Stable aqueous pharmaceutical composition or lyophilized pharmaceutical composition

Abstract

To provide stable aqueous pharmaceutical compositions or lyophilized pharmaceutical compositions containing a protein as an active ingredient.SOLUTION: Provided is an aqueous pharmaceutical composition or lyophilized pharmaceutical composition containing a bioactive protein and 2 nonionic surfactants. For example, the nonionic surfactants are polysorbate 80 and polyoxyethylene (160) polyoxypropylene (30) glycol, and the aqueous pharmaceutical composition or lyophilized pharmaceutical composition further contains sodium chloride as a neutral salt, sucrose as a disaccharide, and a citrate buffer as a buffer.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to an aqueous pharmaceutical composition containing a physiologically active protein and two or more nonionic surfactants, for example, an aqueous pharmaceutical composition or freeze-dried pharmaceutical composition containing a fusion protein obtained by linking an antibody and a lysosomal enzyme as the physiologically active protein, and polysorbate 80 and polyoxyethylene (160) polyoxypropylene (30) glycol as the nonionic surfactants. [Background technology]

[0002] Pharmaceuticals containing proteins as active ingredients were formerly generally supplied as lyophilized preparations (lyophilized pharmaceutical compositions) to ensure the storage stability of the proteins. Currently, many pharmaceuticals containing proteins as their main ingredient, such as lysosomal enzymes like iduronate-2-sulfatase, α-galactosidase A, glucocerebrosidase, and α-L-iduronidase-N-acetylgalactosamine-4-sulfatase, antibodies like anti-human IL-6 receptor antibodies and anti-human PD-1 antibodies, erythropoietin, darbepoetin, and growth hormone, are manufactured and sold in the form of aqueous pharmaceutical compositions. Aqueous pharmaceutical compositions are more convenient than lyophilized pharmaceutical compositions because they do not require dissolution before use. However, some pharmaceuticals containing proteins as their main ingredient are still supplied as lyophilized pharmaceutical compositions.

[0003] Aqueous pharmaceutical compositions often contain nonionic surfactants to enhance the stability of the main protein component or to prevent the protein from adsorbing to the container. Examples of such nonionic surfactants include polysorbate 80. For example, some aqueous pharmaceutical compositions containing darbepoetin or agalsidase as active ingredients contain polysorbate 80 as a nonionic surfactant (Non-Patent Documents 1 and 2). In addition, some aqueous pharmaceutical compositions containing growth hormones contain polyoxyethylene (160) polyoxypropylene (30) glycol as a nonionic surfactant (Non-Patent Document 3).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide an aqueous pharmaceutical composition containing, as an active ingredient, a protein having physiological activity, which contains two or more nonionic surfactants as a surfactant and is stable enough to be distributed in the market.

Means for Solving the Problems

[0006] In the research towards the above object, the present inventors have found that a protein having physiological activity can be stably preserved in the form of an aqueous pharmaceutical composition or a freeze-dried pharmaceutical composition containing sucrose and two nonionic surfactants as excipients, and have completed the present invention. That is, the present invention provides the following. 1. An aqueous pharmaceutical composition or a freeze-dried pharmaceutical composition containing a protein having physiological activity and two nonionic surfactants. 2. The aqueous pharmaceutical composition or the freeze-dried pharmaceutical composition according to 1 above, further containing at least one of a neutral salt, a disaccharide, and a buffer. 3. The aqueous pharmaceutical composition or the freeze-dried pharmaceutical composition according to 1 or 2 above, wherein the nonionic surfactant includes polysorbate and poloxamer. 4. The polysorbate is polysorbate 20 or polysorbate 80, The aqueous pharmaceutical composition or lyophilized pharmaceutical composition according to item 3 above, wherein the poloxamer is selected from the group consisting of polyoxyethylene (42) polyoxypropylene (67) glycol, 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. 5. The aqueous pharmaceutical composition or freeze-dried pharmaceutical composition according to item 3 above, wherein the polysorbate is polysorbate 80 and the poloxamer is polyoxyethylene (160) polyoxypropylene (30) glycol. 6. An aqueous pharmaceutical composition according to any one of 3 to 5 above, 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, or the concentration of the polysorbate is 0.005 to 1.5 mg / mL and the concentration of the poloxamer is 0.05 to 0.6 mg / mL. 7. An aqueous pharmaceutical composition according to any one of 3 to 5 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, or the concentration of the polysorbate is 0.025 to 1.0 mg / mL and the concentration of the poloxamer is 0.1 to 0.5 mg / mL. 8. An aqueous pharmaceutical composition according to any one of 3 to 5 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, or the concentration of the polysorbate is 0.05 to 0.15 mg / mL and the concentration of the poloxamer is 0.15 to 0.45 mg / mL. 9. The aqueous pharmaceutical composition according to any one of items 1 to 8 above, wherein the neutral salt is sodium chloride. 10. The aqueous pharmaceutical composition according to any one of items 1 to 9 above, wherein the disaccharide is selected from the group consisting of trehalose, sucrose, maltose, lactose, and combinations of two or more of these. 11. The aqueous pharmaceutical composition according to any one of items 1 to 10 above, 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. 12. An aqueous pharmaceutical composition according to any one of 3 to 5 above, wherein 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. 13. The aqueous pharmaceutical composition according to any one of 3 to 5 above, wherein the concentration of the neutral salt is 0.5 to 1.0 mg / mL, the concentration of the disaccharide is 55 to 95 mg / mL, the concentration of the buffer is 15 to 25 mM, the concentration of the polysorbate is 0.05 to 1.0 mg / mL, and the concentration of the poloxamer is 0.25 to 0.45 mg / mL. 14. The aqueous pharmaceutical composition according to any one of 3 to 5 above, wherein 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. 15. An aqueous pharmaceutical composition according to any one of items 1 to 14 above, wherein the pH is 4.5 to 6.5. 16. An aqueous pharmaceutical composition according to any one of items 1 to 14 above, wherein the pH is 5.0 to 6.0. 17. An aqueous pharmaceutical composition according to any one of items 1 to 14 above, wherein the pH is 5.2 to 5.8. 18. The aqueous pharmaceutical composition according to any one of items 1 to 17 above, wherein the physiologically active protein is a fusion protein of an antibody and a lysosomal enzyme. 19. The aqueous pharmaceutical composition according to 18, wherein the fusion protein is formed by the lysosomal enzyme being bonded by a peptide bond to either the C-terminal or N-terminal side of either the light chain or heavy chain of the antibody. 20. The aqueous pharmaceutical composition according to 18, wherein the fusion protein is formed by the lysosomal enzyme being bonded to the C-terminal side of the heavy chain of the antibody by a peptide bond. 21. The aqueous pharmaceutical composition according to 18, wherein the fusion protein is bonded to either the C-terminal or N-terminal side of either the light chain or heavy chain of the antibody via a linker consisting of at least one amino acid. 22. The aqueous pharmaceutical composition according to 18, wherein the fusion protein is formed by the lysosomal enzyme being linked to the C-terminal side of the heavy chain of the antibody via a linker consisting of at least one amino acid. 23. The aqueous pharmaceutical composition according to 21 or 22, wherein the linker has an amino acid sequence selected from the group consisting of Gly-Ser, Gly-Gly-Ser, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and an amino acid sequence consisting of 1 to 10 consecutive amino acid sequences of these. 24. The aqueous pharmaceutical composition according to any one of the above 18 to 23, wherein the lysosomal enzyme is a human lysosomal enzyme. 25. The lysosomal enzymes include α-L-iduronidase, iduronic acid-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase BN-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, α-galactosidase An aqueous pharmaceutical composition according to any one of the above 18 to 24, selected from the group consisting of ze, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase-N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amyl-1-6-glucosidase, sialidase, aspartylglucosaminidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2. 26. The aqueous pharmaceutical composition according to 24, wherein the human lysosomal enzyme is α-L-iduronidase. 27. The aqueous pharmaceutical composition according to any one of items 18 to 26 above, wherein the antibody is a human antibody or a humanized antibody. 28. The aqueous pharmaceutical composition according to any one of items 18 to 27 above, wherein the antibody is a Fab antibody, an F(ab')2 antibody, or an F(ab') antibody. 29. The aqueous pharmaceutical composition according to any one of claims 18 to 28, wherein the antibody recognizes molecules present on the surface of vascular endothelial cells as antigens. 30. The aqueous pharmaceutical composition according to paragraph 29, wherein the vascular endothelial cells are human vascular endothelial cells. 31. The aqueous pharmaceutical composition according to 29 or 30 above, wherein the vascular endothelial cells are cerebral vascular endothelial cells. 32. The aqueous pharmaceutical composition according to 31, wherein the molecule present on the surface of the cerebral vascular endothelial cell is selected from the group consisting of transferrin receptor (TfR), insulin receptor, leptin receptor, lipoprotein receptor, IGF receptor, OATP-F, organic anion transporter, and monocarboxylic acid transporter. 33. The aqueous pharmaceutical composition according to 28 above, wherein the antibody is a humanized anti-human transferrin receptor (hTfR) antibody. 34. The antibody is a humanized anti-human transferrin receptor (hTfR) antibody which is a Fab antibody, the human lysosomal enzyme is human α-L-iduronidase, and the fusion protein is a fusion protein of the antibody and the human α-L-iduronidase, and in the fusion protein, (1) The light chain of the antibody contains the amino acid sequence of SEQ ID NO: 22, and (2) The heavy chain of the antibody binds to human α-L-iduronidase at its C-terminus via the amino acid sequence shown in SEQ ID NO: 4, thereby forming the amino acid sequence shown in SEQ ID NO: 27. The aqueous pharmaceutical composition described in item 28 above. 35. The antibody is a humanized anti-human transferrin receptor (hTfR) antibody which is a Fab antibody, the human lysosomal enzyme is human α-L-iduronidase, and the fusion protein is a fusion protein of the antibody and the human α-L-iduronidase, and in the fusion protein, (1) The light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 22, and (2) The heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 23, and the heavy chain is bound at its C-terminus to a human α-L-iduronidase having the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, via the amino acid sequence shown in SEQ ID NO: 4. The aqueous pharmaceutical composition described in item 28 above. 36. An aqueous pharmaceutical composition according to any one of items 1 to 35 above, which is sealed in a container made of borosilicate glass or hydrophobic resin. 37. The aqueous pharmaceutical composition according to 36, wherein the container is formed using a cycloolefin copolymer, a cycloolefin ring-opening polymer, or a cycloolefin ring-opening polymer that has been hydrogenated. 38. A pharmaceutical composition comprising a freeze-dried aqueous pharmaceutical composition as described in any of items 1 to 35 above. 39. The freeze-dried pharmaceutical composition described in 38 above, which is sealed in a container made of borosilicate glass or a hydrophobic resin. 40. The freeze-dried pharmaceutical composition according to 39 above, wherein the material of the container includes a cycloolefin copolymer, a ring-opening polymer of cycloolefins, or a hydrogenated ring-opening polymer of cycloolefins. Aqueous pharmaceutical compositions or freeze-dried pharmaceutical compositions according to items 1 to 40 above, wherein the polymer content after storage in a dark place at a temperature of 41.2 to 8°C for 36 months is 0.5% or less. The aqueous pharmaceutical compositions according to items 1 to 37 above, wherein the polymer content and the degradation product content after storage in a dark place at a temperature of 42.2 to 8°C for 36 months are 0.5% or less and 1% or less, respectively. The freeze-dried pharmaceutical compositions according to 39-41 above, wherein the polymer content and the degradation product content after storage in a dark place at a temperature of 42.2-8°C for 36 months are 0.5% or less and 0.1% or less, respectively. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide an aqueous pharmaceutical composition or a freeze-dried pharmaceutical composition that contains a physiologically active protein as an active ingredient and is stable enough to be distributed on the market. [Brief explanation of the drawing]

