The three CDRs in the immunoglobulin heavy chain are called CDR1, CDR2, and CDR3, in order from the N-terminus. An antibody whose antigen specificity, affinity, etc., have been modified by replacing the CDR of one human antibody with the CDR of another human antibody is also considered a human antibody.

[0014] In the present invention, the term "humanized antibody" refers to an antibody in which the amino acid sequence of a part of the variable region (for example, all or part of the CDR) is derived from a mammal other than a human, and the remaining region is derived from a human. For example, a humanized antibody may be an antibody produced by replacing three complementarity-determining regions (CDRs) of the immunoglobulin light chain and three complementarity-determining regions (CDRs) of the immunoglobulin heavy chain that constitute a human antibody with CDRs of another mammal. The species of the other mammal from which the CDRs transplanted to the appropriate positions in the human antibody are derived is not particularly limited as long as it is a mammal other than a human, but is preferably a mouse, rat, rabbit, horse, or a primate other than a human, more preferably a mouse and a rat, for example a mouse.

[0015] Human antibodies and humanized antibodies have λ and κ chains in their light chains. The light chain constituting an antibody may be either a λ or κ chain. Human antibodies and humanized antibodies also have γ, μ, α, σ, and ε chains, corresponding to IgG, IgM, IgA, IgD, and IgE, respectively. The heavy chain constituting an antibody may be any of the γ, μ, α, σ, and ε chains, but a γ chain is preferred. Furthermore, the γ chain of the antibody's heavy chain may be γ1, γ2, γ3, and γ4 chains, corresponding to IgG1, IgG2, IgG3, and IgG4, respectively. When the heavy chain constituting an antibody is a γ chain, it may be any of the γ1, γ2, γ3, and γ4 chains, but a γ1 or γ4 chain is preferred. If the antibody is a humanized antibody or a human antibody and is IgG, the light chain of the antibody may be either a lambda chain or a kappa chain, and the heavy chain of the antibody may be any of gamma1, gamma2, gamma3, or gamma4 chains, but preferably gamma1 or gamma4. For example, one preferred embodiment of the antibody is one in which the light chain is a kappa chain and the heavy chain is a gamma1 chain, or one in which the light chain is a lambda chain and the heavy chain is a gamma1 chain.

[0016] In one embodiment of the present invention, the anti-hTfR antibody comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 as CDR1 in the variable region of the light chain, the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or the amino acid sequence Lys-Val-Ser as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3, and the amino acid sequence of SEQ ID NO: 9 or 10 as CDR1 in the variable region of the heavy chain, the amino acid sequence of SEQ ID NO: 11 or 12 as CDR2, and the amino acid sequence of SEQ ID NO: 13 or 14 as CDR3, respectively. Furthermore, the anti-human transferrin receptor antibody comprises, for example, the amino acid sequence of SEQ ID NO: 15 in the framework region 3 of the heavy chain.

[0017] In one embodiment of the present invention, the anti-hTfR antibody comprises a variable region of the heavy chain containing the amino acid sequence of SEQ ID NO: 16, and a variable region of the light chain containing the amino acid sequence of SEQ ID NO: 17.

[0018] In a preferred embodiment of the present invention, the anti-hTfR antibody is a Fab, in which the light chain comprises the amino acid sequence of SEQ ID NO: 18 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 19.

[0019] In one embodiment of the present invention, an anti-hTfR antibody may have mutations such as substitutions, deletions, or additions added to the amino acid sequence of the variable region or other parts containing the variable region, as long as the amino acid sequence of each CDR is preserved and affinity to hTfR is maintained. When an amino acid in the amino acid sequence of the variable region of an anti-hTfR antibody is substituted with another amino acid, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of the variable region of an anti-hTfR antibody is deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Furthermore, mutations combining these amino acid substitutions and deletions can also be added. When adding amino acids to the variable region of an anti-hTfR antibody, preferably 1 to 10 amino acids, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of the variable region of the anti-hTfR antibody, either at the N-terminus or C-terminus. Mutations combining these amino acid additions, substitutions, and deletions can also be introduced. The amino acid sequence of the variable region of the mutated anti-hTfR antibody preferably has 80% or more identity with the amino acid sequence of the variable region of the original anti-hTfR antibody, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 99% or more identity. The same applies when adding mutations to the amino acid sequence of other parts, including the variable region. Furthermore, the Kd value for hTfR of the mutated anti-hTfR antibody is preferably 50 times or less than that of the original anti-hTfR antibody, and for example, preferably 10 times or less.

[0020] In one aspect of the present invention, the anti-hTfR antibody has an affinity for both the extracellular region of hTfR and the extracellular region of monkey TfR. In that case, the dissociation constant of the anti-hTfR antibody with the extracellular region of hTfR is 1×10 -10 M or less, and the dissociation constant with the extracellular region of monkey TfR is preferably 5×10 -9 M or less.

[0021] In one aspect of the present invention, the term "human α-L-iduronidase" or "hIDUA" refers specifically to hIDUA having the same amino acid sequence as wild-type hIDUA. Wild-type hIDUA has an amino acid sequence composed of 628 amino acids represented by SEQ ID NO: 20. An hIDUA variant having an amino acid sequence composed of 626 amino acids represented by SEQ ID NO: 21 is also hIDUA. However, not limited thereto, as long as it has IDUA activity, those with mutations such as substitution, deletion, addition, etc. to the amino acid sequence of wild-type hIDUA are also included in hIDUA. When substituting an amino acid in the amino acid sequence of hIDUA with another amino acid, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, still more preferably 1 to 3, and even more preferably 1 to 2. When deleting an amino acid in the amino acid sequence of hIDUA, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, still more preferably 1 to 3, and even more preferably 1 to 2. Also, mutations combining these amino acid substitutions and deletions can be added. When adding an amino acid to hIDUA, preferably 1 to 10, more preferably 1 to 5, still more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of hIDUA or to the N-terminal side or C-terminal side. Mutations combining these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of hIDUA with mutations preferably has 80% or more identity with the original amino acid sequence of hIDUA, more preferably shows 85% or more identity, still more preferably shows 90% or more identity, even more preferably shows 95% or more identity, and even more preferably shows 99% or more identity.

[0022] In this invention, when hIDUA is said to have IDUA activity, it means that when hIDUA is fused with an antibody to form a fusion protein, it has 3% or more of the activity inherently possessed by the native type of hIDUA. However, it is preferable that the activity be 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, compared to the activity inherently possessed by the native type of hIDUA. The same applies when the hIDUA fused with the antibody has been mutated. The antibody is, for example, an anti-hTfR antibody.

[0023] In this invention, the identity between the amino acid sequence of the original protein (including the antibody) and the amino acid sequence of the mutated protein can be easily calculated using well-known identity calculation algorithms. Examples of such algorithms include BLAST (Altschul SF. J Mol . Biol. 215. 403-10, (1990)), the Pearson and Lipman similarity search method (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), and the Smith and Waterman local identity algorithm (Adv. Appl. Math. 2. 482-9 (1981)).

[0024] Furthermore, substitutions of amino acids in the amino acid sequence of the original protein (including antibodies) occur, for example, within amino acid families that are related by their side chains and chemical properties. Such substitutions within amino acid families are expected not to significantly alter the function of the original protein (i.e., they are conservative amino acid substitutions). Examples of such amino acid families include: (1) Aspartic acid and glutamic acid, which are acidic amino acids, (2) Basic amino acids histidine, lysine, and arginine, (3) Aromatic amine acids such as phenylalanine, tyrosine, and tryptophan, (4) Serine and threonine, which are amino acids having a hydroxyl group (hydroxy amino acids) (5) Hydrophobic amino acids methionine, alanine, valine, leucine, and isoleucine, (6) Neutral hydrophilic amino acids such as cysteine, serine, threonine, asparagine, and glutamine, (7) Glycine and proline, amino acids that affect the orientation of peptide chains, (8) Asparagine and glutamine, which are amide-type amino acids (polar amino acids) (9) Aliphatic amino acids, alanine, leucine, isoleucine, and valine, (10) Alanine, glycine, serine, and threonine, which are amino acids with small side chains. (11) Alanine and glycine, which are amino acids with particularly small side chains, (12) Valine, leucine, and isoleucine, which are branched amino acids.

[0025] In one embodiment of the present invention, when referring to a fusion protein of an anti-hTfR antibody and hIDUA, it means a substance in which an anti-hTfR antibody and hIDUA are linked via a peptide linker or directly. For example, a fusion protein of an anti-hTfR antibody and hIDUA is a substance in which the N-terminus or C-terminus of the heavy chain or light chain of the antibody is linked via a linker or directly to the N-terminus or C-terminus of the hIDUA by a peptide bond.

[0026] A fusion protein of the type in which hIDUA is bound to the C-terminus of the light chain of an hTfR antibody is one in which the antibody contains an amino acid sequence that includes all or part of the variable region of the light chain and an amino acid sequence that includes all or part of the variable region of the heavy chain (e.g., a Fab heavy chain), and hIDUA is bound to the C-terminus of the light chain of this antibody. Here, the antibody light chain and hIDUA may be bound directly or via a linker.

[0027] A fusion protein of the type in which hIDUA is bound to the C-terminus of the heavy chain of an hTfR antibody is one in which the antibody contains an amino acid sequence that includes all or part of the variable region of the light chain and an amino acid sequence that includes all or part of the variable region of the heavy chain (e.g., a Fab heavy chain), and hIDUA is bound to the C-terminus of the heavy chain of this antibody. Here, the antibody heavy chain and hIDUA may be bound directly or via a linker.

[0028] A fusion protein of the type in which hIDUA is bound to the N-terminus of the light chain of an hTfR antibody is one in which the antibody contains an amino acid sequence that includes all or part of the variable region of the light chain and an amino acid sequence that includes all or part of the variable region of the heavy chain (e.g., a Fab heavy chain), and hIDUA is bound to the N-terminus of the light chain of this antibody. Here, the antibody light chain and hIDUA may be bound directly or via a linker.

[0029] A fusion protein of the type in which hIDUA is bound to the N-terminus of the heavy chain of an hTfR antibody is one in which the antibody contains an amino acid sequence that includes all or part of the variable region of the light chain and an amino acid sequence that includes all or part of the variable region of the heavy chain (e.g., a Fab heavy chain), and hIDUA is bound to the N-terminus of the heavy chain of this antibody. Here, the heavy chain of the antibody and hIDUA may be bound directly or via a linker.

[0030] When a linker is placed between the hTfR antibody and hIDUA, its sequence is preferably composed of 1 to 50 amino acids, more preferably 1 to 20, even more preferably 10 to 17, and even more preferably 13 to 17, for example, 15 amino acids. Such linkers are not limited in their amino acid sequence, as long as the linked antibody maintains affinity to hTfR and the linked hIDUA exhibits the physiological activity of the protein under physiological conditions. However, they are preferably composed of glycine and serine, and may have, for example, one amino acid of either glycine or serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence shown in SEQ ID NO: 3, or sequences of 1 to 10 or 2 to 5 consecutive amino acids totaling 1 to 150 amino acids, sequences of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, or 25 to 29 amino acids. For example, those consisting of the amino acid sequence Gly-Ser or those having the amino acid sequence shown in SEQ ID NO: 3 can be suitably used as linkers.