[0008] [Figure 1] This figure shows the measured number of particles per unit volume (200 μL) contained in aqueous pharmaceutical compositions (formulations A-C) after 24 hours of shaking. Black bars indicate the number of particles with a particle size of less than 10 μm, and white bars indicate the number of particles with a particle size of 10 μm or more. The vertical axis shows the number of particles (particles / 200 μL), and the horizontal axis shows the concentration of poloxamer 188 (mg / mL). [Figure 2] This figure shows the polymer content of humanized anti-hTfR antibody-hIDUA in aqueous pharmaceutical compositions (Formulations A-C) 24 hours after shaking. The vertical axis represents the polymer content (%), and the horizontal axis represents the concentration of poloxamer 188 (mg / mL). [Figure 3] This figure shows the content of degradation products of humanized anti-hTfR antibody-hIDUA in aqueous pharmaceutical compositions (formulations A-C) 24 hours after shaking. The vertical axis shows the polymer content (%), and the horizontal axis shows the concentration of poloxamer 188 (mg / mL). [Figure 4] This figure shows the measured number of particles per unit volume (200 μL) contained in aqueous pharmaceutical compositions (formulations D-I) after 24 hours of shaking. Black bars indicate the number of particles with a particle size of less than 10 μm, and white bars indicate the number of particles with a particle size of 10 μm or more. The vertical axis shows the number of particles (particles / 200 μL), and the horizontal axis shows the concentration of polysorbate 80 (mg / mL). [Figure 5] This figure shows the polymer content of humanized anti-hTfR antibody-hIDUA in aqueous pharmaceutical compositions (formulations D-I) after 24 hours of shaking. The vertical axis represents the polymer content (%), and the horizontal axis represents the concentration of polysorbate 80 (mg / mL). [Figure 6] This figure shows the content of degradation products of humanized anti-hTfR antibody-hIDUA in aqueous pharmaceutical compositions (formulations D-I) after 24 hours of shaking. The vertical axis represents the polymer content (%), and the horizontal axis represents the concentration of polysorbate 80 (mg / mL). [Figure 7] This figure shows the measured number of particles per unit volume (200 μL) contained in aqueous pharmaceutical compositions (formulations J-N) after 24 hours of shaking. Black bars indicate the number of particles with a particle size of less than 10 μm, and white bars indicate the number of particles with a particle size of 10 μm or more. The vertical axis shows the number of particles (particles / 200 μL), and the horizontal axis shows the concentration of polysorbate 80 (mg / mL). [Figure 8] This figure shows the polymer content of humanized anti-hTfR antibody-hIDUA in aqueous pharmaceutical compositions (formulations J-N) after 24 hours of shaking. The vertical axis represents the polymer content (%), and the horizontal axis represents the concentration of polysorbate 80 (mg / mL). [Figure 9]This figure shows the content of degradation products of humanized anti-hTfR antibody-hIDUA in aqueous pharmaceutical compositions (formulations J-N) after 24 hours of shaking. The vertical axis shows the polymer content (%), and the horizontal axis shows the concentration of polysorbate 80 (mg / mL). [Modes for carrying out the invention]

[0009] This invention relates to a pharmaceutical composition containing a physiologically active protein as an active ingredient that is stable for storage in solution or freeze-dried state. Here, the physiologically active protein includes a protein to which an antibody and a physiologically active substance are bound. In this case, the antibody to which the physiologically active substance is bound is not particularly limited in terms of the animal species of the antibody, as long as it has the property of specifically binding to the antigen, but it is particularly human antibody or humanized antibody. For example, the antibody may be an antibody of a mammal other than a human, or it may be a chimeric antibody of a human antibody and an antibody of another mammal other than a human.

[0010] Human antibodies are antibodies whose entirety is encoded by genes derived from humans. However, antibodies encoded by genes that have been mutated from the original human gene for purposes such as increasing gene expression efficiency are also considered human antibodies. Furthermore, antibodies created by combining two or more genes that encode human antibodies, in which a part of one human antibody is replaced with a part of another human antibody, are also considered human antibodies. Human antibodies have 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. Antibodies whose antigen specificity, affinity, etc., has been modified by replacing the CDR of one human antibody with the CDR of another human antibody are also considered human antibodies.

[0011] In one embodiment of the present invention, an antibody to which mutations such as substitution, deletion, or addition have been introduced into the amino acid sequence of the original antibody by modifying the gene of the original human antibody is also called a human antibody. When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. When amino acids are deleted from the amino acid sequence of the original antibody, the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, an antibody to which mutations combining these amino acid substitutions and deletions have been introduced is also a human antibody. When amino acids are added, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence of the original antibody or to the N-terminus or C-terminus. An antibody to which mutations combining these amino acid additions, substitutions, and deletions have been introduced is also a human antibody. The amino acid sequence of the mutated antibody preferably exhibits 80% or more identity with the amino acid sequence of the original antibody, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and still more preferably 98% or more identity. In other words, in this invention, when we refer to "human-derived genes," we include not only the original human-derived genes but also genes obtained by modifying the original human-derived genes.

[0012] 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.

[0013] In the present invention, the case where the antibody is a human antibody or a humanized antibody will be described in detail below. The light chain of human antibodies and humanized antibodies has a λ chain and a κ chain. The light chain constituting the antibody may be either a λ chain or a κ chain. Furthermore, the heavy chain of human antibodies and humanized antibodies has a γ chain, a μ chain, an α chain, a σ chain and an ε chain, which correspond to IgG, IgM, IgA, IgD and IgE, respectively. The heavy chain constituting the antibody may be any of the γ chain, μ chain, α chain, σ chain and ε chain, but a γ chain is preferred. Moreover, the γ chain of the antibody's heavy chain has γ1 chain, γ2 chain, γ3 chain and γ4 chain, which correspond to IgG1, IgG2, IgG3 and IgG4, respectively. When the heavy chain constituting the antibody is a γ chain, the γ chain may be any of the γ1 chain, γ2 chain, γ3 chain and γ4 chain, but a γ1 chain or a 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 the gamma1, gamma2, gamma3, and 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.

[0014] In the present invention, the term "chimeric antibody" refers to an antibody formed by linking together fragments of two or more different antibodies originating from two or more different species.

[0015] A chimeric antibody is an antibody in which a portion of a human antibody is replaced by a portion of an antibody from a non-human mammal. The antibody consists of an Fc region, a Fab region, and a hinge region, as described below. A specific example of such a chimeric antibody is one in which the Fc region originates from a human antibody while the Fab region originates from an antibody from another mammal. The hinge region originates from either the human antibody or the antibody from the other mammal. Conversely, an example of such a chimeric antibody is one in which the Fc region originates from another mammal while the Fab region originates from a human antibody. The hinge region may originate from either the human antibody or the antibody from the other mammal. The same applies to humanized antibodies.

[0016] Furthermore, antibodies can be said to consist of a variable region and a constant region. Another specific example of a chimeric antibody is the constant region of the heavy chain (C H ) and the steady region of the light chain (C L ) is derived from human antibodies, while the variable region of the heavy chain (V H ) and the variable region of the light chain (V L ) are derived from antibodies of other mammals, or conversely, the constant region of the heavy chain (C H ) and the steady region of the light chain (C L ) is derived from antibodies of other mammals, while the variable region of the heavy chain (V H ) and the variable region of the light chain (V L Examples include those derived from human antibodies. Here, the other mammalian species are not particularly limited as long as they are mammals other than humans, but preferably they are mice, rats, rabbits, horses, or primates other than humans, and more preferably mice. The same applies to humanized antibodies.

[0017] Chimeric antibodies of human and mouse antibodies are particularly referred to as "human / mouse chimeric antibodies". Human / mouse chimeric antibodies include chimeric antibodies in which the Fc region is derived from a human antibody while the Fab region is derived from a mouse antibody, and conversely, chimeric antibodies in which the Fc region is derived from a mouse antibody while the Fab region is derived from a human antibody. The hinge region is derived from either a human antibody or a mouse antibody. As other specific examples of human / mouse chimeric antibodies, the constant region (C H ) of the heavy chain and the constant region (C L ) of the light chain are derived from a human antibody, while the variable region (V H ) of the heavy chain and the variable region (V L ) of the light chain are derived from a mouse antibody, and conversely, those in which the constant region (C H ) of the heavy chain and the constant region (C L ) of the light chain are derived from a mouse antibody, while the variable region (V H ) of the heavy chain and the variable region (V L ) of the light chain are derived from a human antibody are also included. The same can be said for humanized antibodies.

[0018] Antibodies originally have a basic structure consisting of a total of four polypeptide chains, namely two immunoglobulin light chains and two immunoglobulin heavy chains. However, in the present invention, when referring to "antibodies", in addition to those having this basic structure, (1) those consisting of a total of two polypeptide chains, one immunoglobulin light chain and one immunoglobulin heavy chain, and as described in detail below, (2) single-chain antibodies formed by binding a linker to the C-terminal side of an immunoglobulin light chain and further binding an immunoglobulin heavy chain to the C-terminal side thereof, (3) single-chain antibodies formed by binding a linker to the C-terminal side of an immunoglobulin heavy chain and further binding an immunoglobulin light chain to the C-terminal side thereof, (4) single-chain antibodies (scFv) formed by binding a linker to the C-terminal side of the variable region of an immunoglobulin heavy chain and further binding the variable region of an immunoglobulin light chain to the C-terminal side thereof, and (5) This also includes single-chain antibodies (scFv) in which a linker is attached to the C-terminal side of the variable region of an immunoglobulin light chain, and the variable region of an immunoglobulin heavy chain is further attached to the C-terminal side of the linker. (6) Those consisting of a Fab region, which is the basic structure of an antibody in the original sense of which the Fc region has been deleted, and those consisting of all or part of the Fab region and the hinge region (including Fab,F(ab') and F(ab')2), and (7) Single-domain antibodies are also included in the definition of "antibody" in this invention. Furthermore, scFv, which are single-chain antibodies formed by linking the variable region of the light chain and the variable region of the heavy chain via a linker, are also included in the definition of antibody in this invention.

[0019] In this invention, the term "linker" refers, for example, to a peptide chain in which multiple amino acids are linked by peptide bonds. Such a linker consisting of a peptide chain can also be called a "peptide linker." In the context of this specification, "linker" can also be replaced with "linker sequence." The N-terminus of this linker is linked to the C-terminus of another protein by a peptide bond, and the N-terminus of yet another protein is further linked to the C-terminus of the linker, thereby forming a conjugate between the two proteins via the linker.

[0020] Antibodies, which have a basic structure consisting of a total of four polypeptide chains, two light chains and two heavy chains, have a variable region (V) in the light chain. L ) has three complementarity determination regions (CDRs) and a variable region of the heavy chain (V H The light chain has three complementarity-determining regions (CDRs). The three CDRs on the light chain are called CDR1, CDR2, and CDR3, in order from the N-terminus. The three CDRs on the heavy chain are also called CDR1, CDR2, and CDR3, in order from the N-terminus. However, even if some or all of these CDRs are incomplete or absent, the antibody is still included as long as it has the property of specifically binding to a particular antigen. Variable regions (V) of the light and heavy chains L and V HThe region of the antibody other than the CDR is called the framework region (FR). The FRs are numbered FR1, FR2, FR3, and FR4, starting from the N-terminus. Typically, the CDR and FRs exist in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, starting from the N-terminus. The same applies to heavy chain antibodies, which are composed solely of heavy chains.

[0021] In one embodiment of the present invention, 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 the 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 H It may also include a portion of the hinge region in addition to one region. 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. In F(ab'), the heavy chain consists of a variable region and C HIn addition to one region, it includes all or part of the hinge region containing cysteine ​​residues that link the heavy chains together. F(ab')2 refers to a molecule in which two F(ab') molecules are linked by disulfide bonds between cysteine ​​residues present in their hinge regions. The heavy chain that forms F(ab') or F(ab')2 is called the Fab' heavy chain. Furthermore, polymers such as dimers and trimers formed by the direct or linker linkage of multiple antibodies are also antibodies. Moreover, not limited to these, anything that includes a part of an immunoglobulin molecule and has the property of specifically binding to an antigen is included in the definition of "antibody" in this invention. That is, in this invention, when we refer to an immunoglobulin light chain, it includes those derived from an immunoglobulin light chain and having all or part of the amino acid sequence of its variable region. Similarly, when we refer to an immunoglobulin heavy chain, it includes those derived from an immunoglobulin heavy chain and having all or part of the amino acid sequence of its variable region. Therefore, as long as it has all or part of the amino acid sequence of the variable region, even if, for example, the Fc region is deleted, it is still an immunoglobulin heavy chain.

[0022] Furthermore, here, Fc or the Fc region refers to the C region in the antibody molecule. H This refers to a region containing a fragment consisting of two regions (part 2 of the steady-state region of the heavy chain) and a CH3 region (part 3 of the steady-state region of the heavy chain).

[0023] Furthermore, the antibody in the present invention is (8) The present invention also includes scFab, scF(ab'), and scF(ab')2, which are single-chain antibodies obtained by linking the light chain and heavy chain that constitute Fab, F(ab'), or F(ab')2 as shown in (6) above via a linker sequence. Here, in the case of scFab, scF(ab'), and scF(ab')2, the linker sequence may be linked to the C-terminus of the light chain, and the heavy chain may be linked to the C-terminus of the light chain, or the linker may be linked to the C-terminus of the heavy chain, and the light chain may be linked to the C-terminus of the heavy chain. Furthermore, scFv, which is a single-chain antibody obtained by linking the variable region of the light chain and the variable region of the heavy chain via a linker, is also included in the antibodies of the present invention. In the case of scFv, a linker array may be attached to the C-terminal side of the variable region of the light chain, and the variable region of the heavy chain may be attached to the C-terminal side of that linker array, or a linker array may be attached to the C-terminal side of the variable region of the heavy chain, and the variable region of the light chain may be attached to the C-terminal side of that linker array.

[0024] Furthermore, the term "antibody" as used herein includes not only full-length antibodies and those described in (1) to (8) above, but also a broader concept encompassing (1) to (8), which includes antigen-binding fragments (antibody fragments) in which a portion of a full-length antibody is missing. Antigen-binding fragments include heavy-chain antibodies, light-chain antibodies, VHH, VNAR, and those in which a portion of these is missing.

[0025] The term "antigen-binding fragment" refers to a fragment of an antibody that retains at least some of its specific binding activity to an antigen. Examples of binding fragments include, for example, those shown in (4) and (5) above, Fab, Fab', F(ab')2, variable region (Fv), and variable region of the heavy chain (V H ) and the variable region of the light chain (V L A single-chain antibody (scFv) linked with a suitable linker, and a variable region (V) of the heavy chain. H ) and the variable region of the light chain (V L Diabody, scFv, is a polypeptide dimer containing a portion of the constant region (C) in its heavy chain (H chain). H3) This includes minibodies, which are dimers of the bound substance, and other low-molecular-weight antibodies. However, it is not limited to these molecules as long as they have the ability to bind to antigens. Furthermore, these binding fragments also include antibodies treated with appropriate enzymes, and proteins produced in appropriate host cells using genetically modified antibody genes.

[0026] In one embodiment of the present invention, the term "single-chain antibody" refers to a protein in which a linker is bound to the C-terminus of an amino acid sequence containing all or part of the variable region of an immunoglobulin light chain, and an amino acid sequence containing all or part of the variable region of an immunoglobulin heavy chain is further bound to the C-terminus of that linker, and which is capable of specifically binding to a specific antigen. Furthermore, a protein in which a linker is bound to the C-terminus of an amino acid sequence containing all or part of the variable region of an immunoglobulin heavy chain, and an amino acid sequence containing all or part of the variable region of an immunoglobulin light chain is further bound to the C-terminus of that linker, and which is capable of specifically binding to a specific antigen, is also a "single-chain antibody" in the present invention. For example, those shown in (2) and (3) above are included in single-chain antibodies. In a single-chain antibody in which an immunoglobulin light chain is bound to the C-terminus of an immunoglobulin heavy chain via a linker, the immunoglobulin heavy chain usually lacks an Fc region. The variable region of the immunoglobulin light chain has three complementarity-determining regions (CDRs) that are involved in the antigen specificity of the antibody. Similarly, the variable region of the immunoglobulin heavy chain also has three CDRs. These CDRs are the main regions that determine the antigen specificity of the antibody. Therefore, it is preferable that single-chain antibodies contain all three CDRs of the immunoglobulin heavy chain and all three CDRs of the immunoglobulin light chain. However, single-chain antibodies can also be made by deleting one or more CDRs, as long as the antigen-specific affinity of the antibody is maintained.

[0027] In single-chain antibodies, the linker positioned between the light and heavy chains of immunoglobulin is preferably a peptide chain consisting of 2 to 50 amino acid residues, more preferably 8 to 50, even more preferably 10 to 30, and even more preferably 30 to 30 or 30 to 30, for example, 30 or 30 amino acid residues. Such a linker is not limited in its amino acid sequence as long as the anti-hTfR antibody formed by linking the two chains maintains affinity for hTfR, but is preferably composed of glycine alone or glycine and serine, for example, having the amino acid sequence Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly, 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, the amino acid sequence shown in SEQ ID NO: 4, or sequences in which these amino acid sequences are repeated 2 to 10 times or 2 to 5 times. For example, when forming an ScFV by linking the variable region of an immunoglobulin light chain to the C-terminal side of the amino acid sequence consisting of the entire variable region of an immunoglobulin heavy chain via a linker, a linker having the amino acid sequence shown in SEQ ID NO: 4 is preferably used.

[0028] The antibody in one embodiment of the present invention is an antibody derived from a camelid (including alpacas). Some antibodies from camelids consist of two heavy chains linked by disulfide bonds. Antibodies consisting of these two heavy chains are called heavy chain antibodies. VHH is an antibody consisting of one heavy chain that includes the variable region of the heavy chain constituting the heavy chain antibody, or an antibody consisting of one heavy chain that lacks the constant region (CH) constituting the heavy chain antibody. VHH is also one of the antibodies in the embodiments of the present invention. Antibodies in one embodiment of the present invention that have had mutations added to the amino acid sequence of camelid antibodies (including VHH) in order to reduce their antigenicity when administered to humans are also antibodies. When adding mutations to the amino acids of camelid antibodies, the same mutations that can be added to the antibodies described herein can be added. In addition, antibodies consisting of two light chains linked by disulfide bonds are also one of the antibodies in the embodiments of the present invention. Antibodies consisting of these two light chains are called light chain antibodies.

[0029] The antibody in one embodiment of the present invention is a shark-derived antibody. A shark antibody consists of two heavy chains linked by a disulfide bond. An antibody consisting of these two heavy chains is called a heavy chain antibody. A VNAR is an antibody consisting of one heavy chain that includes the variable region of the heavy chain constituting the heavy chain antibody, or an antibody consisting of one heavy chain that lacks the constant region (CH) constituting the heavy chain antibody. A VNAR is also one of the antibodies in the embodiments of the present invention. An antibody in one embodiment of the present invention is a shark antibody in which a mutation has been made in the amino acid sequence of the shark antibody in order to reduce its antigenicity when administered to a human. When a mutation is made in the amino acid sequence of the shark antibody, the same mutations as those that can be made in the antibodies described herein can be made. A humanized shark antibody is also one of the antibodies in the embodiments of the present invention.

[0030] In one embodiment of the present invention, a single-domain antibody is an antibody that has the property of specifically binding to an antigen with a single variable region. Single-domain antibodies include antibodies in which the variable region consists only of the variable region of the heavy chain (heavy chain single-domain antibodies) and antibodies in which the variable region consists only of the variable region of the light chain (light chain single-domain antibodies). VHH and VNAR are types of single-domain antibodies.

[0031] In the present invention, the antigen specifically recognized by the antibody is, for example, a molecule present on the surface of vascular endothelial cells (surface antigen). Examples of such surface antigens include, but are not limited to, transferrin receptors (TfR), insulin receptors, leptin receptors, lipoprotein receptors, IGF receptors, organic anion transporters such as OATP-F, monocarboxylic acid transporters, and Fc receptors. The monocarboxylic acid transporter (MCT) may be any of the 14 subtypes (MCT1 to MCT14), but MCT1, MCT2, MCT4, or MCT8 are particularly preferred, for example, MCT8. The antigen is preferably one of these molecules (surface antigens) present on the surface of human vascular endothelial cells.

[0032] Among the surface antigens mentioned above, transferrin receptors (TfR), insulin receptors, leptin receptors, lipoprotein receptors, IGF receptors, organic anion transporters such as OATP-F, and monocarboxylic acid transporters such as MCT-8 are present on the surface of brain capillary endothelial cells (cerebral vascular endothelial cells) that form the blood-brain barrier. Antibodies that can recognize these antigens can bind to brain capillary endothelial cells via the antigen. Antibodies bound to brain capillary endothelial cells can then cross the blood-brain barrier and reach the central nervous system. Therefore, by binding the target protein to such an antibody, it can reach the central nervous system. Target proteins include proteins that have a function that should exert their pharmacological effect in the central nervous system. For example, lysosomal enzymes that are deficient or dysfunctional in patients with lysosomal storage diseases accompanied by central nervous system disorders are examples of target proteins. These lysosomal enzymes cannot reach the central nervous system on their own and therefore do not exert any therapeutic effect on central nervous system disorders in patients. However, by binding them with these antibodies, they can cross the blood-brain barrier, thereby improving central nervous system disorders seen in patients with lysosomal storage diseases.

[0033] In the present invention, the terms "human transferrin receptor" or "hTfR" refer to a membrane protein having the amino acid sequence shown in SEQ ID NO: 5. In one embodiment, the anti-hTfR antibody of the present invention specifically binds to the portion of the amino acid sequence shown in SEQ ID NO: 5 from the 89th cysteine ​​residue from the N-terminus to the phenylalanine residue at the C-terminus (the extracellular region of hTfR), but is not limited thereto.

[0034] The method for producing antibodies will be explained below, using antibodies against hTfR as an example. The general method for producing antibodies against hTfR involves creating recombinant human transferrin receptors (rhTfR) using cells into which an expression vector incorporating the hTfR gene has been introduced, and then immunizing animals such as mice with these rhTfR. By extracting antibody-producing cells against hTfR from the immunized animals and fusing them with myeloma cells, hybridoma cells capable of producing antibodies against hTfR can be produced.

[0035] Furthermore, cells that produce antibodies against hTfR can also be obtained by immunizing immune system cells obtained from animals such as mice with rhTfR using in vitro immunization. When immunizing by in vitro immunization, there are no particular limitations on the animal species from which the immune system cells originate, but preferably they are primates including mice, rats, rabbits, guinea pigs, dogs, cats, horses, and humans, more preferably mice, rats, and humans, and even more preferably mice and humans. As mouse immune system cells, for example, splenocytes prepared from the spleen of a mouse can be used. As human immune system cells, cells prepared from human peripheral blood, bone marrow, spleen, etc. can be used. When human immune system cells are immunized by in vitro immunization, human antibodies against hTfR can be obtained.

[0036] In the present invention, there are no particular limitations on the human lysosomal enzyme to be bound to the antibody, but examples include α-L-iduronidase, iduronic acid-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2-activated protein, β-hexosaminidase A, β-hexosaminidase BN-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, and acid sphingomyelinase. Examples include α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase-N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amyl-1-6-glucosidase, sialidase, aspartylglucosaminidase, palmitoyl protein thioesterase-1 (PPT-1), tripeptidyl peptidase-1 (TPP-1), hyaluronidase-1, CLN1, CLN2, CLN3, CLN6, and CLN8, which are lysosomal enzymes.

[0037] When an antibody specifically recognizes molecules (surface antigens) present on the surface of vascular endothelial cells, the human lysosomal enzymes conjugated to the antibody are used as follows: α-L-iduronidase as a treatment for central nervous system disorders in Hurler syndrome, Hurler-Schaye syndrome, and Schayet syndrome; iduronate-2-sulfatase as a treatment for central nervous system disorders in Hunter syndrome; glucocerebrosidase as a treatment for central nervous system disorders in Gaucher disease; β-galactosidase as a treatment for central nervous system disorders in GM1 gangliosidosis types 1-3; and GM2 activating protein White matter is used as a treatment for central nervous system disorders in GM2-gangliosidosis AB variant, β-hexosaminidase A as a treatment for central nervous system disorders in Sandhoff disease and Tisachs disease, β-hexosaminidase B as a treatment for central nervous system disorders in Sandhoff disease, N-acetylglucosamine-1-phosphotransferase as a treatment for central nervous system disorders in I-cell diseases, α-mannosidase as a treatment for central nervous system disorders in α-mannosidosis, β-mannosidase as a treatment for central nervous system disorders in β-mannosidosis, and galactosyl Ceramidase is used as a treatment for central nervous system disorders in Krabbe disease, saposin C as a treatment for central nervous system disorders in Gaucher disease-like storage, arylsulfatase A as a treatment for central nervous system disorders in metachromatic leukodystrophy, α-L-fucosidase as a treatment for central nervous system disorders in fucosidosis, aspartylglucosaminidase as a treatment for central nervous system disorders in aspartylglucosamiuria, α-N-acetylgalactosaminidase as a treatment for central nervous system disorders in Schindler's disease and Kawasaki disease, and acid sphingomyelia Erinase is used as a treatment for central nervous system disorders in Niemann-Pick disease, α-galactosidase A as a treatment for central nervous system disorders in Fabry disease, β-glucuronidase as a treatment for central nervous system disorders in Sly syndrome, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, and N-acetylglucosamine-6-sulfate sulfatase as treatments for central nervous system disorders in Sanfilippo syndrome, and acidic ceramidase as a treatment for central nervous system disorders in Faber disease.Amylo-1,6-glucosidase can be used as a treatment for central nervous system disorders in Forbes-Koli's disease, sialidase as a treatment for central nervous system disorders in sialidase deficiency, aspartylglucosaminidase as a treatment for central nervous system disorders in aspartylglucosamineuria, palmitoylprotein thioesterase-1 (PPT-1) as a treatment for central nervous system disorders in neuronal ceroid lipofuscinosis or Santavuori-Haltia disease, tripeptidyl peptidase-1 (TPP-1) as a treatment for central nervous system disorders in neuronal ceroid lipofuscinosis or Jansky-Bielschowsky disease, hyaluronidase-1 as a treatment for central nervous system disorders in hyaluronidase deficiency, and CLN1, CLN2, CLN3, CLN6, and CLN8 as treatments for central nervous system disorders in Batten disease.