[0031] A preferred example of the fusion protein of the anti-hTfR antibody and hIDUA in the present invention is one in which the light chain of the anti-hTfR antibody contains the amino acid sequence of SEQ ID NO: 18, the heavy chain of the anti-hTfR antibody contains the amino acid sequence of SEQ ID NO: 19, and the heavy chain is bound at its C-terminus to a human α-L-iduronidase having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21 via a linker of the amino acid sequence of SEQ ID NO: 3.

[0032] A further preferred example of the fusion protein of the anti-hTfR antibody and hIDUA in the present invention is one in which the light chain of the anti-hTfR antibody comprises the amino acid sequence of SEQ ID NO: 18, and the heavy chain of the anti-hTfR antibody comprises the amino acid sequence of SEQ ID NO: 19, wherein the heavy chain, at its C-terminus, is linked to human α-L-iduronidase having the amino acid sequence of SEQ ID NO: 20 via a linker of the amino acid sequence of SEQ ID NO: 3, thereby forming the amino acid sequence of SEQ ID NO: 24.

[0033] Further preferred examples of the fusion protein of anti-hTfR antibody and hIDUA in the present invention include a light chain of anti-hTfR antibody comprising the amino acid sequence of SEQ ID NO: 18, a heavy chain of anti-hTfR antibody comprising the amino acid sequence of SEQ ID NO: 19, wherein the heavy chain is conjugated at its C-terminus to human α-L-iduronidase having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21 via a linker of the amino acid sequence of SEQ ID NO: 3.

[0034] The pharmaceutical composition of the present invention contains a fusion protein of an anti-hTfR antibody and hIDUA as an active ingredient. The pharmaceutical composition may be a freeze-dried agent or an aqueous solution.

[0035] A pharmaceutical composition in one embodiment of the present invention comprises at least one of a neutral salt, a disaccharide, a nonionic surfactant, and a buffer. The pharmaceutical may further contain polysorbate and / or poloxamer as the nonionic surfactant.

[0036] The nonionic surfactant included in the pharmaceutical composition is not particularly limited as long as it is pharmaceutically acceptable, but polysorbates and poloxamers are preferred as such nonionic surfactants. Here, polysorbate 20 and polysorbate 80 can be given as examples of polysorbates. As for poloxamers, polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, polyoxyethylene (42) polyoxypropylene (67) glycol, polyoxyethylene (3) polyoxypropylene (17) glycol, polyoxyethylene (20) polyoxypropylene (20) glycol, and polyoxyethylene (120) polyoxypropylene (40) glycol can be given as examples, with polyoxyethylene (160) polyoxypropylene (30) glycol being particularly preferred. Polyoxyethylene (160) polyoxypropylene (30) glycol is synonymous with poloxamer 188.

[0037] The pharmaceutical composition may contain two types of nonionic surfactants. When the pharmaceutical composition contains two types of nonionic surfactants, a preferred combination of nonionic surfactants is one being polysorbate and the other being poloxamer. For example, the combination of polysorbate 20 and poloxamer 188, and polysorbate 80 and poloxamer 188 are preferred, and the combination of polysorbate 80 and poloxamer 188 is particularly preferred. These combinations can also be further combined with other types of polysorbate, poloxamer, etc.

[0038] When the pharmaceutical composition is an aqueous solution, and the aqueous solution contains polysorbate and poloxamer as two nonionic surfactants, the concentration of polysorbate is preferably 0.005 to 1.5 mg / mL, more preferably 0.025 to 1.0 mg / mL, even more preferably 0.05 to 1.0 mg / mL, and even more preferably 0.05 to 0.15 mg / mL, for example, 0.075 mg / mL. The concentration of poloxamer in this case is preferably 0.1 to 0.6 mg / mL, more preferably 0.2 to 0.5 mg / mL, and even more preferably 0.25 to 0.45 mg / mL, for example, 0.325 mg / mL.

[0039] When the pharmaceutical composition is an aqueous solution, and the aqueous solution contains polysorbate 80 and poloxamer 188 as two nonionic surfactants, the concentration of polysorbate 80 is preferably 0.005 to 1.5 mg / mL, more preferably 0.025 to 1.0 mg / mL, and even more preferably 0.05 to 1.0 mg / mL, for example, 0.075 mg / mL. The concentration of poloxamer 188 in this case is preferably 0.1 to 0.6 mg / mL, more preferably 0.2 to 0.5 mg / mL, and even more preferably 0.25 to 0.45 mg / mL, for example, 0.325 mg / mL. For example, the concentration of polysorbate 80 is 0.05 to 1.0 mg / mL and the concentration of poloxamer 188 is 0.25 to 0.45 mg / mL. Furthermore, for example, the concentration of polysorbate 80 is 0.075 mg / mL, and the concentration of poloxamer 188 is 0.325 mg / mL.

[0040] The neutral salt contained in the pharmaceutical composition is not particularly limited as long as it is pharmaceutically acceptable, but sodium chloride and magnesium chloride are preferred as such neutral salts, and sodium chloride is particularly preferred.

[0041] The disaccharides included in the pharmaceutical composition are not particularly limited as long as they are pharmaceutically acceptable, but trehalose, sucrose, maltose, lactose, or combinations thereof are preferred, with sucrose being particularly preferred.

[0042] When the pharmaceutical composition is an aqueous solution, the concentration of disaccharides in the aqueous solution is preferably 50 to 100 mg / mL, more preferably 55 to 95 mg / mL, and even more preferably 60 to 90 mg / mL, for example, 75 mg / mL.

[0043] The buffering agent included in the pharmaceutical composition is not particularly limited as long as it is pharmaceutically acceptable, but citrate buffers, phosphate buffers, glycine buffers, histidine buffers, carbonate buffers, acetate buffers, or combinations thereof are preferred. When the pharmaceutical composition is an aqueous solution, the concentration of the buffering agent included in the aqueous solution is preferably 3 to 30 mM, more preferably 10 to 30 mM, even more preferably 15 to 25 mM, for example 20 mM. When a citrate buffering agent is used as the buffering agent in the aqueous solution, the concentration of the citrate buffering agent included in the aqueous solution is preferably 3 to 30 mM, more preferably 10 to 30 mM, even more preferably 15 to 25 mM, for example 20 mM. Furthermore, the pH of the aqueous solution adjusted by the buffering agent is preferably 4.5 to 7.0, more preferably 4.5 to 6.5, even more preferably 5.0 to 6.0, even more preferably 5.2 to 5.8, for example 5.5. Furthermore, the pH of the aqueous solution adjusted with the citrate buffer is preferably 4.5 to 7.0, more preferably 4.5 to 6.5, even more preferably 5.0 to 6.0, and even more preferably 5.2 to 5.8, for example 5.5.

[0044] The following (1) to (3) are suitable compositions for a pharmaceutical composition that is an aqueous solution: (1) The fusion protein concentration is 1 to 10 mg / mL, the neutral salt concentration is 0.3 to 1.2 mg / mL, the disaccharide concentration is 50 to 100 mg / mL, the buffer concentration is 10 to 30 mM, the polysorbate concentration is 0.005 to 1.5 mg / mL, and the poloxamer concentration is 0.1 to 0.6 mg / mL; (2) The fusion protein concentration is 2-8 mg / mL, the neutral salt concentration is 0.5-1.0 mg / mL, the disaccharide concentration is 55-95 mg / mL, the buffer concentration is 15-25 mM, the polysorbate concentration is 0.05-1.0 mg / mL, and the poloxamer concentration is 0.25-0.45 mg / mL; and (3) The fusion protein concentration is 4-6 mg / mL, the neutral salt concentration is 0.7-0.9 mg / mL, the disaccharide concentration is 60-90 mg / mL, the buffer concentration is 15-25 mM, the polysorbate concentration is 0.05-0.15 mg / mL, and the poloxamer concentration is 0.25-0.45 mg / mL.

[0045] The pH of the aqueous solutions described in (1) to (3) above is adjusted to, for example, 4.5 to 6.5, 5.0 to 6.0, or 5.2 to 5.8.

[0046] The following (1) to (3) are suitable compositions for the pharmaceutical composition which is a freeze-drying agent: (1) When dissolved in pure water, the concentration of the fusion protein is 1 to 10 mg / mL, the concentration of the neutral salt is 0.3 to 1.2 mg / mL, the concentration of the disaccharide is 50 to 100 mg / mL, the concentration of the buffer is 10 to 30 mM, the concentration of the polysorbate is 0.005 to 1.5 mg / mL, and the concentration of the poloxamer is 0.1 to 0.6 mg / mL; (2) When dissolved in pure water, the concentration of the fusion protein is 2-8 mg / mL, the concentration of the neutral salt is 0.5-1.0 mg / mL, the concentration of the disaccharide is 55-95 mg / mL, the concentration of the buffer is 15-25 mM, the concentration of the polysorbate is 0.05-1.0 mg / mL, and the concentration of the poloxamer is 0.25-0.45 mg / mL; and (3) When dissolved in pure water, the concentration of the fusion protein in the neutral salt is 4 to 6 mg / mL, the concentration is 0.7 to 0.9 mg / mL, the concentration of the disaccharide is 60 to 90 mg / mL, the concentration of the buffer is 15 to 25 mM, the concentration of the polysorbate is 0.05 to 0.15 mg / mL, and the concentration of the poloxamer is 0.25 to 0.45 mg / mL.

[0047] The pH of the freeze-drying agents described in (1) to (3) above when dissolved in pure water is, for example, 4.5 to 6.5, 5.0 to 6.0, or 5.2 to 5.8.

[0048] The aqueous pharmaceutical composition may be supplied in the form of a vial, or as a pre-filled formulation in a syringe. There are no particular limitations on the material of the syringe, vial, or other container for filling the aqueous pharmaceutical composition, but borosilicate glass is preferred. In addition, hydrophobic resins such as cycloolefin copolymers, cycloolefin ring-opening polymers, or hydrogenated cycloolefin ring-opening polymers are also preferred.

[0049] The lyophilized pharmaceutical composition can also be supplied as a kit along with a special solution for dissolving it. The lyophilized agent is used by dissolving it in a special solution, pure water, etc., before use. There are no particular limitations on the material of the syringes, vials, etc., used for encapsulating and filling the lyophilized preparation, but borosilicate glass is preferred. In addition, hydrophobic resins such as cycloolefin copolymers, cycloolefin ring-opening polymers, or hydrogenated cycloolefin ring-opening polymers are also preferred.