[0038] When an antibody specifically recognizes molecules (surface antigens) present on the surface of vascular endothelial cells, human α-L-iduronidase (hIDUA) is a suitable lysosomal enzyme to bind to the antibody. hIDUA is a type of lysosomal enzyme that has the activity to hydrolyze iduronic acid bonds present in glycosaminoglycan (GAG) molecules such as heparan sulfate and dermatan sulfate. Mucopolysaccharidosis type I is a genetic disorder caused by a mutation in the gene encoding this enzyme. Mucopolysaccharidosis type I is classified into Hurler syndrome, Hurler-Scheille syndrome, and Schayle syndrome, with Hurler syndrome being the severe form, Hurler-Scheille syndrome being the intermediate form, and Schayle syndrome being the mild form. In these patients, heparan sulfate and dermatan sulfate accumulate in the tissues, resulting in various symptoms such as corneal opacity and intellectual disability. However, intellectual disability may not be observed in the mild form. The fusion protein of this antibody and hIDUA can degrade GAGs accumulated in brain tissue by crossing the blood-brain barrier (BBB), and therefore can be used as a treatment for central nervous system disorders when administered to patients with Hurler syndrome who exhibit intellectual disability.

[0039] In the present invention, the terms "human α-L-idulonidase" or "hIDUA" specifically refer to hIDUA having the same amino acid sequence as wild-type hIDUA. Wild-type hIDUA has an amino acid sequence consisting of 628 amino acids, as shown in SEQ ID NO: 6. A variant of hIDUA having an amino acid sequence consisting of 626 amino acids, as shown in SEQ ID NO: 7, is also considered hIDUA. However, it is not limited to these; as long as it has IDUA activity, any variant of wild-type hIDUA with mutations such as substitution, deletion, or addition to its amino acid sequence is also included as hIDUA. When substituting amino acids in the amino acid sequence of hIDUA with other amino acids, 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 deleting amino acids from the amino acid sequence of hIDUA, the number of amino acids deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations combining these amino acid substitutions and deletions can also be introduced. When adding amino acids to hIDUA, preferably 1 to 10, 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 hIDUA or to 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 mutated hIDUA preferably has 80% or more identity with the original hIDUA amino acid sequence, 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.

[0040] 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)).

[0041] 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 amino acid families include the following: (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, tryptophan, (4) Serine and threonine, which are 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 amino acids, (9) Alanine and glycine are amino acids with small side chains.

[0042] 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 is 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, relative 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.

[0043] In this invention, "fusion protein" refers to a substance obtained by directly linking an antibody and a human lysosomal enzyme via a non-peptide linker or a peptide linker. The method for linking the antibody and the human lysosomal enzyme is described in detail below.

[0044] Methods for conjugating antibodies to human lysosomal enzymes include methods using non-peptide linkers or methods using peptide linkers. Non-peptide linkers can include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethers, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, or derivatives thereof, or combinations thereof. A peptide linker is a peptide chain or derivative thereof consisting of 1 to 50 amino acids linked by peptide bonds, and its N-terminus and C-terminus form covalent bonds with either the antibody or the human lysosomal enzyme, thereby conjugating the antibody to the human lysosomal enzyme.

[0045] When using biotin-streptavidin as a non-peptide linker, the antibody may be conjugated with biotin, and the human lysosomal enzyme may be conjugated with streptavidin, and the antibody and human lysosomal enzyme may be conjugated via the binding of biotin and streptavidin. Conversely, the antibody may be conjugated with streptavidin, and the human lysosomal enzyme may be conjugated with biotin, and the antibody and human lysosomal enzyme may be conjugated via the binding of biotin and streptavidin.

[0046] The antibody of the present invention, conjugated with a human lysosomal enzyme using PEG as a non-peptide linker, is specifically called antibody-PEG-human lysosomal enzyme. Antibody-PEG-human lysosomal enzyme can be produced by conjugating an antibody with PEG to create antibody-PEG, and then conjugating antibody-PEG with human lysosomal enzyme. Alternatively, antibody-PEG-human lysosomal enzyme can also be produced by conjugating a human lysosomal enzyme with PEG to create human lysosomal enzyme-PEG, and then conjugating human lysosomal enzyme-PEG with antibody. When conjugating PEG with antibodies and human lysosomal enzyme, PEG modified with functional groups such as carbonate, carbonylimidazole, active carboxylic acid esters, azulactone, cyclic imidothion, isocyanate, isothiocyanate, imidate, or aldehyde is used. These functional groups introduced into PEG react mainly with amino groups in the antibody and human lysosomal enzyme molecules, resulting in covalent bonding between PEG and the antibody and human lysosomal enzyme. There are no particular limitations on the molecular weight and shape of the PEG used in this case, but its average molecular weight (MW) is preferably MW = 300 to 60000, and more preferably MW = 500 to 20000. For example, PEG with average molecular weights of approximately 300, 500, 1000, 2000, 4000, 10000, and 20000 can be suitably used as a non-peptide linker.

[0047] For example, antibody-PEG can be obtained by mixing an antibody with polyethylene glycol having an aldehyde group as a functional group (ALD-PEG-ALD) so that the molar ratio of ALD-PEG-ALD to the antibody is 1:11, 1:12.5, 1:15, 1:110, 1:120, etc., and then adding a reducing agent such as NaCNBH3 and reacting the mixture. Next, antibody-PEG-human lysosomal enzyme can be obtained by reacting antibody-PEG with human lysosomal enzyme in the presence of a reducing agent such as NaCNBH3. Conversely, antibody-PEG-human lysosomal enzyme can also be obtained by first conjugating human lysosomal enzyme with ALD-PEG-ALD to produce human lysosomal enzyme-PEG, and then conjugating human lysosomal enzyme-PEG with an antibody.

[0048] Antibodies and human lysosomal enzymes can also be linked to the N-terminus or C-terminus of the N-terminus or C-terminus of the antibody's heavy or light chain via a linker or directly, using peptide bonds. A fusion protein formed by linking an antibody and a human lysosomal enzyme in this way can be obtained by incorporating a DNA fragment in which the cDNA encoding the human lysosomal enzyme is positioned in-frame, either directly or with a linker-encoding DNA fragment in between, at the 3' or 5' end of the cDNA encoding the antibody's heavy or light chain, into an expression vector for eukaryotes such as mammalian cells or yeast, and then culturing mammalian cells into which this expression vector has been introduced. In the case where the DNA fragment encoding the human lysosomal enzyme is linked to the heavy chain, an expression vector for mammalian cells incorporating the cDNA fragment encoding the antibody's light chain is also introduced into the same host cells. Similarly, in the case where the DNA fragment encoding the human lysosomal enzyme is linked to the light chain, an expression vector for mammalian cells incorporating the cDNA fragment encoding the antibody's heavy chain is also introduced into the same host cells. When the antibody is a single-chain antibody, a fusion protein formed by linking the antibody to a human lysosomal enzyme can be obtained by incorporating a DNA fragment into an expression vector for mammalian cells, yeast, or other eukaryotic cells. This fragment consists of a single-chain antibody-encoding cDNA directly linked to the 5' or 3' end of the cDNA encoding the human lysosomal enzyme, or linked via a DNA fragment encoding a linker. The expression vector is then introduced into these cells.

[0049] A fusion protein of the type in which a human lysosomal enzyme is bound to the C-terminus of the antibody's light chain 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, and the human lysosomal enzyme is bound to the C-terminus of the antibody's light chain. Here, the antibody's light chain and the human lysosomal enzyme may be bound directly or via a linker.

[0050] A fusion protein of the type in which a human lysosomal enzyme is bound to the C-terminus of the antibody's heavy chain 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, and the human lysosomal enzyme is bound to the C-terminus of the antibody's heavy chain. Here, the antibody's heavy chain and the human lysosomal enzyme may be bound directly or via a linker.

[0051] A fusion protein of the type in which a human lysosomal enzyme is bound to the N-terminus of the antibody light chain 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, and the human lysosomal enzyme is bound to the N-terminus of the antibody light chain. Here, the antibody light chain and the human lysosomal enzyme may be bound directly or via a linker.

[0052] A fusion protein of the type in which a human lysosomal enzyme is bound to the N-terminus of the antibody's heavy chain 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, and the human lysosomal enzyme is bound to the N-terminus of the antibody's heavy chain. Here, the antibody's heavy chain and the human lysosomal enzyme may be bound directly or via a linker.

[0053] When a linker is placed between the antibody and the human lysosomal enzyme, its sequence is preferably composed of 1 to 50 amino acids, more preferably 1 to 17, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids. However, the number of amino acids constituting the linker can be appropriately adjusted to 1, 2, 3, 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, etc., depending on the human lysosomal enzyme to be bound to the antibody. Such linkers are not limited in their amino acid sequence, as long as the linked antibody maintains affinity to hTfR and the linked human lysosomal enzyme exhibits physiological activity 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, the amino acid sequence shown in SEQ ID NO: 4, or sequences of 1 to 10 or 2 to 5 consecutive amino acids, 1 to 50 amino acids, 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: 4 can be suitably used as linkers. The same applies even if the antibody is a single-chain antibody.

[0054] Furthermore, if a single peptide chain in the fusion protein contains multiple linkers, for convenience, each linker will be named sequentially from the N-terminus as the first linker, the second linker, and so on.

[0055] As a preferred embodiment of the antibody when the antibody is an anti-human transferrin receptor antibody, In the variable region of the light chain: (a) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 8 or 9, (b) CDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or 11, and (c) CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12; In the variable region of the heavy chain: (d) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 13 or 14, (e) CDR2 comprises the amino acid sequence shown in SEQ ID NO: 15 or 16, and (f) An example of an antibody is one in which CDR3 comprises the amino acid sequence shown in SEQ ID NO: 17 or 18. Here, the antibody is preferably a human antibody or a humanized antibody.

[0056] The amino acid sequence combinations of each CDR shown in (a) to (f) above may be any combination, for example, those shown in Table 1.

[0057] [Table 1]

[0058] As a further preferred embodiment of the antibody, when the antibody is an anti-human transferrin receptor antibody, (x) An example of an antibody is one in which the variable region of the light chain has the amino acid sequence shown in SEQ ID NO: 20 and the variable region of the heavy chain includes the amino acid sequence shown in SEQ ID NO: 21. Here, the antibody is preferably a human antibody or a humanized antibody. Here, the amino acid sequence of the variable region of the light chain, indicated by SEQ ID NO: 20, includes the amino acid sequences indicated by SEQ ID NOs: 8 and 9 as CDR1, the amino acid sequences indicated by SEQ ID NOs: 10 and 11 as CDR2, and the amino acid sequence indicated by SEQ ID NO: 12 as CDR3. Furthermore, the amino acid sequence of the variable region of the heavy chain, indicated by SEQ ID NO: 21, includes the amino acid sequences indicated by SEQ ID NOs: 13 and 14 as CDR1, the amino acid sequences indicated by SEQ ID NOs: 15 and 16 as CDR2, and the amino acid sequences indicated by SEQ ID NOs: 17 and 18 as CDR3. In addition, the amino acid sequence indicated by SEQ ID NO: 19 is included as the heavy chain framework region 3.

[0059] However, the preferred embodiment of the antibody when the antibody is a humanized antibody and an anti-human transferrin receptor antibody is not limited to (x) above. For example, an antibody in which the amino acid sequence of the variable region of the light chain has 80% or more identity with the amino acid sequence of the variable region of the light chain in (x) above, and the amino acid sequence of the variable region of the heavy chain has 80% or more identity with the amino acid sequence of the variable region of the heavy chain in (x) above, can also be used in the present invention as long as it has affinity for hTfR.

[0060] Furthermore, an antibody in which the amino acid sequence of the variable region of the light chain has 85% or more identity with the amino acid sequence of the variable region of the light chain in (x) above, and the amino acid sequence of the variable region of the heavy chain has 85% or more identity with the amino acid sequence of the variable region of the heavy chain in (x) above, An antibody in which the amino acid sequence of the variable region of the light chain has 90% or more identity with the amino acid sequence of the variable region of the light chain in (x) above, and the amino acid sequence of the variable region of the heavy chain has 90% or more identity with the amino acid sequence of the variable region of the heavy chain in (x) above, and Antibodies in which the amino acid sequence of the variable region of the light chain has 95% or more identity with the amino acid sequence of the variable region of the light chain in (x) above, and the amino acid sequence of the variable region of the heavy chain has 95% or more identity with the amino acid sequence of the variable region of the heavy chain in (x) above, can also be used in the present invention, as long as they have affinity for hTfR.

[0061] As an antibody in which the amino acid sequence of the variable region of the light chain is identical to the amino acid sequence of the variable region of the light chain in (x) above, and the amino acid sequence of the variable region of the heavy chain is identical to the amino acid sequence of the variable region of the heavy chain in (x) above, (xa) The variable region of the light chain contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 20, and CDR1 contains the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 contains the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 contains the amino acid sequence of SEQ ID NO: 12; the variable region of the heavy chain contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21, and CDR1 contains the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 contains the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17 or 18. (xb) The variable region of the light chain contains an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 20, and CDR1 contains the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 contains the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 contains the amino acid sequence of SEQ ID NO: 12; the variable region of the heavy chain contains an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21, and CDR1 contains the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 contains the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17 or 18. (xc) The variable region of the light chain contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 20, and CDR1 contains the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 contains the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 contains the amino acid sequence of SEQ ID NO: 12; the variable region of the heavy chain contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21, and CDR1 contains the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 contains the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17 or 18. (xd) The variable region of the light chain contains an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 20, and CDR1 contains the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 contains the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 contains the amino acid sequence of SEQ ID NO: 12; the variable region of the heavy chain contains an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21, and CDR1 contains the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 contains the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17 or 18. These are some examples.

[0062] Furthermore, as a preferred embodiment of the antibody when the antibody is also an anti-human transferrin receptor antibody, In the amino acid sequence of the variable region of the light chain in (x) above, 1 to 5 amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, and in the amino acid sequence of the variable region of the heavy chain in (x) above, 1 to 5 amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, In the amino acid sequence of the variable region of the light chain in (x) above, 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, and in the amino acid sequence of the variable region of the heavy chain in (x) above, 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, and In the above (x), an amino acid sequence in the variable region of the light chain in which one or two amino acids are substituted, deleted, or added, and in the above (x), an amino acid sequence in the variable region of the heavy chain in which one or two amino acids are substituted, deleted, or added, can also be used in the present invention as long as it has affinity for hTfR. Here, the antibody is preferably a human antibody or a humanized antibody.

[0063] As an antibody in which the amino acid sequence of the variable region of the light chain of the antibody is such that the amino acids in the amino acid sequence constituting the variable region of the light chain in (x) above are substituted, deleted, or added, and the amino acid sequence of the variable region of the heavy chain in (x) above is such that the amino acids in the amino acid sequence constituting it are substituted, deleted, or added, (xe) In the variable region of the light chain, the amino acid sequence is such that 1 to 5 amino acids are substituted, deleted, or added, and CDR1 contains the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 contains the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 contains the amino acid sequence of SEQ ID NO: 12; and in the variable region of the heavy chain, the amino acid sequence is such that 1 to 5 amino acids are substituted, deleted, or added, and CDR1 contains the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 contains the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17 or 18. (xf) In the variable region of the light chain, the amino acid sequence is such that 1 to 3 amino acids are substituted, deleted, or added, and CDR1 contains the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 contains the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 contains the amino acid sequence of SEQ ID NO: 12; and in the variable region of the heavy chain, the amino acid sequence is such that 1 to 3 amino acids are substituted, deleted, or added, and CDR1 contains the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 contains the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17 or 18. (xg) In the variable region of the light chain, one or two amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, and CDR1 includes the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 includes the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 includes the amino acid sequence of SEQ ID NO: 12; and in the variable region of the heavy chain, one or two amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, and CDR1 includes the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 includes the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 includes the amino acid sequence of SEQ ID NO: 17 or 18. These are some examples.

[0064] In (xa) to (xg) above, the combination of amino acid sequences for each CDR can be any combination, for example, those shown in Table 2. The same applies to (ya) to (yg) below.

[0065] [Table 2]

[0066] In the present invention, as a preferred embodiment in which the antibody is a humanized antibody and is an anti-human transferrin receptor antibody, (y) An example of a Fab antibody is one in which the light chain contains the amino acid sequence shown in SEQ ID NO: 22 and the heavy chain contains the amino acid sequence shown in SEQ ID NO: 23. Here, the light chain contains the amino acid sequence shown in SEQ ID NO: 20 as a variable region, and the heavy chain contains the amino acid sequence shown in SEQ ID NO: 21 as a variable region. In the present invention, the heavy chain constituting the Fab is called the Fab heavy chain. That is, the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 23 is the Fab heavy chain.

[0067] When the antibody is a humanized antibody and an anti-human transferrin receptor antibody, the preferred embodiment of the antibody is not limited to (y) above. For example, an antibody in which the amino acid sequence of the light chain has 80% or more identity with the amino acid sequence of the light chain in (y) above, and the amino acid sequence of the heavy chain has 80% or more identity with the amino acid sequence of the heavy chain in (y) above, can also be used in the present invention, as long as it has affinity for hTfR.

[0068] Furthermore, an antibody in which the amino acid sequence of the light chain has 85% or more identity with the amino acid sequence of the light chain in (y) above, and the amino acid sequence of the heavy chain has 85% or more identity with the amino acid sequence of the heavy chain in (y) above, An antibody in which the amino acid sequence of the light chain has 90% or more identity with the amino acid sequence of the light chain in (y) above, and the amino acid sequence of the heavy chain has 90% or more identity with the amino acid sequence of the heavy chain in (y) above, and Antibodies having a light chain amino acid sequence that is 95% or more identical to the light chain amino acid sequence in (y) above, and a heavy chain amino acid sequence that is 95% or more identical to the heavy chain amino acid sequence in (y) above, can also be used in the present invention, as long as they have affinity for hTfR.

[0069] As an antibody in which the amino acid sequence of the light chain is identical to the amino acid sequence of the light chain in (y) above, and the amino acid sequence of the heavy chain is identical to the amino acid sequence of the heavy chain in (y) above, (ya) The light chain contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, and comprises the amino acid sequence of SEQ ID NO: 8 or 9 as CDR1, the amino acid sequence of SEQ ID NO: 10 or 11 as CDR2, and the amino acid sequence of SEQ ID NO: 12 as CDR3, and the heavy chain contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and comprises the amino acid sequence of SEQ ID NO: 13 or 14 as CDR1, the amino acid sequence of SEQ ID NO: 15 or 16 as CDR2, and the amino acid sequence of SEQ ID NO: 17 or 18 as CDR3, (yb) The light chain contains an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, and comprises the amino acid sequence of SEQ ID NO: 8 or 9 as CDR1, the amino acid sequence of SEQ ID NO: 10 or 11 as CDR2, and the amino acid sequence of SEQ ID NO: 12 as CDR3, and the heavy chain contains an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and comprises the amino acid sequence of SEQ ID NO: 13 or 14 as CDR1, the amino acid sequence of SEQ ID NO: 15 or 16 as CDR2, and the amino acid sequence of SEQ ID NO: 17 or 18 as CDR3, (yc) The light chain contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, and comprises the amino acid sequence of SEQ ID NO: 8 or 9 as CDR1, the amino acid sequence of SEQ ID NO: 10 or 11 as CDR2, and the amino acid sequence of SEQ ID NO: 12 as CDR3, and the heavy chain contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and comprises the amino acid sequence of SEQ ID NO: 13 or 14 as CDR1, the amino acid sequence of SEQ ID NO: 15 or 16 as CDR2, and the amino acid sequence of SEQ ID NO: 17 or 18 as CDR3, (yd) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, and comprising the amino acid sequence of SEQ ID NO: 8 or 9 as CDR1, the amino acid sequence of SEQ ID NO: 10 or 11 as CDR2, and the amino acid sequence of SEQ ID NO: 12 as CDR3, and a heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and comprising the amino acid sequence of SEQ ID NO: 13 or 14 as CDR1, the amino acid sequence of SEQ ID NO: 15 or 16 as CDR2, and the amino acid sequence of SEQ ID NO: 17 or 18 as CDR3.

[0070] Furthermore, in the case where the antibody is a humanized antibody and is an anti-human transferrin receptor antibody, a preferred embodiment of the antibody is that the amino acid sequence of the antibody is In the amino acid sequence of the light chain in (y) above, 1 to 5 amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, and in the amino acid sequence of the heavy chain in (y) above, 1 to 5 amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, In the amino acid sequence of the light chain in (y) above, 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, and in the amino acid sequence of the heavy chain in (y) above, 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence that constitutes it, and In the above (y), an amino acid sequence of the light chain in which one or two amino acids are substituted, deleted, or added, and in the above (y), an amino acid sequence of the heavy chain in which one or two amino acids are substituted, deleted, or added, can also be used in the present invention as long as it has affinity for hTfR.

[0071] As an antibody in which the amino acid sequence of the light chain is such that the amino acids in the amino acid sequence constituting the light chain in (y) above are substituted, deleted, or added, and the amino acid sequence of the heavy chain in (y) above is such that the amino acids in the amino acid sequence constituting the heavy chain are substituted, deleted, or added, (ye) In the light chain amino acid sequence, 1 to 5 amino acids are substituted, deleted, or added, and CDR1 contains the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 contains the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 contains the amino acid sequence of SEQ ID NO: 12; and in the heavy chain amino acid sequence, 1 to 5 amino acids are substituted, deleted, or added, and CDR1 contains the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 contains the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17 or 18. (yf) In the amino acid sequence of the light chain, 1 to 3 amino acids are substituted, deleted, or added, and CDR1 contains the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 contains the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 contains the amino acid sequence of SEQ ID NO: 12; and in the amino acid sequence of the heavy chain, 1 to 3 amino acids are substituted, deleted, or added, and CDR1 contains the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 contains the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17 or 18. (yg) In the light chain amino acid sequence, one or two amino acids are substituted, deleted, or added to the amino acid sequence constituting it, and CDR1 contains the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 contains the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 contains the amino acid sequence of SEQ ID NO: 12; and in the heavy chain amino acid sequence, one or two amino acids are substituted, deleted, or added to the amino acid sequence constituting it, and CDR1 contains the amino acid sequence of SEQ ID NO: 13 or 14, CDR2 contains the amino acid sequence of SEQ ID NO: 15 or 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17 or 18. These are some examples.

[0072] When the antibody is a humanized anti-human transferrin receptor antibody and the human lysosomal enzyme is human α-L-iduronidase (hIDUA), the following are preferred forms of the fusion protein: A fusion protein comprising a light chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 22, and a Fab heavy chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 23, with human α-L-iduronidase, shown in SEQ ID NO: 6, bound to the C-terminus via a linker, shown in SEQ ID NO: 4.

[0073] Furthermore, when the antibody is a humanized anti-human transferrin receptor antibody and the human lysosomal enzyme is human α-L-iduronidase (hIDUA), the following are considered preferred forms of the fusion protein. That is, The light chain of the humanized anti-hTfR antibody has the amino acid sequence shown in SEQ ID NO: 22, A fusion protein in which the Fab heavy chain of the humanized anti-hTfR antibody is bound at its C-terminus to human α-L-idulonidase shown in SEQ ID NO: 6 via a linker having the amino acid sequence shown in SEQ ID NO: 4, and the entire protein has the amino acid sequence shown in SEQ ID NO: 27.

[0074] In the present invention, physiologically active proteins can be produced, for example, by culturing mammalian cells that have been artificially manipulated to produce the protein by expressing or strongly expressing the gene encoding the protein. In this case, the gene to be strongly expressed in the mammalian cells that produce the protein is generally introduced into the mammalian cells by transforming them with an expression vector into which the gene is incorporated. There are no particular limitations on the mammalian cells used in this process, but human, mouse, and Chinese hamster-derived cells are preferred, and CHO cells derived from Chinese hamster ovary cells are particularly preferred. In the present invention, when referring to proteins, it specifically refers to recombinant proteins secreted into the culture medium when mammalian cells that produce such proteins are cultured.

[0075] An aqueous pharmaceutical composition according to one embodiment of the present invention contains a physiologically active protein as an active ingredient and two or more nonionic surfactants. The aqueous pharmaceutical composition may further contain at least one of a neutral salt, a disaccharide, and a buffering agent.

[0076] The two or more nonionic surfactants contained in the aqueous pharmaceutical composition are not particularly limited as long as they are 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 poloxamers, polyoxyethylene (42) polyoxypropylene (67) glycol, 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.

[0077] When an aqueous 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 combinations 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.

[0078] In the aqueous pharmaceutical composition, the concentrations of two or more nonionic surfactants are preferably 0.105 to 0.6 mg / mL and 0.005 to 1.5 mg / mL, more preferably 0.1 to 0.5 mg / mL and 0.025 to 1.0 mg / mL, even more preferably 0.15 to 0.45 mg / mL and 0.05 to 1.0 mg / mL, and even more preferably 0.25 to 0.45 mg / mL and 0.05 to 0.15 mg / mL, for example 0.325 mg / mL and 0.075 mg / mL, for example 0.162 mg / mL and 0.075 mg / mL, for example 0.130 mg / mL and 0.075 mg / mL, for example 0.108 mg / mL and 0.075 mg / mL, for example 0.08 mg / mL and 0.075 mg / mL, for example 0.08 mg / mL and 0.075 mg / mL.

[0079] When an aqueous pharmaceutical composition 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 0.105 to 0.6 mg / mL, more preferably 0.1 to 0.5 mg / mL, and even more preferably 0.15 to 0.45 mg / mL, for example, 0.325 mg / mL. Other examples include 0.162 mg / mL, 0.130 mg / mL, 0.108 mg / mL, and 0.08 mg / mL.