[0050] Whether in aqueous solution or freeze-dried form, the pharmaceutical composition is usually added to a dialysis bag containing physiological saline solution or the like, diluted, and then injected into the patient.

[0051] In one embodiment of the present invention, the pharmaceutical composition is used as a therapeutic agent for mucopolysaccharidosis type I. Mucopolysaccharidosis type I is classified into the severe form Hurler syndrome (MPS IH), the intermediate form Hurler-Scheille syndrome (MPS IH-S), and the mild form Schayle syndrome (MPS IS), and the pharmaceutical composition can be used for any type of mucopolysaccharidosis type I.

[0052] Mucopolysaccharidosis type 1 is caused by a genetic deficiency of some or all hIDUA, which has the activity to hydrolyze the non-sulfated α-L-iduronosidic bond present in glycosaminoglycans (GAGs) such as heparan sulfate and dermatan sulfate. Due to the hIDUA deficiency in mucopolysaccharidosis type 1, patients experience abnormal accumulation of heparan sulfate and dermatan sulfate in tissues throughout the body, including the brain, leading to symptoms such as skeletal deformities and severe intellectual disability.

[0053] The fusion protein of anti-hTfR antibody and hIDUA, which is the active ingredient of the pharmaceutical composition, can cross the blood-brain barrier (BBB) ​​and exert IDUA activity within brain tissue, thereby degrading heparan sulfate and dermatan sulfate. Therefore, this pharmaceutical composition is particularly effective as a therapeutic agent for enzyme replacement therapy in patients with mucopolysaccharidosis type 1 who have central nervous system disorders.

[0054] In one embodiment of the present invention, the drug is administered to a patient with mucopolysaccharidosis type 1 via a parenteral route, for example, by intravenous infusion, and is administered by intravenous infusion, for example, by drip infusion.

[0055] The pharmaceutical composition is administered intravenously to patients with mucopolysaccharidosis type 1 in doses of 0.1 to 10 mg / kg body weight, for example, 1 to 10 mg / kg body weight, 1 to 6 mg / kg body weight, 2 to 6 mg / kg body weight, 2 mg / kg body weight, 4 mg / kg body weight, 6 mg / kg body weight, or 8 mg / kg body weight. When the pharmaceutical composition is administered intravenously by drip infusion, the administration rate is adjusted so that the fusion protein is infused at a rate of 0.33 mg to 200 mg per hour. Typically, the administration time is 30 minutes to 4 hours, for example, 3 hours.

[0056] Mucopolysaccharidosis type 1 is a genetic disease, and medication is a symptomatic treatment; therefore, the pharmaceutical composition needs to be administered continuously. The pharmaceutical composition is preferably administered at intervals of 3 to 21 days, more preferably 5 to 14 days, for example, at intervals of 7 days, 14 days, etc. There are no particular restrictions on the duration of administration of the pharmaceutical composition, but it is preferably at least 1 month, more preferably at least 3 months. The pharmaceutical composition is intended to be administered throughout the patient's life.

[0057] The dosage of the pharmaceutical composition can be kept low for the initial administration and then gradually increased. Since the fusion protein of the active ingredient, anti-hTfR antibody, and hIDUA is a foreign substance to the patient, there is a risk of adverse reactions such as immune responses. By adopting a method of gradually increasing the dosage, the risk of sudden adverse reactions in the patient can be reduced.

[0058] Examples of administration schedules when employing a method of gradually increasing the dosage include the following (1) to (8). (1) The initial dose should be 0.1-2 mg / kg body weight, and thereafter the dose should be increased compared to the initial dose. (2) The initial dose should be 0.1 to 2 mg / kg body weight, and then the dose should be increased to 2 mg / kg body weight, 4 mg / kg body weight, or 6 mg / kg body weight. (3) The first dose is 0.1 to 0.5 mg / kg body weight, the second and third doses are 1 to 2 mg / kg body weight, and from the fourth dose onward, 4 mg / kg body weight is administered as a maintenance dose. (4) The first dose should be 0.1-0.2 mg / kg body weight, the second dose 2 mg / kg body weight, and the third dose onward 4 mg / kg body weight (maintenance dose). (5) The first dose is 0.1 mg / kg body weight, and subsequent doses are 2 mg / kg body weight (maintenance dose). (6) The first dose is 0.1 mg / kg body weight, the second dose is 1 mg / kg body weight, the third dose is 2 mg / kg body weight, and from the fourth dose onward the dose is 4 mg / kg body weight (maintenance dose). (7) The first dose is 0.1 mg / kg body weight, the second dose is 2 mg / kg body weight, and from the fourth dose onward, the dose is 4 mg / kg body weight (maintenance dose). (8) The first dose is 1.0 mg / kg body weight, the second dose is 2 mg / kg body weight, and from the fourth dose onward, the dose is 4 mg / kg body weight (maintenance dose).

[0059] The preferred dosage and administration of the pharmaceutical composition is to administer it intravenously to the patient by drip infusion over at least 1 or 2 hours, for example, 3 hours, at intervals of 7 days for at least 1 month, at a dose of 4 mg / kg body weight.

[0060] The pharmaceutical composition, when administered to a patient according to the above-described dosage and administration instructions, does not cause serious adverse events in the patient. However, because it suppresses the immune response, the pharmaceutical composition may be used in combination with an immunosuppressant. There are no particular limitations on the immunosuppressants that can be used in combination; for example, alkylating agents such as cyclophosphamide, antimetabolites such as azathioprine, mycophenolate mofetil, methotrexate, and mizoribine, and intracellular signal transduction inhibitors such as cyclosporine and tacrolimus can be used.

[0061] When the pharmaceutical composition is used as a therapeutic agent for enzyme replacement therapy in patients with mucopolysaccharidosis type 1, the effect of administering the pharmaceutical composition can be evaluated, for example, by collecting cerebrospinal fluid (CSF), serum, and / or urine from the patient before and after administration and measuring the concentrations of heparan sulfate and / or dermatan sulfate in these samples. Since the accumulation of heparan sulfate in the central nervous system is thought to cause central nervous system dysfunction, it is expected that a decrease in the concentration of heparan sulfate in the cerebrospinal fluid will improve the function of the patient's central nervous system. The concentration of heparan sulfate and / or dermatan sulfate can be measured by the method described in the examples.

[0062] When administered to a patient with mucopolysaccharidosis type 1, the concentration of heparan sulfate in the cerebrospinal fluid collected from the patient after administration is lower than the concentration of heparan sulfate in the cerebrospinal fluid collected from the patient before the initial administration of the pharmaceutical composition. The concentration of heparan sulfate after administration is preferably 2 / 3 or less, 1 / 2 or less, or 1 / 3 or less compared to the concentration of heparan sulfate before administration.

[0063] For example, the pharmaceutical composition can reduce the concentration of heparan sulfate and / or dermatan sulfate in the patient's cerebrospinal fluid (CSF), serum, and / or urine by administering it to a patient with mucopolysaccharidosis type 1 at a dose of 2 mg / kg body weight or 4 mg / kg body weight, or initially at a dose of 1.0 mg / kg body weight, then at a dose of 2 mg / kg body weight, and then gradually increasing the dose to 4 mg / kg body weight (maintenance dose) from the fourth dose onward, with a 7-day interval between doses, for 3 months (12 weeks). When administered in this manner, the concentration of heparan sulfate in the cerebrospinal fluid collected from the patient after administration is preferably 2 / 3 or less, more preferably 1 / 2 or less, and even more preferably 1 / 3 or less, compared to the concentration of heparan sulfate in the cerebrospinal fluid collected from the patient before the initial administration of the pharmaceutical composition. Alternatively, the concentration of heparan sulfate in the cerebrospinal fluid collected from the patient after administration is preferably 2000 ng / mL or less, more preferably 1800 ng / mL or less, even more preferably 1600 ng / mL or less, even more preferably 1500 ng / mL or less, and even more preferably 1400 ng / mL or less.

[0064] The pharmaceutical composition is expected to improve the functional impairments of patients with mucopolysaccharidosis type 1 when administered to them. Expected improvements in functional impairments include, but are not limited to, the ability to converse for longer periods, the ability to write more, increased vitality, reduced joint pain in the lower back and knees, reduced pain and muscle and joint stiffness associated with walking, easier execution of fine motor skills such as opening cans and full-body exercises such as basketball, and improved language ability. [Examples]

[0065] The present invention will be described in more detail below with reference to examples, but the present invention is not intended to be limited to these examples.

[0066] [Example 1] Construction of a vector for humanized anti-hTfR antibody-hIDUA fusion protein expression The vector for expressing the humanized anti-hTfR antibody-hIDUA fusion protein was constructed using genes encoding a light chain having the amino acid sequence shown in SEQ ID NO: 18 as the antibody portion, and a heavy chain Fab region having the amino acid sequence shown in SEQ ID NO: 19.

[0067] [Construction of pE-neo vectors and pE-hygr vectors] The pEF / myc / nuc vector (Invitrogen) was digested with KpnI and NcoI, and the region containing the EF-1 promoter and its first intron was excised and blunt-ended with T4 DNA polymerase. Separately, the pCI-neo (Invitrogen) was digested with BglII and EcoRI, and the region containing the CMV enhancer / promoter and intron was excised, followed by blunt-ending with T4 DNA polymerase. The region containing the EF-1α promoter and its first intron (after blunt-ending) was inserted into this to construct the pE-neo vector. The pE-neo vector was digested with SfiI and BstXI, and a region of approximately 1 kbp containing the neomycin resistance gene was excised. Using pcDNA3.1 / Hygro(+) (Invitrogen) as a template, the hygromycin gene was amplified by PCR using primers Hyg-Sfi5' (SEQ ID NO: 22) and Hyg-BstX3' (SEQ ID NO: 23). The amplified hygromycin gene was digested with SfiI and BstXI and inserted into the above-mentioned pE-neo vector to construct the pE-hygr vector. The construction of the pE-neo vector and pE-hygr vector was carried out with reference to patent document (Japanese Patent No. 6279466).

[0068] [Construction of pE-IRES-GS-puro] The expression vector pPGKIH (Miyahara M. et.al., J. Biol. Chem. 275,613-618(2000)) was digested with restriction enzymes (XhoI and BamHI), and the internal ribosome binding site (IRES) and hygromycin resistance gene (Hyg) derived from mouse encephalomyocarditis virus (EMCV) were extracted. r A DNA fragment containing the gene and the polyadenylated region (mPGKpA) of mouse phosphoglycerate kinase (mPGK) was excised. This DNA fragment was inserted between the XhoI and BamHI sites of pBluescript SK(-) (Stratagene), and this was named pBSK(IRES-Hygr-mPGKpA).