[0080] When the aqueous pharmaceutical composition contains polysorbate 80 and poloxamer 188 as two nonionic surfactants, the concentration of polysorbate 80 is preferably 0.005 to 0.15 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 0.105 to 0.6 mg / mL, more preferably 0.1 to 0.5 mg / mL, and even more preferably 0.15 to 0.45 mg / mL, for example, 0.325 mg / mL, and also for example, 0.162 mg / mL, 0.130 mg / mL, 0.108 mg / mL, and 0.08 mg / mL. For example, the concentration of polysorbate 80 is 0.05 to 1.0 mg / mL, and the concentration of poloxamer 188 is 0.15 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. For example, the concentration of polysorbate 80 is 0.075 mg / mL and the concentration of poloxamer 188 is 0.162 mg / mL; for example, the concentration of polysorbate 80 is 0.075 mg / mL and the concentration of poloxamer 188 is 0.130 mg / mL; for example, the concentration of polysorbate 80 is 0.075 mg / mL and the concentration of poloxamer 188 is 0.108 mg / mL; for example, the concentration of polysorbate 80 is 0.075 mg / mL and the concentration of poloxamer 188 is 0.08 mg / mL.

[0081] The neutral salt contained in the aqueous 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.

[0082] The concentration of the neutral salt in the aqueous pharmaceutical composition is preferably 0.3 to 1.2 mg / mL, more preferably 0.5 to 1.0 mg / mL, and even more preferably 0.7 to 0.9 mg / mL, for example, 0.8 mg / mL.

[0083] The disaccharides contained in the aqueous pharmaceutical composition are not particularly limited as long as they are pharmaceutically acceptable, but trehalose, sucrose, maltose, lactose, or combinations thereof are preferred as such disaccharides, with sucrose being particularly preferred.

[0084] The concentration of disaccharides in the aqueous pharmaceutical composition 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.

[0085] The buffering agent included in the aqueous 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. The concentration of the buffering agent included in the aqueous pharmaceutical composition is preferably 3 to 30 mM, more preferably 10 to 30 mM, even more preferably 15 to 25 mM, for example 20 mM. The pH of the aqueous pharmaceutical composition 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. When a citrate buffering agent is used as the buffering agent, the concentration of the citrate buffering agent included in the aqueous pharmaceutical composition 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 pharmaceutical composition 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.

[0086] A suitable composition for the aqueous pharmaceutical composition of the present invention is, (A) A solution in which the concentration of physiologically active protein is 0.5 to 20 mg / mL, the concentration of neutral salt is 0.3 to 1.2 mg / mL, the concentration of disaccharide is 50 to 100 mg / mL, the concentrations of two or more nonionic surfactants are 0.105 to 0.6 mg / mL and 0.005 to 1.5 mg / mL, the concentration of buffer is 3 to 30 mM, and the pH is 4.5 to 6.5. (B) A solution having a concentration of physiologically active protein of 1.0 to 10 mg / mL, a concentration of neutral salt of 0.5 to 1.0 mg / mL, a concentration of disaccharides of 55 to 95 mg / mL, and of two or more nonionic surfactants, the concentrations of two nonionic surfactants being 0.1 to 0.5 mg / mL and 0.025 to 1.0 mg / mL, respectively, with a buffer concentration of 10 to 30 mM and a pH of 5.0 to 6.0. (C) A solution is provided in which the concentration of physiologically active protein is 2.0 to 10 mg / mL, the concentration of neutral salt is 0.7 to 0.9 mg / mL, the concentration of disaccharide is 60 to 90 mg / mL, the concentrations of two or more nonionic surfactants are 0.15 to 0.45 mg / mL and 0.05 to 0.15 mg / mL, the concentration of buffer is 15 to 25 mM, and the pH is 5.2 to 5.8.

[0087] In the aqueous pharmaceutical compositions shown in (A) to (C) above, the physiologically active protein is, for example, a fusion protein of a humanized anti-hTfR antibody and hIDUA. A preferred form of such a fusion protein of a humanized anti-hTfR antibody and hIDUA is as follows: a fusion protein comprising a light chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 22, and a human α-L-iduronidase, shown in SEQ ID NO: 6, bound to the C-terminal side of the Fab heavy chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 23, via a linker shown in SEQ ID NO: 4.

[0088] Furthermore, in the aqueous pharmaceutical compositions shown in (A) to (C) above, if the physiologically active protein is a fusion protein of a humanized anti-hTfR antibody and hIDUA, the following is a further preferred form of the fusion protein: a fusion protein in which the light chain of the humanized anti-hTfR antibody has the amino acid sequence shown in SEQ ID NO: 22, and the Fab heavy chain of the humanized anti-hTfR antibody is bound at its C-terminus to human α-L-iduronidase shown in SEQ ID NO: 6 via a linker having the amino acid sequence shown in SEQ ID NO: 4, and the fusion protein as a whole has the amino acid sequence shown in SEQ ID NO: 27.

[0089] In the aqueous pharmaceutical compositions shown in (A) to (C) above, the preferred concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 0.5 to 20 mg / mL, 1.0 to 10 mg / mL, 2.0 to 10 mg / mL, or 2.0 to 6.0 mg / mL, and can be adjusted as appropriate to 2.5 mg / mL, 5.0 mg / mL, etc.

[0090] In the aqueous pharmaceutical compositions shown in (A) to (C) above, the neutral salt is not particularly limited as long as it is pharmaceutically acceptable, but sodium chloride is preferred. When sodium chloride is used as the neutral salt, its concentration is preferably 0.3 to 1.2 mg / mL, more preferably 0.5 to 1.0 mg / mL, and even more preferably 0.7 to 0.9 mg / mL, for example, 0.8 mg / mL.

[0091] In the aqueous pharmaceutical compositions shown in (A) to (C) above, there are no particular limitations on the disaccharide as long as it is pharmaceutically acceptable, but sucrose is preferred. When sucrose is used as the disaccharide, its concentration 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.

[0092] In the aqueous pharmaceutical compositions shown in (A) to (C) above, there are no particular limitations on the two or more nonionic surfactants as long as they are 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 poloxamers, polyoxyethylene (42) polyoxypropylene (67) glycol, 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. When the aqueous 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 combinations 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. The concentrations of two or more nonionic surfactants in the aqueous pharmaceutical composition are preferably 0.105 to 0.6 mg / mL and 0.005 to 1.5 mg / mL, more preferably 0.1 to 0.5 mg / mL and 0.025 to 1.0 mg / mL, even more preferably 0.15 to 0.45 mg / mL and 0.05 to 1.0 mg / mL, and even more preferably 0.25 to 0.45 mg / mL and 0.05 to 0.15 mg / mL, for example, 0.325 mg / mL and 0.075 mg / mL, respectively.For example, these are 0.162 mg / mL and 0.075 mg / mL, 0.130 mg / mL and 0.075 mg / mL, 0.108 mg / mL and 0.075 mg / mL, and 0.08 mg / mL and 0.075 mg / mL, respectively. When the aqueous pharmaceutical composition contains polysorbate and poloxamer as two nonionic surfactants, the concentration of polysorbate is 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. Furthermore, the concentration of poloxamer at this time is preferably 0.105 to 0.6 mg / mL, more preferably 0.1 to 0.5 mg / mL, and even more preferably 0.15 to 0.45 mg / mL, for example, 0.325 mg / mL. Other examples include 0.162 mg / mL, 0.130 mg / mL, 0.108 mg / mL, and 0.08 mg / mL.

[0093] The buffer used in the aqueous pharmaceutical compositions shown in (A) to (C) above is not particularly limited as long as it is pharmaceutically acceptable, but a citrate buffer is preferred. When a citrate buffer is used as the buffer, its concentration is preferably 3 to 30 mM, more preferably 10 to 30 mM, even more preferably 15 to 25 mM, for example 20 mM. The pH of the aqueous pharmaceutical composition adjusted by the buffer is preferably 4.5 to 6.5, more preferably 5.0 to 6.0, even more preferably 5.2 to 5.8, for example 5.5.

[0094] As an example of a suitable aqueous pharmaceutical composition in which the physiologically active protein is a fusion protein of an antibody and a human lysosomal enzyme, the fusion protein is a fusion protein of a humanized anti-hTfR antibody and hIDUA, with a concentration of 1 to 10 mg / mL, a concentration of neutral salt (especially sodium chloride) of 0.3 to 1.2 mg / mL, a concentration of disaccharide (especially sucrose) of 50 to 100 mg / mL, a concentration of polysorbate (especially polysorbate 80) of 0.025 to 1.0 mg / mL, a concentration of poloxamer (especially poloxamer 188) of 0.1 to 0.5 mg / mL, and a concentration of citrate buffer of 10 to 30 mM. More specifically, examples include a fusion protein of humanized anti-hTfR antibody and hIDUA with a concentration of 1-10 mg / mL, a sodium chloride concentration of 0.5-1.0 mg / mL, a sucrose concentration of 55-95 mg / mL, a polysorbate (especially polysorbate 80) concentration of 0.05-1.0 mg / mL, a poloxamer (especially poloxamer 188) concentration of 0.15-0.45 mg / mL, and a citrate buffer concentration of 15-25 mM. More specifically, examples include formulations in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 1-10 mg / mL, the concentration of sodium chloride is 0.7-0.9 mg / mL, the concentration of sucrose is 60-90 mg / mL, the concentration of polysorbate (especially polysorbate 80) is 0.05-0.15 mg / mL, the concentration of poloxamer (especially poloxamer 188) is 0.15-0.45 mg / mL, and the concentration of citrate buffer is 15-25 mM. More specifically, a solution may be provided in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.325 mg / mL, and the concentration of citrate buffer is 20 mM.More specifically, an example is a formulation in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.162 mg / mL, and the concentration of citrate buffer is 20 mM. More specifically, an example is a formulation in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.130 mg / mL, and the concentration of citrate buffer is 20 mM. More specifically, an example is a formulation in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.108 mg / mL, and the concentration of citrate buffer is 20 mM. More specifically, an example is a formulation in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.08 mg / mL, and the concentration of citrate buffer is 20 mM.

[0095] The aqueous pharmaceutical composition of the present invention, which contains a physiologically active protein as an active ingredient, can be stably stored in a dark place at a temperature of 2 to 8°C for a period of preferably 3 months, more preferably 1 year, even more preferably 2 years, and even more preferably 3 years or longer, for example, 4 years, 5 years, 6 years, etc. Furthermore, the aqueous pharmaceutical composition of the present invention can be stably stored in a dark place at a temperature of 22 to 28°C for preferably 3 months, more preferably 6 months.

[0096] The aqueous pharmaceutical composition of the present invention, which contains a physiologically active protein as an active ingredient, can be administered intravenously, intramuscularly, intraperitoneally, or subcutaneously as an injectable preparation. The aqueous pharmaceutical composition of the present invention may be in the form of being filled in a vial, or it may be supplied 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, and hydrophobic resins such as cycloolefin copolymers, cycloolefin ring-opening polymers, or hydrogenated cycloolefin ring-opening polymers are also preferred.

[0097] The freeze-dried pharmaceutical composition in one embodiment of the present invention contains a physiologically active protein as an active ingredient and two or more nonionic surfactants. The freeze-dried pharmaceutical composition may further contain at least one of a neutral salt, a disaccharide, and a buffering agent.

[0098] The two or more nonionic surfactants contained in the freeze-dried pharmaceutical composition are not particularly limited as long as they are 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 poloxamers, polyoxyethylene (42) polyoxypropylene (67) glycol, 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.

[0099] When a freeze-dried pharmaceutical composition contains two nonionic surfactants, a preferred combination of nonionic surfactants is one polysorbate and the other poloxamer. For example, the combinations 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 polysorbates, poloxamers, etc.

[0100] In one embodiment of the present invention, the freeze-dried pharmaceutical composition is used after being dissolved in an aqueous solvent. Suitable aqueous solvents used at this time include, but are not limited to, pure water and physiological saline. When a freeze-dried pharmaceutical composition is dissolved in an aqueous solvent, a problem may occur in which some of the freeze-dried components do not dissolve, but this problem does not occur with the freeze-dried pharmaceutical composition in question. The freeze-dried pharmaceutical composition dissolved in an aqueous solvent is also an aqueous pharmaceutical composition of the present invention. In this case, the aqueous pharmaceutical composition can be stored stably in a dark place at a temperature of 2 to 8°C, preferably for 1 day, more preferably for 1 week. The freeze-dried pharmaceutical composition can be stored stably in a dark place at a temperature of 2 to 8°C for a period of time of 3 months, more preferably for 1 year, even more preferably for 2 years, even more preferably for 3 years, or longer, for example, 4 years, 5 years, 6 years, etc. The freeze-dried pharmaceutical composition can also be stored stably in a dark place at a temperature of 22 to 28°C for 3 months, more preferably for 6 months.

[0101] In the aqueous pharmaceutical composition obtained by dissolving the freeze-dried pharmaceutical composition, the concentrations of two or more nonionic surfactants are preferably 0.105 to 0.6 mg / mL and 0.005 to 1.5 mg / mL, more preferably 0.1 to 0.5 mg / mL and 0.025 to 1.0 mg / mL, even more preferably 0.15 to 0.45 mg / mL and 0.05 to 1.0 mg / mL, and even more preferably 0.15 to 0.45 mg / mL and 0.05 to 0.15 mg / mL, for example, 0.325 mg / mL and 0.075 mg / mL, respectively. For example, these are 0.162 mg / mL and 0.075 mg / mL, respectively; 0.130 mg / mL and 0.075 mg / mL, respectively; 0.108 mg / mL and 0.075 mg / mL, respectively; and 0.08 mg / mL and 0.075 mg / mL, respectively.