[0069] Using pBSK(IRES-Hygr-mPGKpA) as a template, a DNA fragment containing a portion of the EMCV IRES was amplified by PCR using primers IRES5' (SEQ ID NO: 24) and IRES3' (SEQ ID NO: 25). This DNA fragment was digested with restriction enzymes (XhoI and HindIII) and inserted between the XhoI and HindIII sites of pBSK(IRES-Hygr-mPGKpA) to obtain pBSK(NotI-IRES-Hygr-mPGKpA). pBSK(NotI-IRES-Hygr-mPGKpA) was digested with restriction enzymes (NotI and BamHI) and inserted between the NotI and BamHI sites of the pE-hygr vector to obtain plasmid pE-IRES-Hygr.

[0070] The expression vector pPGKIH was digested with EcoRI, and a DNA fragment containing the mPGK promoter region (mPGKp) was excised. This DNA fragment was inserted into the EcoRI site of pBluescript SK(-) (Stratagene), and this was designated as mPGK promoter / pBS(-). Using mPGK promoter / pBS(-) as a template, the DNA fragment containing the mPGK promoter region (mPGKp) was amplified by PCR using primers mPGKP5' (SEQ ID NO: 26) and mPGKP3' (SEQ ID NO: 27). This DNA fragment was digested with restriction enzymes (BglII and EcoRI) and inserted between the BglII and EcoRI sites of pCI-neo (Promega), and this was designated as pPGK-neo. pE-IRES-Hygr was digested with restriction enzymes (NotI and BamHI) to excavate DNA fragments (IRES-Hygr), which were then inserted between the NotI and BamHI sites of pPGK-neo to form pPGK-IRES-Hygr.

[0071] cDNA was prepared from CHO-K1 cells and used as a template. Using primers GS5' (SEQ ID NO: 28) and GS3' (SEQ ID NO: 29), a DNA fragment containing the GS gene was amplified by PCR. This DNA fragment was digested with restriction enzymes (BalI and BamHI) and inserted between the BalI and BamHI sites of pPGK-IRES-Hygr to form pPGK-IRES-GS-ΔpolyA.

[0072] Using pCAGIPuro (Miyahara M. et.al., J. Biol. Chem. 275,613-618(2000)) as a template, and primers puro5' (SEQ ID NO: 30) and puro3' (SEQ ID NO: 31), the puromycin resistance gene (puro r A DNA fragment containing a gene was amplified. This DNA fragment was inserted into a pT7Blue T-Vector (Novagen), and this was designated as pT7-puro. pT7-puro was digested with restriction enzymes (AflII and BstXI) and inserted between the AflII and BstXI sites of the expression vector pE-neo to create pE-puro.

[0073] Using pE-puro as a template, a DNA fragment containing the late polyadenylation region of SV40 was amplified by PCR using primers SV40polyA5' (SEQ ID NO: 32) and SV40polyA3' (SEQ ID NO: 33). This DNA fragment was digested with restriction enzymes (NotI and HpaI) and inserted between the NotI and HpaI sites of the expression vector pE-puro to form pE-puro(XhoI). pPGK-IRES-GS-ΔpolyA was digested with restriction enzymes (NotI and XhoI) to excavate a DNA fragment containing the IRES-GS region, and this was inserted between the NotI and XhoI sites of the expression vector pE-puro(XhoI) to form pE-IRES-GS-puro. The construction of pE-IRES-GS-puro was performed with reference to patent document (JP No. 6279466).

[0074] [Construction of pE-mIRES-GS-puro (ΔE)] Using the expression vector pE-IRES-GS-puro as a template, the region from IRES to GS of EMCV was amplified by PCR using primers mIRES-GS5' (SEQ ID NO: 34) and mIRES-GS3' (SEQ ID NO: 35). A DNA fragment in which the start codon (ATG) located second from the 5' end of IRES in EMCV was mutated and disrupted was amplified. Using the expression vector pE-IRES-GS-puro as a template, a DNA fragment containing the above region from IRES to GS was amplified by PCR using this DNA fragment and the above-mentioned primer IRES5'. This DNA fragment was digested with restriction enzymes (NotI and PstI), and the excised DNA fragment was inserted between the NotI and PstI sites of pBluescript SK(-) (Stratagene), resulting in mIRES / pBlueScript SK(-).

[0075] The expression vector pE-IRES-GS-puro was digested with SphI, and the SV40 enhancer region was excised. The remaining DNA fragment was self-ligated, and this was obtained as pE-IRES-GS-puro(ΔE). mIRES / pBlueScript SK(-) was digested with NotI and PstI, and a region containing the modified IRES (mIRES) and a portion of the GS gene was excised. Separately, pE-IRES-GS-puro(ΔE) was digested with NotI and PstI, and the above-mentioned region containing a portion of the mIRES and GS gene was inserted to construct pE-mIRES-GS-puro(ΔE).

[0076] [Construction of pEM-hygr(LC3) and pE-mIRES-GSp-Fab-IDUA] A DNA fragment (CMVE-EF-1αp-IFNβMAR) containing β-Globin MAR (Matrix Attachment Region), CMV enhancer, human EF-1α promoter, MluI and BamHI cleavage sites, and interferon β Mar was artificially synthesized (SEQ ID NO: 36). The HindIII sequence was introduced into the 5' end of this DNA fragment, and the EcoRI sequence into the 3' end. This DNA fragment was digested with HindIII and EcoRI, and inserted between the HindIII and EcoRI sites in the pUC57 vector to form JCR69 in pUC57. A DNA fragment (IRES-HygroR-mPGKpA) containing MluI and BamHI cleavage sites, IRES, hygromycin resistance gene, and mPGK polyadenylation signal was artificially synthesized (SEQ ID NO: 40). This DNA fragment was inserted into the MluI and BamHI sites of JCR69 in pUC57 to form pEM hygro.

[0077] A DNA fragment (SEQ ID NO: 37) containing the gene encoding the full length of the light chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 14 was artificially synthesized and inserted into pUC57-Amp, resulting in JCR131 in pUC57-Amp. A MluI sequence was introduced into the 5' end of this DNA fragment, and a NotI sequence into the 3' end. This plasmid DNA was digested with MluI and NotI and incorporated between the MluI and NotI sequences in the expression vector pEM hygro. The resulting vector was designated as pEM-hygr(LC3), a vector for expressing the light chain of a humanized anti-hTfR antibody.

[0078] A DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 39 was artificially synthesized. This fragment consisted of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 19, to which a human IDUA having the amino acid sequence shown in SEQ ID NO: 20 was bound via a linker sequence shown in SEQ ID NO: 3 to the C-terminus of the Fab heavy chain, which had the amino acid sequence shown in SEQ ID NO: 3. The entire fragment contained a gene encoding a protein with the amino acid sequence shown in SEQ ID NO: 38. A MluI sequence was introduced to the 5' end of this DNA fragment, and a NotI sequence to the 3' end. This DNA fragment was digested with MluI and NotI and incorporated between MluI and NotI in pE-mIRES-GS-puro(ΔE). The resulting vector was designated as pE-mIRES-GSp-Fab-IDUA, an expression vector for a protein in which hIDUA was bound to the C-terminus of the Fab heavy chain of a humanized anti-hTfR antibody.

[0079] [Example 2] Preparation of a cell line that highly expresses a humanized anti-hTfR antibody-hIDUA fusion protein CHO cells (CHO-K1: obtained from the American Type Culture Collection) were transformed with pEM-hygr(LC3) and pE-mIRES-GSp-Fab-IDUA constructed in Example 1 using NEPA21 (NEPAGENE) by the following method.

[0080] Cell transformation was generally carried out using the following method: 2 × 10⁶ CHO-K1 cells 7 CD OptiCHO TMThe cells were suspended in a 1:1 mixture of culture medium (Thermo Fisher Scientific) and PBS. A 50 μL cell suspension was taken and added to it, and CD OptiCHO TM 50 μL of pEM-hygr(LC3) plasmid DNA solution, diluted to 200 μg / mL, was added to a 1:1 mixture of culture medium and PBS. Electroporation was performed using NEPA21 (NEPAGENE) to introduce pEM-hygr(LC3) plasmid DNA into the cells. After culturing overnight at 37°C and 5% CO2, the cells were treated with CD OptiCHO with 0.5 mg / mL of hygromycin. TM The cells were selectively cultured in culture medium. The resulting cells were then introduced with pE-mIRES-GSp-Fab-IDUA plasmid DNA (digested with AhdI and linearized) using the same method. After culturing overnight at 37°C and 5% CO2, the cells were treated with CD OptiCHO by adding 0.5 mg / mL hygromycin and 10 μg / mL puromycin. TM Cells were selectively cultured in culture medium. During selective culture, the concentration of MSX was gradually increased until the final MSX concentration was 300 μM, which selectively proliferated cells exhibiting drug resistance.

[0081] Next, using the limiting dilution method, the cells selected in selective culture were seeded onto a 96-well plate so that no more than one cell per well proliferated, and the cells were cultured for approximately two weeks until each cell formed a monoclonal colony. The culture supernatant was collected from the wells in which monoclonal colonies had formed, and the humanized antibody content was examined by ELISA to select cell lines that highly expressed humanized anti-hTfR antibody-hIDUA fusion protein.

[0082] The ELISA method used in this study was generally as follows: 100 μL of chicken anti-IDUA polyclonal antibody solution, diluted to 5 μg / mL in 0.05 M bicarbonate buffer, was added to each well of a 96-well microtiter plate (Nunc). The plates were allowed to stand at room temperature or 4°C for at least 1 hour to allow the antibody to adsorb to the plate. Next, each well was washed three times with Tris-buffered saline (pH 8.0) with 0.05% Tween20 added (TBS-T). Finally, 300 μL of Tris-buffered saline (pH 8.0) with 1% BSA added was added to each well, and the plates were allowed to stand at room temperature for 1 hour. Next, each well was washed three times with TBS-T, and then 100 μL of culture supernatant or purified humanized anti-hTfR antibody-hIDUA fusion protein, diluted to an appropriate concentration with Tris-buffered saline (pH 8.0) with 0.1% BSA and 0.05% Tween20 (TBS-BT), was added to each well, and the plate was left to stand at room temperature for 1 hour. Next, the plate was washed three times with TBS-T, and then 50 μL of HRP-labeled anti-human IgG polyclonal antibody solution diluted with TBS-BT was added to each well, and the plate was left to stand at room temperature for 1 hour. After washing each well three times with TBS-T, color development was performed using the ELISA POD substrate TMB kit (Nakalai Tesque). Next, 50 μL of 1 mol / L sulfuric acid was added to each well to stop the reaction, and the absorbance at 450 nm was measured for each well using a 96-well plate reader. Cells corresponding to wells showing high measurement values ​​were selected as cell lines that highly express the humanized anti-hTfR antibody-hIDUA fusion protein. These cell lines that thus obtained highly express the humanized anti-hTfR antibody-hIDUA fusion protein were designated as humanized anti-hTfR antibody-hIDUA expression cells. The fusion protein of the humanized anti-hTfR antibody and hIDUA expressed by this cell line was designated as the humanized anti-hTfR antibody-hIDUA fusion protein (humanized anti-hTfR antibody-hIDUA).