[0102] When a freeze-dried pharmaceutical composition contains polysorbate and poloxamer as two nonionic surfactants, the concentration of polysorbate in the aqueous pharmaceutical composition obtained by dissolving it 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 0.105 to 0.6 mg / mL, more preferably 0.1 to 0.5 mg / mL, and even more preferably 0.15 to 0.45 mg / mL, for example, 0.325 mg / mL. Other examples include 0.162 mg / mL, 0.130 mg / mL, 0.108 mg / mL, and 0.08 mg / mL.

[0103] When a freeze-dried pharmaceutical composition contains polysorbate 80 and poloxamer 188 as two nonionic surfactants, the concentration of polysorbate 80 in the aqueous pharmaceutical composition obtained by dissolving it is preferably 0.005 to 0.15 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 0.105 to 0.6 mg / mL, more preferably 0.1 to 0.5 mg / mL, and even more preferably 0.15 to 0.45 mg / mL, for example, 0.325 mg / mL. Other examples include 0.162 mg / mL, 0.130 mg / mL, 0.108 mg / mL, and 0.08 mg / mL. For example, the concentration of polysorbate 80 is 0.05 to 1.0 mg / mL, and the concentration of poloxamer 188 is 0.15 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. For example, the concentration of polysorbate 80 is 0.075 mg / mL and the concentration of poloxamer 188 is 0.162 mg / mL; for example, the concentration of polysorbate 80 is 0.075 mg / mL and the concentration of poloxamer 188 is 0.130 mg / mL; for example, the concentration of polysorbate 80 is 0.075 mg / mL and the concentration of poloxamer 188 is 0.108 mg / mL; for example, the concentration of polysorbate 80 is 0.075 mg / mL and the concentration of poloxamer 188 is 0.08 mg / mL.

[0104] The neutral salt contained in the freeze-dried 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.

[0105] When a freeze-dried pharmaceutical composition contains a neutral salt, the concentration of the neutral salt in the aqueous pharmaceutical composition obtained by dissolving it is preferably 0.3 to 1.2 mg / mL, more preferably 0.5 to 1.0 mg / mL, and even more preferably 0.7 to 0.9 mg / mL, for example, 0.8 mg / mL.

[0106] The disaccharides contained in the freeze-dried 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.

[0107] When a freeze-dried pharmaceutical composition contains disaccharides, the concentration of disaccharides in the aqueous pharmaceutical composition obtained by dissolving them 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.

[0108] The buffering agent included in the freeze-dried pharmaceutical composition is not particularly limited as long as it is pharmaceutically acceptable, but citrate buffering agents, phosphate buffering agents, glycine buffering agents, histidine buffering agents, carbonate buffering agents, acetate buffering agents, or combinations thereof are preferred. When the freeze-dried pharmaceutical composition contains a buffering agent, the concentration of the buffering agent in the aqueous pharmaceutical composition obtained by dissolving it is preferably 3 to 30 mM, more preferably 10 to 30 mM, even more preferably 15 to 25 mM, for example 20 mM. The pH of the aqueous pharmaceutical composition 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. When citrate buffering agents are used as buffering agents, the concentration of citrate buffering agents included in the aqueous pharmaceutical composition 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 pharmaceutical composition 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.

[0109] A suitable example of the freeze-dried pharmaceutical composition of the present invention is an aqueous pharmaceutical composition obtained by dissolving it, (D) A solution in which the concentration of physiologically active protein is 0.5 to 20 mg / mL, the concentration of neutral salt is 0.3 to 1.2 mg / mL, the concentration of disaccharide is 50 to 100 mg / mL, the concentrations of two or more nonionic surfactants are 0.105 to 0.6 mg / mL and 0.005 to 1.5 mg / mL, the concentration of buffer is 3 to 30 mM, and the pH is 4.5 to 6.5. (E) A solution having a concentration of physiologically active protein of 1.0 to 10 mg / mL, a concentration of neutral salt of 0.5 to 1.0 mg / mL, a concentration of disaccharides of 55 to 95 mg / mL, and of two or more nonionic surfactants, the concentrations of two nonionic surfactants being 0.1 to 0.5 mg / mL and 0.025 to 1.0 mg / mL, respectively, with a buffer concentration of 10 to 30 mM and a pH of 5.0 to 6.0. (F) A solution in which the concentration of physiologically active protein is 2.0 to 10 mg / mL, the concentration of neutral salt is 0.7 to 0.9 mg / mL, the concentration of disaccharide is 60 to 90 mg / mL, the concentrations of two or more nonionic surfactants are 0.15 to 0.45 mg / mL and 0.05 to 0.15 mg / mL, the concentration of buffer is 15 to 25 mM, and the pH is 5.2 to 5.8.

[0110] In (D) to (F) above, the physiologically active protein is, for example, a fusion protein of a humanized anti-hTfR antibody and hIDUA. A preferred form of such a fusion protein of a humanized anti-hTfR antibody and hIDUA is as follows: a fusion protein consisting of a light chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 22, and a human α-L-iduronidase, shown in SEQ ID NO: 6, bound to the C-terminal side of the Fab heavy chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 23, via a linker shown in SEQ ID NO: 4.

[0111] Furthermore, in (D) to (F) above, if the physiologically active protein is a fusion protein of a humanized anti-hTfR antibody and hIDUA, the following is a further preferred form of the fusion protein: a fusion protein in which the light chain of the humanized anti-hTfR antibody has the amino acid sequence shown in SEQ ID NO: 22, and the Fab heavy chain of the humanized anti-hTfR antibody is bound at its C-terminus to human α-L-iduronidase shown in SEQ ID NO: 6 via a linker having the amino acid sequence shown in SEQ ID NO: 4, and the fusion protein as a whole has the amino acid sequence shown in SEQ ID NO: 27.

[0112] In (D) to (F) above, the preferred concentrations of the fusion protein of humanized anti-hTfR antibody and hIDUA are 0.5 to 20 mg / mL, 1.0 to 10 mg / mL, 2.0 to 10 mg / mL, or 2.0 to 6.0 mg / mL, and can be adjusted as appropriate to 2.5 mg / mL, 5.0 mg / mL, etc.

[0113] In (D) to (F) above, the neutral salt is not particularly limited as long as it is pharmaceutically acceptable, but sodium chloride is preferred. When sodium chloride is used as the neutral salt, its concentration is preferably 0.3 to 1.2 mg / mL, more preferably 0.5 to 1.0 mg / mL, and even more preferably 0.7 to 0.9 mg / mL, for example, 0.8 mg / mL.

[0114] In (D) to (F) above, there are no particular limitations on the disaccharide as long as it is pharmaceutically acceptable, but sucrose is preferred. When sucrose is used as the disaccharide, its concentration 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.

[0115] In (D) to (F) above, there are no particular limitations on the two or more nonionic surfactants as long as they are 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 (42) polyoxypropylene (67) glycol, 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. When (D) to (F) above contain two types of nonionic surfactants, a preferred combination of nonionic surfactants is one being polysorbate and the other being poloxamer. For example, the combinations 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. Of the two or more nonionic surfactants in (D) to (F) above, the concentrations of the two are preferably 0.105 to 0.6 mg / mL and 0.005 to 1.5 mg / mL, more preferably 0.1 to 0.5 mg / mL and 0.025 to 1.0 mg / mL, even more preferably 0.15 to 0.45 mg / mL and 0.05 to 1.0 mg / mL, and even more preferably 0.15 to 0.45 mg / mL and 0.05 to 0.15 mg / mL, for example 0.325 mg / mL and 0.075 mg / mL, respectively.For example, these are 0.162 mg / mL and 0.075 mg / mL, respectively, 0.130 mg / mL and 0.075 mg / mL, respectively, 0.108 mg / mL and 0.075 mg / mL, respectively, and 0.08 mg / mL and 0.075 mg / mL, respectively. When (D) to (F) above contain polysorbate and poloxamer as two types of nonionic surfactants, the concentration of polysorbate is 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, even more preferably 0.05 to 0.15 mg / mL, for example, 0.075 mg / mL. Furthermore, the concentration of poloxamer at this time is preferably 0.105 to 0.6 mg / mL, more preferably 0.1 to 0.5 mg / mL, and even more preferably 0.15 to 0.45 mg / mL, for example, 0.325 mg / mL. Other examples include 0.162 mg / mL, 0.130 mg / mL, 0.108 mg / mL, and 0.08 mg / mL.

[0116] The buffer used in (D) to (F) above is not particularly limited as long as it is pharmaceutically acceptable, but citrate buffer is preferred. When citrate buffer is used as the buffer, its concentration is preferably 3 to 30 mM, more preferably 10 to 30 mM, even more preferably 15 to 25 mM, for example 20 mM. The pH of the aqueous pharmaceutical composition adjusted by the buffer is preferably 4.5 to 6.5, more preferably 5.0 to 6.0, even more preferably 5.2 to 5.8, for example 5.5.

[0117] As an example of a suitable composition for a lyophilized pharmaceutical composition in which the physiologically active protein is a fusion protein of an antibody and a human lysosomal enzyme, the fusion protein is a fusion protein of a humanized anti-hTfR antibody and hIDUA, and the aqueous pharmaceutical composition obtained by dissolving the lyophilized pharmaceutical composition has a concentration of the fusion protein of 1 to 10 mg / mL, a concentration of neutral salt (especially sodium chloride) of 0.3 to 1.2 mg / mL, a concentration of disaccharide (especially sucrose) of 50 to 100 mg / mL, a concentration of polysorbate (especially polysorbate 80) of 0.025 to 1.0 mg / mL, a concentration of poloxamer (especially poloxamer 188) of 0.1 to 0.5 mg / mL, and a concentration of citrate buffer of 10 to 30 mM. More specifically, examples include formulations with a fusion protein concentration of humanized anti-hTfR antibody and hIDUA of 1-10 mg / mL, a sodium chloride concentration of 0.5-1.0 mg / mL, a sucrose concentration of 55-95 mg / mL, a polysorbate (especially polysorbate 80) concentration of 0.05-1.0 mg / mL, a poloxamer (especially poloxamer 188) concentration of 0.15-0.45 mg / mL, and a citrate buffer concentration of 15-25 mM. More specifically, examples include formulations in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 1-10 mg / mL, the concentration of sodium chloride is 0.7-0.9 mg / mL, the concentration of sucrose is 60-90 mg / mL, the concentration of polysorbate (especially polysorbate 80) is 0.05-0.15 mg / mL, the concentration of poloxamer (especially poloxamer 188) is 0.15-0.45 mg / mL, and the concentration of citrate buffer is 15-25 mM. More specifically, a solution may be provided in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.325 mg / mL, and the concentration of citrate buffer is 20 mM.More specifically, an example is a formulation in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.162 mg / mL, and the concentration of citrate buffer is 20 mM. More specifically, an example is a formulation in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.130 mg / mL, and the concentration of citrate buffer is 20 mM. More specifically, an example is a formulation in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.108 mg / mL, and the concentration of citrate buffer is 20 mM. More specifically, an example is a formulation in which the concentration of the fusion protein of humanized anti-hTfR antibody and hIDUA is 5 mg / mL, the concentration of sodium chloride is 0.8 mg / mL, the concentration of sucrose is 75 mg / mL, the concentration of polysorbate 80 is 0.075 mg / mL, the concentration of poloxamer 188 is 0.08 mg / mL, and the concentration of citrate buffer is 20 mM.

[0118] The lyophilized formulation of the present invention can also be supplied as a kit together with a special solution for dissolving it. The lyophilized formulation is administered intravenously, intramuscularly, intraperitoneally, or subcutaneously to the patient after being dissolved in a special solution, pure water, etc., before use. There are no particular limitations on the material of the syringes, vials, and other containers used for encapsulating and filling the lyophilized formulation, 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.

[0119] In one embodiment of the present invention, the aqueous pharmaceutical composition has a polymer content of preferably 0.5% or less, more preferably 0.4% or less, and even more preferably 0.3% or less after 36 months of storage in the dark at a temperature of 2 to 8 °C. Furthermore, in one embodiment of the present invention, the polymer content and the decomposition product content of the aqueous pharmaceutical composition have preferably 0.5% or less and 1% or less, more preferably 0.4% or less and 0.8% or less, and even more preferably 0.3% or less and 0.6% or less, respectively.

[0120] The aqueous pharmaceutical composition in one embodiment of the present invention can be stored in a dark place at a temperature of 2 to 8°C for preferably 24 months, more preferably 36 months, even more preferably 48 months, and even more preferably 72 months after manufacture. In other words, it can be stored for a long period of time in a state where it can be administered to patients as an aqueous pharmaceutical composition at the receiving medical institution.