[0083] The resulting humanized anti-hTfR antibody-hIDUA expression strain was mixed with CD OptiCHO containing 10 mg / L insulin, 16 μmol / L thymidine, 100 μmol / L hypoxanthine, 500 μg / mL hygromycin B, 10 μg / mL puromycin, 300 μmol / L MSX, and 10% (v / v) DMSO. TM The cells were suspended in culture medium, then dispensed into cryotubes and stored in liquid nitrogen as seed cells.

[0084] [Example 3] Culture of humanized anti-hTfR antibody-hIDUA expressing strain To obtain humanized anti-hTfR antibody-hIDUA, humanized anti-hTfR antibody-hIDUA-expressing strains were cultured using the following method. The humanized anti-hTfR antibody-hIDUA-expressing strains obtained in Example 2 were cultured to a cell density of approximately 3 x 10⁶ 5 Approximately 170 L of serum-free medium (CD OptiCHO), adjusted to pH 6.9, containing 10 mg / L insulin, 16 μmol / L thymidine, and 100 μmol / L hypoxanthine, to achieve a concentration of 10 cells / mL. TM The cells were suspended in culture medium (ThermoFisher SCIENTIFIC). 170 L of this cell suspension was transferred to a culture vessel. The medium was stirred with an impeller at approximately 100 rpm to maintain a dissolved oxygen saturation of approximately 30%, and the cells were cultured at a temperature range of 34-37°C for approximately 10 days. During the culture period, cell density, cell viability, glucose concentration in the medium, and lactate concentration were monitored. If the glucose concentration in the medium fell below 3.0 g / L, glucose solution was immediately added to the medium to bring the glucose concentration to 3.5 g / L. During the culture period, feed solution (EFFICIENTFEED A+) was used. TM ThermoFisher SCIENTIFIC added appropriate amounts to the culture medium. After the culture was complete, the medium was collected. The collected medium was filtered through Millistak+HC Pod Filter grade D0HC (Merck) and then further filtered through Millistak+ HC grade X0HC (Merck) to obtain a culture supernatant containing humanized anti-hTfR antibody-hIDUA. This culture supernatant was then processed using Pellicon TM3 Cassette w / Ultracel PLCTK Membrane (pore size: 30 kDa, membrane area: 1.14m 2 The solution was ultrafiltered using a Merck filter and concentrated until the volume was reduced to approximately 1 / 14th. Next, this concentrate was filtered using an Opticap XL600 (0.22 μm, Merck). The resulting solution was used as the concentrated culture supernatant.

[0085] [Example 4] Purification of humanized anti-hTfR antibody-hIDUA To the concentrated culture supernatant obtained in Example 3, 0.25 times the volume of 2 M arginine solution (pH 7.0) was added. This solution was equilibrated with 25 mM MES buffer (pH 6.5) containing 400 mM arginine in a volume four times the column volume. Capture Select TM Humanized anti-hTfR antibody-hIDUA was adsorbed onto a CH1-XL column (column volume: approx. 3.1 L, bed height: approx. 20 cm, Thermo Fisher Scientific) at a constant flow rate of 100 cm / hour. TM The CH1-XL column is an affinity column in which a ligand that specifically binds to the CH1 domain of IgG antibodies is immobilized on a support.

[0086] Next, the column was washed by supplying five times the volume of the column buffer at the same flow rate. Then, the column was further washed by supplying three times the volume of the column buffer (25 mM MES buffer, pH 6.5) at the same flow rate. Next, the humanized anti-hTfR antibody-hIDUA adsorbed on the column was eluted with five times the volume of the column buffer (10 mM sodium acetate-HCl buffer, pH 3.5). The eluate was received in a container pre-filled with 250 mM MES buffer (pH 6.0) and immediately neutralized.

[0087] The eluate from the affinity column described above was diluted with 250 mM MES buffer (pH 6.5) to adjust the pH of the eluate to 6.0. This eluate was then filtered using an Opticap XL600 filter (pore size: 0.22 μm, Merck). The filtered solution was then loaded onto a Capto adhere column (column volume: approximately 1.5 L, bed height: approximately 10 cm, GE Healthcare), a multimodal anion exchange column equilibrated with 50 mM MES buffer (pH 6.0) containing 15 mM NaCl in a volume five times the column volume, at a constant flow rate of 300 cm / hour. The loaded solution containing the humanized anti-hTfR antibody-hIDUA was collected. Capto adhere is a strong anion exchanger with N-benzyl-N-methylethanolamine as its ligand, exhibiting selectivity based on electrostatic interactions, hydrogen bonding, hydrophobic interactions, etc.

[0088] Next, the column was washed by supplying five times the volume of the column buffer at the same flow rate, and this washing solution was collected.

[0089] The above loading solution and washing solution were equilibrated with 25 mM MES buffer (pH 6.5) containing 300 mM NaCl in a volume four times the column volume. A Capto MMC column (column volume: approximately 3.1 L, bed height: approximately 20 cm, GE Healthcare), a multimodal weak cation exchange column, was then loaded at a constant flow rate of 200 cm / hour. Capto MMC is a weak cation exchanger that exhibits selectivity based on hydrophobic interactions, hydrogen bond formation, etc.

[0090] Next, the column was washed by supplying five times the volume of the buffer solution at the same flow rate. Then, the humanized anti-hTfR antibody-hIDUA adsorbed on the weak cation exchange column was eluted with 25 mM MES buffer (pH 6.5) containing 1 M NaCl in ten times the volume of the column.

[0091] The eluate from the weak cation exchange column described above was adjusted to pH 5.8 by adding 20 mM citrate buffer (pH 5.5) containing 0.5 times the volume of 0.8 mg / mL NaCl and 75 mg / mL sucrose. Then, Pellicon TM 3 Cassette w / Ultracel PLCTK Membrane (pore size: 30 kDa, membrane area: 0.57 m 2 The solution was ultrafiltered using a Merck filter and concentrated until the concentration of humanized anti-hTfR antibody-hIDUA in the solution was approximately 30 mg / mL. This concentrate was then filtered using an Opticap XL600 (0.22 μm, Merck).

[0092] The above concentrate was loaded onto a BioSEC column (column volume: approximately 9.4 L, bed height: 30 cm, Merck), a size exclusion column, at a constant flow rate of 40 cm / hour, equilibrated with 20 mM citrate buffer (pH 5.5) containing 0.8 mg / mL NaCl and 75 mg / mL sucrose in a volume 1.5 times the column volume. The same buffer was then supplied at the same flow rate. At this time, a spectrophotometer was placed in the flow path of the eluate from the size exclusion column to continuously measure the absorbance of the eluate, and the absorbance at 280 nm was monitored. The fraction showing an absorption peak at 280 nm was collected as the fraction containing humanized anti-hTfR antibody-hIDUA, and this was used as the purified humanized anti-hTfR antibody-hIDUA product.

[0093] [Example 5] Preparation of aqueous pharmaceutical composition Using the purified humanized anti-hTfR antibody-hIDUA fusion protein obtained in Example 4, aqueous pharmaceutical compositions having the compositions shown in Table 1 were prepared. These aqueous pharmaceutical compositions are filled and sealed in glass or plastic vials, ampoules, or syringes in volumes of 1 to 10 mL and stored at low temperatures (e.g., 4°C). When filled and sealed in syringes, they become pre-filled syringe formulations.

[0094] [Table 1]

[0095] [Example 6] Clinical Trial (Phase I) To confirm the safety and efficacy of the aqueous pharmaceutical composition containing the humanized anti-hTfR antibody-hIDUA fusion protein prepared in Example 5 (hereinafter referred to as the "investigation drug") against mucopolysaccharidosis type I, a clinical trial (Phase I) was conducted.

[0096] In the clinical trial (Phase I), subjects were patients with mucopolysaccharidosis type 1 (MPS I) who met at least the selection criteria shown in Table 2. The number of subjects was four. All subjects had been receiving standard enzyme replacement therapy for mucopolysaccharidosis type 1 prior to participating in the trial.

[0097] [Table 2]

[0098] The investigational drug was administered to patients according to the dosage and administration method shown in Table 3. Specifically, each subject received an initial dose of 0.1 mg / kg body weight, followed by a second dose of 1.0 mg / kg body weight, a third dose of 2.0 mg / kg body weight, and a fourth dose of 4.0 mg / kg body weight, for a total of four doses. Each dose was administered at a one-week interval. Each dose was administered by intravenous infusion over approximately three hours while observing the subject's condition. After the completion of the clinical trial, patients were to receive standard enzyme replacement therapy for mucopolysaccharidosis type I. Cerebrospinal fluid was collected from each subject before administration of the investigational drug and within five hours after the fourth dose. Serum and urine were also collected from each subject before administration of the investigational drug and one week after the fourth dose, before standard enzyme replacement therapy for mucopolysaccharidosis type I was administered.

[0099] [Table 3]

[0100] The efficacy of the investigational drug against mucopolysaccharidosis type 1 was evaluated by examining the items shown in Table 4. Table 4 shows some of the efficacy evaluation items described in the clinical trial protocol.

[0101] [Table 4]

[0102] The safety of the investigational drug in patients with mucopolysaccharidosis type 1 was evaluated by examining the items shown in Table 5. Table 5 shows some of the safety evaluation items described in the clinical trial protocol.

[0103] [Table 5]

[0104] [Example 7] Evaluation of efficacy in clinical trial (Phase I) (Concentrations of heparan sulfate and dermatan sulfate in cerebrospinal fluid) Figures 1 and 2 show the results of measuring the concentrations of heparan sulfate and dermatan sulfate in cerebrospinal fluid collected from each subject before administration of the test drug and within 5 hours after the fourth administration in Example 6. Regarding heparan sulfate, the average concentration in the cerebrospinal fluid of the subjects was 1132 ng / mL before administration of the test drug, but this decreased significantly to 467 ng / mL after administration (Figure 1). Since the average concentration of heparan sulfate in cerebrospinal fluid in non-mucopolysaccharidosis patients is approximately 360 ng / mL, it was predicted that the concentration of heparan sulfate in cerebrospinal fluid could be reduced to the level of non-mucopolysaccharidosis patients by further administration of the test drug. The concentrations of heparan sulfate and dermatan sulfate were measured using the methods described in Examples 12-14.

[0105] Regarding dermatan sulfate, the average concentration of dermatan sulfate in the cerebrospinal fluid of subjects was 264 ng / mL before administration of the investigational drug, but this decreased significantly to 213 ng / mL after administration (Figure 2). Similar to heparan sulfate, it was predicted that continuing administration of the investigational drug could further reduce the concentration of dermatan sulfate in the cerebrospinal fluid to levels seen in non-mucopolysaccharidosis patients.

[0106] These results indicate that the test drug administered to patients by intravenous infusion passed through the blood-brain barrier (BBB) ​​to the central nervous system, where it broke down and removed accumulated glycosaminoglycans, demonstrating that the test drug is also effective against central nervous system abnormalities in mucopolysaccharidosis type 1. Furthermore, these results suggest that the test drug will be effective when administered once a week at a dose of 1-6 mg / kg body weight or 1-8 mg / kg body weight.

[0107] (Concentrations of heparan sulfate and dermatan sulfate in serum and urine) Figures 3 and 4 show the results of measuring the concentrations of heparan sulfate and dermatan sulfate in serum collected from each subject in Example 6, before administration of the test drug and one week after the fourth administration, before the usual enzyme replacement therapy for mucopolysaccharidosis type I was administered. Regarding heparan sulfate, the average concentration in the subjects' serum was 359 ng / mL before administration of the test drug, but decreased significantly to 217 ng / mL after administration (Figure 3). Regarding dermatan sulfate, the average concentration in the subjects' serum was 965 ng / mL before administration of the test drug, but decreased to 867 ng / mL after administration (Figure 4).

[0108] Figures 5 and 6 show the results of measuring the concentrations of heparan sulfate and dermatan sulfate in urine collected from each subject in Example 6 before administration of the investigational drug and one week after the fourth administration, before the usual enzyme replacement therapy for mucopolysaccharidosis type I was administered. Regarding heparan sulfate, the average concentration in the subjects' urine was 31.0 μg / mg creatinine before administration of the investigational drug, but decreased significantly to 16.0 μg / mg creatinine after administration (Figure 5). Regarding dermatan sulfate, the average concentration in the subjects' urine was 22.7 μg / mg creatinine before administration of the investigational drug, but decreased significantly to 17.1 μg / mg creatinine after administration (Figure 6). These results indicate that the test drug administered to patients by intravenous infusion broke down and removed glycosaminoglycans accumulated not only in the central nervous system but also in tissues throughout the body, demonstrating that the test drug is effective against central nervous system and other tissue abnormalities in mucopolysaccharidosis type 1. Furthermore, in patients in whom glycosaminoglycans accumulated in the central nervous system and other tissues are broken down and removed, improvements in functional impairments can be expected, such as the ability to converse for longer periods, the ability to write more, increased vitality, reduced joint pain in the lower back and knees, reduced pain and muscle and joint stiffness associated with walking, easier execution of finger movements such as opening cans and full-body exercises such as basketball, and improved language ability.

[0109] [Example 8] Safety evaluation of clinical trial (Phase I) No serious adverse events were observed in any subject during the administration period of the investigational drug. This indicates that the investigational drug can be safely administered once a week at a dose of 0.1–4 mg / kg body weight.

[0110] [Example 9] Clinical trial (Phase II) The clinical trial (Phase II) aims to confirm the safety and efficacy of the investigational drug against mucopolysaccharidosis type I after 3 months of administration.

[0111] The clinical trial (Phase II) included patients with mucopolysaccharidosis type 1 (MPS I) who met at least the selection criteria shown in Table 2. All subjects had been receiving standard enzyme replacement therapy for mucopolysaccharidosis type 1 prior to participating in the trial.

[0112] The clinical trial (Phase II) consists of two trials. In the first trial of the clinical trial (Phase II), the investigational drug will be administered to patients according to the dosage and administration method shown in Table 6. Specifically, each subject will receive an initial dose of 1.0 mg / kg body weight, followed by a second dose of 2.0 mg / kg body weight, and then 10 doses from the third to the twelfth dose at a dose of 4.0 mg / kg body weight. Each dose will be administered at a one-week interval. Each dose will be administered by intravenous infusion over approximately 3 hours while observing the subject's condition. There will be two subjects.

[0113] [Table 6]

[0114] In the second phase of the clinical trial (Phase II), the investigational drug was administered to patients according to the dosage and administration method shown in Table 7. Specifically, subjects were divided into two groups: one group received the investigational drug at a dose of 2.0 mg / kg body weight (2.0 mg / kg body weight group), and the other group received it at a dose of 4.0 mg / kg body weight (4.0 mg / kg body weight group). Each group received the investigational drug 12 times at one-week intervals. Each administration was performed by intravenous infusion over approximately 3 hours while observing the subject's condition. The trial was conducted openly, with 15 subjects.

[0115] [Table 7]

[0116] In both the first and second phases of the clinical trial (Phase II), cerebrospinal fluid (CSF) was collected from each subject before administration of the investigational drug and within 5 hours after the 12th dose. Serum and urine were also collected from each subject before administration of the investigational drug and one week after the 12th dose, prior to the administration of standard enzyme replacement therapy for mucopolysaccharidosis type I. For the second phase, CSF, serum, and urine were collected only from 6 of the planned 15 subjects who completed the treatment.

[0117] The efficacy of the investigational drug for mucopolysaccharidosis type 1 will be evaluated by examining the items shown in Table 4, as well as by administering the Modified Brief Visuospatial Memory Test (BVMT-R), the Modified Hopkins Verbal Learning Test (HVLT-R), and the Attentional Variable Test (TOVA) to the subjects.

[0118] The safety of the investigational drug in patients with mucopolysaccharidosis type 1 will be evaluated by examining the items shown in Table 5. Table 5 shows some of the safety evaluation items described in the clinical trial protocol.

[0119] [Example 10] Evaluation of efficacy in clinical trial (Phase II) (Concentrations of heparan sulfate and dermatan sulfate in cerebrospinal fluid) Figures 7 and 8 show the results of measuring the concentrations of heparan sulfate and dermatan sulfate in cerebrospinal fluid (CSF) collected from each subject in the first trial before administration of the investigational drug and within 5 hours after the 12th administration. Regarding heparan sulfate, in all subjects, the concentration of heparan sulfate in the CSF decreased to less than 1 / 3 of the pre-administration level after administration of the investigational drug (Figure 7). Furthermore, in all subjects, the concentration of heparan sulfate in the CSF was 1400 ng / mL or less, and also below the average value of approximately 360 ng / mL for CSF in non-mucopolysaccharidosis patients. These results suggest that by continuing administration of the investigational drug, the concentration of heparan sulfate in the CSF can be reduced to and maintained at the level of non-mucopolysaccharidosis patients.

[0120] Regarding dermatan sulfate, in all subjects, the concentration of dermatan sulfate in the cerebrospinal fluid decreased after administration of the study drug compared to before administration (Figure 8). The average concentration of dermatan sulfate in cerebrospinal fluid in non-mucopolysaccharidosis patients is approximately 220 ng / mL. Similar to heparan sulfate, it was predicted that continued administration of the study drug could reduce the concentration of dermatan sulfate in cerebrospinal fluid to the level of non-mucopolysaccharidosis patients and maintain that level.

[0121] Figures 9 and 10 show the results of measuring the concentrations of heparan sulfate and dermatan sulfate in cerebrospinal fluid (CSF) collected from each subject in the second trial before administration of the investigational drug and within 5 hours after the 12th administration. Regarding heparan sulfate, in all subjects, the concentration of heparan sulfate in the CSF decreased to less than 1 / 3 of the pre-administration level after administration of the investigational drug (Figure 9). Furthermore, in all subjects, the concentration of heparan sulfate in the CSF became 1400 ng / mL or less, approaching or falling below the average value of approximately 360 ng / mL for CSF in non-mucopolysaccharidosis patients. It was predicted that further administration of the investigational drug could reduce the concentration of heparan sulfate in the CSF to the level of non-mucopolysaccharidosis patients and maintain that level.

[0122] Regarding dermatan sulfate, in all subjects, the concentration of dermatan sulfate in the cerebrospinal fluid decreased after administration of the study drug compared to before administration (Figure 10). The average concentration of dermatan sulfate in cerebrospinal fluid in non-mucopolysaccharidosis patients is approximately 220 ng / mL. Similar to heparan sulfate, it was predicted that continued administration of the study drug could reduce the concentration of dermatan sulfate in cerebrospinal fluid to the level of non-mucopolysaccharidosis patients and maintain that level. The concentrations of heparan sulfate and dermatan sulfate were measured using the methods described in Examples 12-14.

[0123] These results indicate that the test drug administered to patients by intravenous infusion passed through the blood-brain barrier (BBB) ​​to the central nervous system, where it broke down and removed accumulated glycosaminoglycans, demonstrating that the test drug is also effective against central nervous system abnormalities in mucopolysaccharidosis type 1. Furthermore, these results suggest that the test drug will be effective when administered once a week at a dose of 1-6 mg / kg body weight or 1-8 mg / kg body weight.

[0124] (Concentrations of heparan sulfate and dermatan sulfate in serum) Figures 11 and 12 show the results of measuring the concentrations of heparan sulfate and dermatan sulfate in serum collected from each subject in the first study before administration of the investigational drug and one week after the 12th administration, before the administration of standard enzyme replacement therapy for mucopolysaccharidosis type I. For both heparan sulfate and dermatan sulfate, a general decrease in concentration after administration compared to before administration of the investigational drug was observed (Figures 11 and 12).

[0125] Figures 13 and 14 show the results of measuring the concentrations of heparan sulfate and dermatan sulfate in serum collected from each subject in the second study before administration of the investigational drug and one week after the 12th administration, before the administration of standard enzyme replacement therapy for mucopolysaccharidosis type I, respectively. For both heparan sulfate and dermatan sulfate, a general decrease in concentration after administration compared to before administration of the investigational drug was observed (Figures 13 and 14).

[0126] The above results indicate that the test drug administered to patients by intravenous infusion broke down and removed glycosaminoglycans accumulated not only in the central nervous system but also in tissues throughout the body, demonstrating that the test drug is effective against central nervous system and other tissue abnormalities in mucopolysaccharidosis type 1. Furthermore, in patients in whom glycosaminoglycans accumulated in the central nervous system and other tissues have been broken down and removed, it can be expected that improvements in central nervous system dysfunction will lead to effects such as the ability to converse for longer periods, the ability to write more, increased vitality, reduced joint pain in the lower back and knees, reduced pain and muscle and joint stiffness associated with walking, easier execution of finger movements such as opening cans and full-body exercises such as basketball, and improved language ability.

[0127] [Example 11] Safety evaluation of clinical trial (Phase II) No serious adverse events were observed in any subject during the administration period of the investigational drug. This indicates that the investigational drug can be safely administered once a week at a dose of 0.1 mg / kg to 4 mg / kg body weight.

[0128] [Example 12: Method for measuring heparan sulfate and dermatan sulfate (preparation of various solutions)] The solutions (a) to (k) used in the test were prepared according to the following procedure. (a) MeCN / water: 0.5 mL of sterile water for injection and 4.5 mL of acetonitrile were mixed to make MeCN / water. This solution was prepared immediately before use. (b) Deuterium-labeled solvent: Under ice bath conditions, 240 μL of acetyl chloride was added dropwise to 1.5 mL of methanol-d4 (Sigma-Aldrich) to prepare a deuterium-labeled solvent. This solution was prepared immediately before use. (c)PBS / citric acid solution: A 10 mM citric acid solution was prepared by dissolving citric acid in pure water. Furthermore, a 10 mM sodium citrate solution was prepared by dissolving trisodium citrate dihydrate in pure water. The sodium citrate solution was added dropwise to the citric acid solution to adjust the pH to 3.0. This solution was prepared as 10 mM citrate buffer (pH 3.0). Additionally, a PBS / citric acid solution was prepared by adding 2 mL of PBS to 18 mL of 10 mM citrate buffer (pH 3.0). (d) Mobile phase A: Mobile phase A was prepared by adding 2.5 mL of 1 M aqueous ammonium formate solution and 400 μL of aqueous ammonium hydroxide solution (25% NH4OH) to 247.5 mL of pure water and mixing. This solution was prepared immediately before use. (e) Mobile phase B: Mobile phase B was prepared by mixing 5 mL of 1 M aqueous ammonium formate solution, 450 mL of acetonitrile, and 800 μL of aqueous ammonium hydroxide solution (25% NH4OH) in 45 mL of pure water. This solution was prepared immediately before use. (f) Heparan sulfate standard stock solution (HS standard stock solution): Heparan sulfate (Iduron) was weighed into a 1.5 mL microcentrifuge tube and dissolved in sterile water for injection to prepare a solution with a concentration of 5.0 mg / mL. The prepared solution was dispensed in 15 μL portions into 0.5 mL screw-cap tubes and stored frozen (-15°C or below) until use. This solution was used as the HS standard stock solution. (g) Dermatan sulfate standard stock solution (DS standard stock solution): Chondroitin sulfate B sodium salt from porcine intestinal mucosa (Sigma-Aldrich) was weighed into a 1.5 mL microtube, dissolved in sterile water for injection, and a solution with a concentration of 5.0 mg / mL was prepared. The prepared solution was dispensed in 15 μL portions into 0.5 mL screw-cap tubes and stored frozen (-15°C or below) until use. This solution was used as the DS standard stock solution. (h) Dermatan sulfate internal standard solution (DS internal standard solution): 40 μL of the DS standard stock solution was measured into a borosilicate screw-cap test tube, and the solvent was removed under a nitrogen stream. 400 μL of deuterium-labeled solvent was added to the dry material, and after stirring, deuteriomethanolysis was carried out at 65°C for 75 minutes. After the reaction, the solvent was removed under a nitrogen stream. 500 μL of MeCN / water was added to the dry material, and sonication was performed for 30 minutes. The prepared solution was dispensed in 20 μL portions into 0.5 mL screw-cap tubes and stored frozen (-15°C or below). This solution was designated as the dermatan sulfate internal standard solution (DS internal standard solution). (i) Heparan sulfate internal standard solution (HS internal standard solution): 40 μL of HS standard stock solution was measured into a borosilicate screw-cap test tube, and the solvent was removed under a nitrogen stream. 400 μL of deuterium-labeled solvent was added to the dry solution, and after stirring, deuteriomethanolysis was carried out at 65°C for 75 minutes. After the reaction, the solvent was removed under a nitrogen stream. 500 μL of MeCN / water was added to the dry solution, and sonication was performed for 30 minutes. The prepared solution was dispensed in 20 μL portions into 0.5 mL screw-cap tubes and stored frozen (-15°C or below). This solution was designated as the internal standard solution of heparan sulfate (HS internal standard solution). (j) Solution for dissolving the sample: 5 mL of MeCN / water was mixed with 1 μL of HS internal standard solution and 1 μL of DS internal standard solution, and then sonicated for 30 minutes. This solution was used as the sample dissolution solution. This solution was prepared immediately before use. (k) Solution for creating the calibration curve: 480 μL of PBS / citric acid solution was measured out, and 10 μL each of HS standard stock solution and DS standard stock solution were added to it to prepare a solution containing 100 μg / mL each of dermatan sulfate and heparan sulfate. This solution was diluted with PBS / citric acid solution to prepare a solution containing 2500 ng / mL each of dermatan sulfate and heparan sulfate. This solution was then diluted twice with PBS / citric acid solution to prepare solutions containing dermatan sulfate and heparan sulfate at concentrations of 25 to 2500 ng / mL each. This solution was used for creating the calibration curve.

[0129] [Example 13: Method for measuring heparan sulfate and dermatan sulfate (methanolysis reaction)] 20 μL of each calibration curve solution prepared in Example 12 was measured out and individually placed in borosilicate screw-cap test tubes. A PBS / citric acid solution was also placed in a borosilicate screw-cap test tube as a blank. The solvent in the test tubes was removed under a nitrogen stream (N=1). 20 μL of 2,2-dimethoxypropane and 200 μL of methanol hydrochloride (3N concentration) were added to the dry material and stirred. A methanolysis reaction was then carried out in a constant temperature water bath at 70°C for 90 minutes. After the reaction, the solvent was removed under a nitrogen stream. 50 μL of sample dissolution solution was added to the dry material to dissolve it, then centrifuged (15000 rpm, 10 minutes, room temperature), and the supernatant was collected in a vial.

[0130] Furthermore, the dermatan sulfate standard stock solution and heparan sulfate standard stock solution prepared in Example 12 above were diluted with PBS / citric acid solution to prepare four types of quality control samples (QC samples) containing the dermatan sulfate standard stock solution and heparan sulfate standard stock solution at concentrations of 25.0 ng, 50.0 ng / mL, 500 ng / mL, and 2000 ng / mL, respectively, and were designated QC-LL, QC-L, QC-M, and QC-H. 20 μL of each was measured out and individually placed into borosilicate screw-cap test tubes, and the solvent in the test tubes was removed under a nitrogen stream. To the dry material, 20 μL of 2,2-dimethoxypropane and 200 μL of methanol hydrochloride with a concentration of 3N were added and stirred, and then a methanolysis reaction was carried out in a constant temperature water bath at 70°C for 90 minutes. After the reaction, the solvent was removed under a nitrogen stream. 50 μL of sample dissolution solution was added to the dry material to dissolve it, then the mixture was centrifuged (15000 rpm, 10 minutes, room temperature), and the supernatant was collected in a vial.

[0131] Furthermore, the cerebrospinal fluid, serum, and urine collected in Example 6 or 9 were each divided into borosilicate screw-cap test tubes, and the solvent in the test tubes was removed under a nitrogen stream. To the dry material, 20 μL of 2,2-dimethoxypropane and 200 μL of methanol hydrochloride (3N concentration) were added and stirred, and then the methanolysis reaction was carried out in a constant temperature water bath at 70°C for 90 minutes. After the reaction, the solvent was removed under a nitrogen stream. 50 μL of sample dissolution solution was added to the dry material to dissolve it, and then the mixture was centrifuged (15000 rpm, 10 minutes, room temperature), and the supernatant was collected in a vial.

[0132] [Example 14: Method for measuring heparan sulfate and dermatan sulfate (LC / MS / MS analysis)] LC / MS / MS analysis was performed using a combination of hydrophilic interaction ultra-high performance liquid chromatography (HMS) and tandem quadrupole mass spectrometry. A QTRAP5500 (AB Sciex Co., Ltd.) was used as the mass spectrometer (MS / MS), paired with a Nexera X2 (Shimadzu Corporation) HPLC system. Acquity UPLC was used as the LC column. TMA BEH Amid 1.7 μm (2.1 x 50 mm, Waters) was used. Mobile phases A and B prepared in Example 12 were used as the mobile phases. The column temperature was set to 50°C.

[0133] The column was equilibrated with a mixture of mobile phase A (6% v / v) and mobile phase B (94% v / v). Then, 10 μL of the sample was injected, and chromatography was performed under the mobile phase gradient conditions shown in Table 8. The mobile phase flow rate was 0.4 mL / min.

[0134] [Table 8]

[0135] The ion source parameters of the MS / MS instrument were set according to the user manual for QTRAP5500 (AB Sciex Corporation), as shown in Table 9.

[0136] [Table 9]

[0137] By measuring the calibration curve preparation solutions and QC samples, the area of ​​the peaks (detection peaks) detected on the chromatograms of product ions derived from dermatan sulfate and heparan sulfate contained in each calibration curve preparation solution was determined. In addition, the area of ​​the detection peaks of product ions derived from DS internal standard solution and HS internal standard solution was determined. Measurements were performed with N=1 for the calibration curve preparation solutions and N=3 for the QC samples (QC-LL, QC-L, QC-M, and QC-H).

[0138] The dermatan sulfate and heparan sulfate contained in the DS and HS internal standard solutions are deuterium-labeled. Therefore, the mass-to-charge ratios (m / z) of the precursor ions derived from these are 432 and 390, respectively, which are larger than those of unlabeled precursor ions. The mass-to-charge ratios (m / z) of the precursor ions derived from unlabeled dermatan sulfate and heparan sulfate are 426 and 384, respectively. Therefore, these precursor ions are separated at the quadrupole (Q1) based on their mass-to-charge ratios (m / z), making them individually detectable. Table 10 summarizes the (m / z) values ​​of the precursor ions and product ions.

[0139] [Table 10]

[0140] The ratio of the area of ​​the detection peak derived from the DS internal standard solution (DS-IS detection peak area) to the area of ​​the detection peak derived from the dermatan sulfate contained in each calibration solution (DS detection peak area) was calculated (DS detection peak area / DS-IS detection peak area). This value was plotted on the vertical axis, and the concentration of dermatan sulfate in each calibration solution was plotted on the horizontal axis. A regression equation was calculated using quadratic programming to create a calibration curve.

[0141] Furthermore, the ratio of the area of ​​the detection peak derived from the HS internal standard solution (HS-IS detection peak area) to the area of ​​the detection peak derived from the heparan sulfate contained in each calibration curve preparation solution (HS detection peak area) was calculated (HS detection peak area / HS-IS detection peak area). This value was plotted on the vertical axis, and the concentration of heparan sulfate in each calibration curve preparation solution was plotted on the horizontal axis. A regression equation was calculated using quadratic programming to create a calibration curve.

[0142] The measured values ​​of cerebrospinal fluid, serum, and urine from each subject were interpolated into the calibration curve described above to determine the concentrations of DS and HS in each sample. The measured values ​​of DS and HS in urine were corrected for creatinine to calculate the amount contained in urine containing 1 mg of creatinine (μg / mg creatinine). [Industrial applicability]

[0143] According to the present invention, for example, a novel drug can be provided that can be used in enzyme replacement therapy for patients with mucopolysaccharidosis type 1. [Sequence Listing Free Text]

[0144] Sequence ID 1: Amino acid sequence of linker example 1 Sequence ID 2: Amino acid sequence of linker example 2 Sequence ID 3: Amino acid sequence of linker example 3 Sequence ID 4: Amino acid sequence 1 of light chain CDR1 Sequence ID 5: Amino acid sequence 2 of light chain CDR1 Sequence ID 6: Amino acid sequence 1 of light chain CDR2 Sequence ID 7: Amino acid sequence 2 of light chain CDR2 Sequence ID 8: Amino acid sequence 1 of light chain CDR3 SEQ ID NO: 9: Amino acid sequence 1 of heavy chain CDR1 SEQ ID NO: 10: Amino acid sequence 2 of heavy chain CDR1 Sequence ID 11: Amino acid sequence 1 of heavy chain CDR2 Sequence ID 12: Amino acid sequence 2 of heavy chain CDR2 SEQ ID NO: 13: Amino acid sequence 1 of heavy chain CDR3 Sequence ID 14: Amino acid sequence 2 of heavy chain CDR3 Sequence ID 15: Amino acid sequence of heavy chain framework region 3 Sequence ID 16: Amino acid sequence of the variable region of the heavy chain Sequence ID 17: Amino acid sequence of the variable region of the light chain Sequence ID 18: Amino acid sequence of the light chain Sequence ID 19: Amino acid sequence of the Fab heavy chain Sequence ID 20: Amino acid sequence 1 of human IDUA Sequence ID 21: Amino acid sequence 2 of human IDUA Sequence ID 22: Primer Hyg-Sfi5', synthetic sequence Sequence ID 23: Primer Hyg-BstX3', synthetic sequence Sequence ID 24: Primer IRES5', synthetic sequence Sequence ID 25: Primer IRES3', synthetic sequence Sequence ID 26: Primer mPGKP5', synthetic sequence Sequence ID 27: Primer mPGKP3', synthetic sequence Sequence ID 28: Primer GS5', synthetic sequence Sequence ID 29: Primer GS3', synthetic sequence Sequence ID 30: Primer puro5', synthetic sequence Sequence ID 31: Primer puro3', synthetic sequence Sequence ID 32: Primer SV40polyA5', synthetic sequence Sequence ID 33: Primer SV40polyA3', synthetic sequence Sequence ID 34: Primer mIRES-GS5', synthetic sequence Sequence ID 35: Primer mIRES-GS3', synthetic sequence Sequence ID 36: CMVE-EF-1αp-IFNβMAR, synthetic sequence Sequence ID 37: Base sequence and synthetic sequence encoding the amino acid sequence of the light chain Sequence ID 38: Amino acid sequence of a fusion protein of Fab heavy chain and human IDUA Sequence ID 39: Base sequence and synthetic sequence containing the gene encoding the amino acid sequence of the fusion protein of Fab heavy chain and human IDUA. Sequence ID 40: IRES-HygroR-mPGKpA, synthetic sequence

Claims

1. A pharmaceutical composition comprising an anti-human transferrin receptor antibody and human α-L-iduronidase fusion protein as an active ingredient, the fusion protein being administered to a patient suffering from mucopolysaccharidosis type I by intravenous infusion at a dose of 0.1 to 10 mg / kg body weight, A pharmaceutical composition, wherein the anti-human transferrin receptor antibody is a Fab and the fusion protein is represented by the following (a) or (b): (a) the light chain of the anti-human transferrin receptor antibody comprises the amino acid sequence of SEQ ID NO:18, the heavy chain of the anti-human transferrin receptor antibody comprises the amino acid sequence of SEQ ID NO:19, and the heavy chain is linked at its C-terminus to human α-L-iduronidase having the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21 via a linker having the amino acid sequence of SEQ ID NO:3; (b) the light chain of the anti-human transferrin receptor antibody comprises the amino acid sequence of SEQ ID NO:18, the heavy chain of the anti-human transferrin receptor antibody comprises the amino acid sequence of SEQ ID NO:19, and the heavy chain is bound to human α-L-iduronidase having the amino acid sequence of SEQ ID NO:20 at its C-terminus via a linker having the amino acid sequence of SEQ ID NO:3, thereby forming the amino acid sequence of SEQ ID NO:

24.

2. The pharmaceutical composition according to claim 1, wherein the fusion protein is administered at a dose of 0.1 to 8 mg / kg body weight.

3. The pharmaceutical composition according to claim 1, wherein the fusion protein is administered at a dose of 1 to 6 mg / kg body weight.

4. 2. The pharmaceutical composition according to claim 1, wherein the fusion protein is administered at a dose of 2 mg / kg body weight or 4 mg / kg body weight.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the fusion protein is administered at a rate of 0.33 mg / hour to 200 mg / hour.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the fusion protein is administered over a period of 3 hours.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the fusion protein is administered by intravenous infusion.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the administration is carried out at intervals of 5 to 21 days for at least 3 consecutive months.

9. The pharmaceutical composition according to any one of claims 1 to 7, wherein the administration is carried out at intervals of 7 days for at least one month.

10. The pharmaceutical composition according to claim 8 or 9, wherein the fusion protein is administered at a dose of 0.1 to 2 mg / kg body weight in the first administration, and the dose is increased in the second and subsequent administrations.

11. The pharmaceutical composition according to claim 8 or 9, wherein the fusion protein is administered at a dose of 0.1 to 2 mg / kg body weight at the initial administration, and then at a maintenance dose of 2 to 6 mg / kg body weight.

12. 11. The pharmaceutical composition according to claim 8 or 10, which is administered at a maintenance dose of 2 mg / kg body weight or 4 mg / kg body weight.

13. The pharmaceutical composition according to any one of claims 1 to 12, which is a lyophilized agent or an aqueous liquid agent, further comprising a neutral salt, a disaccharide, a non-ionic surfactant, and a buffering agent, and the non-ionic surfactant includes polysorbate and poloxamer.

14. A pharmaceutical composition which is an aqueous liquid formulation as described in claim 13, wherein the polysorbate is polysorbate 80, the poloxamer is polyoxyethylene (160) polyoxypropylene (30) glycol, the concentration of the polysorbate is 0.005 to 1.5 mg / mL, and the concentration of the poloxamer is 0.1 to 0.6 mg / mL.

15. A pharmaceutical composition which is an aqueous liquid formulation as described in claim 13, wherein the polysorbate is polysorbate 80, the poloxamer is polyoxyethylene (160) polyoxypropylene (30) glycol, the concentration of the polysorbate is 0.025 to 1.0 mg / mL, and the concentration of the poloxamer is 0.2 to 0.5 mg / mL.

16. A pharmaceutical composition which is an aqueous liquid formulation as described in claim 13, wherein the polysorbate is polysorbate 80, the poloxamer is polyoxyethylene (160) polyoxypropylene (30) glycol, the concentration of the polysorbate is 0.05 to 0.15 mg / mL, and the concentration of the poloxamer is 0.25 to 0.45 mg / mL.

17. The pharmaceutical composition according to claim 13, wherein the polysorbate is polysorbate 80, the poloxamer is polyoxyethylene (160) polyoxypropylene (30) glycol, the neutral salt is sodium chloride, the disaccharide is sucrose, and the buffer is a citrate buffer, and the pharmaceutical composition is an aqueous liquid preparation selected from the group consisting of the following (1) to (3): (1) The concentration of the fusion protein is 1-10 mg / mL, the concentration of the neutral salt is 0.3-1.2 mg / mL, the concentration of the disaccharide is 50-100 mg / mL, the concentration of the buffer is 10-30 mM, the concentration of the polysorbate is 0.005-1.5 mg / mL, and the concentration of the poloxamer is 0.1-0.6 mg / mL; (2) the fusion protein has a concentration of 2-8 mg / mL, the neutral salt has a concentration of 0.5-1.0 mg / mL, the disaccharide has a concentration of 55-95 mg / mL, the buffer has a concentration of 15-25 mM, the polysorbate has a concentration of 0.05-1.0 mg / mL, and the poloxamer has a concentration of 0.25-0.45 mg / mL; and (3) The concentration of the fusion protein is 4 to 6 mg / mL, the concentration of the neutral salt is 0.7 to 0.9 mg / mL, the concentration of the disaccharide is 60 to 90 mg / mL, the concentration of the buffer is 15 to 25 mM, the concentration of the polysorbate is 0.05 to 0.15 mg / mL, and the concentration of the poloxamer is 0.25 to 0.45 mg / mL.

18. 18. The pharmaceutical composition according to claim 17, which is an aqueous solution having a pH of 4.5 to 6.5, 5.0 to 6.0, or 5.2 to 5.

8.

19. The pharmaceutical composition according to claim 13, wherein the polysorbate is polysorbate 80, the poloxamer is polyoxyethylene (160) polyoxypropylene (30) glycol, the neutral salt is sodium chloride, the disaccharide is sucrose, and the buffer is a citrate buffer, and the lyophilization agent is selected from the group consisting of (1) to (3) below: (1) When dissolved in pure water, the fusion protein has a concentration of 1 to 10 mg / mL, the neutral salt has a concentration of 0.3 to 1.2 mg / mL, the disaccharide has a concentration of 50 to 100 mg / mL, the buffer has a concentration of 10 to 30 mM, the polysorbate has a concentration of 0.005 to 1.5 mg / mL, and the poloxamer has a concentration of 0.1 to 0.6 mg / mL; (2) When dissolved in pure water, the fusion protein has a concentration of 2 to 8 mg / mL, the neutral salt has a concentration of 0.5 to 1.0 mg / mL, the disaccharide has a concentration of 55 to 95 mg / mL, the buffer has a concentration of 15 to 25 mM, the polysorbate has a concentration of 0.05 to 1.0 mg / mL, and the poloxamer has a concentration of 0.25 to 0.45 mg / mL; and (3) When dissolved in pure water, the fusion protein has a concentration of 4 to 6 mg / mL, the neutral salt has a concentration of 0.7 to 0.9 mg / mL, the disaccharide has a concentration of 60 to 90 mg / mL, the buffer has a concentration of 15 to 25 mM, the polysorbate has a concentration of 0.05 to 0.15 mg / mL, and the poloxamer has a concentration of 0.25 to 0.45 mg / mL.

20. The pharmaceutical composition according to claim 19, which is a freeze-dried agent having a pH of 4.5 to 6.5, 5.0 to 6.0, or 5.2 to 5.8 when dissolved in pure water.

21. The pharmaceutical composition according to any one of claims 1 to 12, wherein the patient has a disorder in the central nervous system.

22. The pharmaceutical composition according to claim 21, which has the effect of reducing the concentrations of dermatan sulfate and heparan sulfate in cerebrospinal fluid, serum, and urine.

23. The pharmaceutical composition according to claim 22, for use in enzyme replacement therapy for patients with mucopolysaccharidosis type I.

24. The pharmaceutical composition according to claim 23, which is used in combination with an immunosuppressant.

25. 25. Enzyme replacement therapy for patients with mucopolysaccharidosis type I using the pharmaceutical composition of claim 24.

26. The enzyme replacement therapy of claim 25, wherein the patient has a central nervous system disorder.

Images