[0121] In one embodiment of the present invention, the freeze-dried pharmaceutical composition has a polymer content of preferably 0.5% or less, more preferably 0.4% or less, and even more preferably 0.3% or less after 36 months of storage in the dark at a temperature of 2 to 8 °C. In another embodiment of the present invention, the aqueous pharmaceutical composition has a polymer content of preferably 0.5% or less and a degradation product content of preferably 0.1% or less, and more preferably 0.4% or less and 0.07% or less, and even more preferably 0.3% or less and 0.05%, respectively, after 36 months of storage in the dark at a temperature of 2 to 8 °C.

[0122] The lyophilized pharmaceutical composition in one embodiment of the present invention can be stored in a dark place at a temperature of 2 to 8°C for preferably 24 months, more preferably 36 months, even more preferably 48 months, and even more preferably 72 months after manufacture. In other words, it can be stored for a long period of time in a state where it can be administered to patients as a lyophilized pharmaceutical composition at the receiving medical institution. [Examples]

[0123] 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.

[0124] [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: 22 and a heavy chain Fab region having the amino acid sequence shown in SEQ ID NO: 23.

[0125] [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: 13) and Hyg-BstX3' (SEQ ID NO: 14). 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).

[0126] [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).

[0127] 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: 29) and IRES3' (SEQ ID NO: 30). 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.

[0128] 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: 31) and mPGKP3' (SEQ ID NO: 32). 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.

[0129] cDNA was prepared from CHO-K1 cells and used as a template. Using primers GS5' (SEQ ID NO: 33) and GS3' (SEQ ID NO: 34), 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.

[0130] Using pCAGIPuro (Miyahara M. et.al., J. Biol. Chem. 275,613-618(2000)) as a template, and primers puro5' (SEQ ID NO: 35) and puro3' (SEQ ID NO: 36), 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.

[0131] 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: 37) and SV40polyA3' (SEQ ID NO: 38). 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 excise 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 (Japanese Patent No. 6279466).

[0132] [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: 39) and mIRES-GS3' (SEQ ID NO: 40). A DNA fragment was then amplified by disrupting the EMCV by adding a mutation to the start codon (ATG) located second from the 5' end of IRES. 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 aforementioned 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(-).

[0133] 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).

[0134] [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: 41). 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: 42). This DNA fragment was inserted into the MluI and BamHI sites of JCR69 in pUC57 to form pEM hygro.

[0135] A DNA fragment (SEQ ID NO: 26) 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: 22 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), an expression vector for the light chain of the humanized anti-hTfR antibody.

[0136] A DNA fragment containing the base sequence shown in SEQ ID NO: 28 was artificially synthesized. This fragment consisted of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 23, to which a human IDUA having the amino acid sequence shown in SEQ ID NO: 6 was bound via a linker sequence shown in SEQ ID NO: 4 to the C-terminal side of the Fab region of the heavy chain, and the entire resulting DNA fragment containing the amino acid sequence shown in SEQ ID NO: 27 was then synthesized. 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, a vector for expressing a protein in which hIDUA is bound to the C-terminal side of the Fab heavy chain of a humanized anti-hTfR antibody.

[0137] [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.

[0138] Cell transformation was generally carried out using the following method: 2 × 10⁶ CHO-K1 cells 7 CD OPTI-CHO TM The cells were suspended in a 1:1 mixture of culture medium (Thermo Fisher Scientific) and PBS. A 50 μL cell suspension was taken and treated with CD OPTI-CHO. 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 OPTI-CHO with 0.5 mg / mL of hygromycin. TMThe cells were selectively cultured in culture medium. The resulting cells were then introduced with the pE-mIRES-GSp-Fab-IDUA plasmid DNA (digested with AhdI and linearized) using the same method. 37 o After culturing the cells overnight under conditions of 1C, 5% CO2, the cells were treated with 0.5 mg / mL hygromycin and 0.8 mg / mL puromycin in CD OPTI-CHO. 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.

[0139] Next, cells selected by selective culture were seeded onto a 96-well plate using the limiting dilution method 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 formed, and the humanized antibody content was examined by ELISA to select cell lines that highly expressed humanized anti-hTfR antibody-hIDUA fusion protein.

[0140] 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). Then, 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).

[0141] The resulting humanized anti-hTfR antibody-hIDUA expression strain was mixed with CD OPTI-CHO 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.

[0142] [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 OPTI-CHO), 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 a speed of approximately 80-100 rpm to maintain the dissolved oxygen saturation of the medium at approximately 30%, and 34-37 o Cells were cultured for approximately 10 days at a temperature range of 10°C. During the culture period, cell density, cell viability, glucose concentration in the culture medium, and lactate concentration were monitored. If the glucose concentration in the culture medium fell below 3.0 g / L, glucose solution was immediately added to the medium to bring the glucose concentration up to 3.5 g / L. During the culture period, the feed solution (EFFICIENTFEED A+) was used. TM ThermoFisher SCIENTIFIC added appropriate amounts to the culture medium. After the culture was complete, the culture medium was collected. The collected culture medium was then mixed with MILLISTAK+HC. TM Filtered with Pod Filter grade D0HC (Merck), and further filtered with MILLISTAK+ TMThe culture supernatant containing humanized anti-hTfR antibody-hIDUA was obtained by filtering with HCgrade X0HC (Merck). This culture supernatant was then converted to PELLICON. TM 3 Cassette w / Ultracel PLCTK Membrane (pore size: 30 kDa, membrane area: 1.14 m 2 The solution was ultrafiltered using a Merck (or similar) and concentrated until the volume was reduced to approximately 1 / 14th. Next, this concentrated solution was subjected to OPTICAP (or similar). TM The solution was filtered using an XL600 (0.22 μm, Merck). The resulting liquid was used as the concentrated culture supernatant.

[0143] [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. CAPTURE SELECT 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.

[0144] 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.

[0145] To the eluate from the affinity column mentioned above, 250 mM MES buffer (pH 6.5) was added to adjust the pH of the eluate to 6.0. Then, this eluate was subjected to OPTICAP TM The solution was filtered using an XL600 filter (pore size: 0.22 μm, Merck). The filtered solution was then equilibrated with 50 mM MES buffer (pH 6.0) containing 15 mM NaCl in a volume five times the column volume, using a CAPTO multimodal anion exchange column. TM An adhere column (column volume: approx. 1.5 L, bed height: approx. 10 cm, GE Healthcare) was loaded at a constant flow rate of 300 cm / hour. The loading solution containing this humanized anti-hTfR antibody-hIDUA was collected. CAPTO TM Adhesion uses N-benzyl-N-methylethanolamine as a ligand and is a strong anion exchanger with selectivity based on electrostatic interactions, hydrogen bonding, hydrophobic interactions, etc.

[0146] 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.

[0147] 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, forming a multimodal weak cation exchange column called CAPTO. TM An MMC column (column volume: approx. 3.1 L, bed height: approx. 20 cm, GE Healthcare) was loaded at a constant flow rate of 200 cm / hour. CAPTO TM MMC is a weak cation exchanger that exhibits selectivity based on hydrophobic interactions, hydrogen bond formation, and other factors.

[0148] 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.

[0149] The eluate from the weak cation exchange column described above was adjusted to pH 5.8 by adding 0.5 times the volume of 20 mM citrate buffer (pH 5.5) containing 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 (Merck) and concentrated until the concentration of humanized anti-hTfR antibody-hIDUA in the solution was approximately 30 mg / mL. This concentrate was then OPTICAP TM The sample was filtered using an XL600 (0.22 μm, Merck).

[0150] 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.

[0151] [Example 5] Stability study of aqueous pharmaceutical composition containing humanized anti-hTfR antibody-hIDUA 1 Aqueous pharmaceutical compositions often contain nonionic surfactants to enhance the stability of the main protein component or to prevent the protein from adsorbing to the container. For example, some growth hormone aqueous pharmaceutical compositions contain poloxamer 188 as a nonionic surfactant. Therefore, the following study was conducted to investigate the effect of adding poloxamer 188 on the stability of proteins in aqueous pharmaceutical compositions containing humanized anti-hTfR antibody-hIDUA.

[0152] Using the purified humanized anti-hTfR antibody-hIDUA product obtained in Example 4, three aqueous pharmaceutical compositions were prepared as shown in Table 3, each containing sodium chloride, citrate buffer, sucrose, poloxamer 188, and humanized anti-hTfR antibody-hIDUA, differing only in the concentration of poloxamer 188. Each of these three aqueous pharmaceutical compositions (Formulas A-C) was filled into 2 mL glass vials, sealed, and shaken continuously at room temperature for 24 hours using a shaker (SR-2S, Taitec Co., Ltd.) (shaking speed: 240 reciprocations / min, amplitude: 40 mm). The number of particles per unit volume (200 μL) (particle size: 1-100 μm) in the aqueous pharmaceutical compositions after shaking was measured using the method described in Example 8. Furthermore, the content of the humanized anti-hTfR antibody-hIDUA polymer in the aqueous pharmaceutical compositions after shaking was measured using the method described in Example 9. Furthermore, the content of degradation products of humanized anti-hTfR antibody-hIDUA contained in each aqueous pharmaceutical composition was measured using the method described in Example 10.

[0153] [Table 3]

[0154] Figure 1 shows the measurement results of the number of particles contained in the aqueous pharmaceutical composition. It was found that, regardless of the concentration of poloxamer 188, the aqueous pharmaceutical composition after shaking contained a large number of particles with a particle size of less than 10 μm.

[0155] Next, we examine the measurement results for the content of the humanized anti-hTfR antibody-hIDUA polymer in the aqueous pharmaceutical composition. The measurement results are shown in Figure 2. Regardless of the concentration of poloxamer 188, almost no polymer was present in the aqueous pharmaceutical composition after shaking.

[0156] Next, we examine the measurement results of the content of degradation products of the humanized anti-hTfR antibody-hIDUA contained in the aqueous pharmaceutical composition. The measurement results are shown in Figure 3. Regardless of the concentration of poloxamer 188, almost no degradation products were present in the aqueous pharmaceutical composition after shaking.

[0157] [Example 6] Stability study of aqueous pharmaceutical composition containing humanized anti-hTfR antibody-hIDUA 2 Furthermore, regarding aqueous pharmaceutical compositions to which nonionic surfactants are added, for example, in the case of darbepoetin and agalsidase, there are aqueous pharmaceutical compositions to which polysorbate 80 is added as the nonionic surfactant. Therefore, in order to investigate the effect of adding polysorbate 80 on the stability of proteins in aqueous pharmaceutical compositions containing humanized anti-hTfR antibody-hIDUA, the following study was conducted.

[0158] Using the purified humanized anti-hTfR antibody-hIDUA product obtained in Example 4, six aqueous pharmaceutical compositions were prepared as shown in Table 4, each containing sodium chloride, citrate buffer, sucrose, polysorbate 80, and humanized anti-hTfR antibody-hIDUA, differing only in the concentration of polysorbate 80. Each of these six aqueous pharmaceutical compositions (Formulas D to I) was filled into 2 mL glass vials, sealed, and shaken continuously at room temperature for 24 hours using a shaker (SR-2S, Taitec Co., Ltd.) (shaking speed: 240 reciprocations / min, amplitude: 40 mm). The number of particles per unit volume (200 μL) contained in the aqueous pharmaceutical composition after shaking (particle size: 1 to 100 μm) was measured using the method described in Example 8. The content of the humanized anti-hTfR antibody-hIDUA polymer contained in the aqueous pharmaceutical composition after shaking was measured using the method described in Example 9. Furthermore, the content of degradation products of humanized anti-hTfR antibody-hIDUA contained in each aqueous pharmaceutical composition was measured using the method described in Example 10.

[0159] [Table 4]

[0160] Figure 4 shows the measurement results of the number of particles contained in the aqueous pharmaceutical composition. It was found that when the concentration of polysorbate 80 was 0.25 mg / mL or higher, the number of particles with a particle size of less than 10 μm contained in the aqueous pharmaceutical composition after shaking was significantly lower compared to when the concentration of polysorbate 80 was 0.1 mg / mL or lower.

[0161] Next, we examine the measurement results for the content of the humanized anti-hTfR antibody-hIDUA polymer contained in the aqueous pharmaceutical composition. The measurement results are shown in Figure 5. The higher the concentration of polysorbate 80, the less polymer was found in the aqueous pharmaceutical composition after shaking. When the concentration of polysorbate 80 was 0.5 mg / mL or higher, almost no polymer was detected in the aqueous pharmaceutical composition.

[0162] Next, we examine the measurement results of the content of degradation products of humanized anti-hTfR antibody-hIDUA contained in the aqueous pharmaceutical composition. The measurement results are shown in Figure 6. Regardless of the concentration of polysorbate 80, almost no degradation products were observed in the aqueous pharmaceutical composition after shaking.

[0163] From Example 5 and the results described above, it was concluded that adding poloxamer 188 is effective in suppressing the generation of polymers of humanized anti-hTfR antibody-hIDUA in aqueous pharmaceutical compositions, and adding polysorbate 80 is effective in suppressing the increase of fine particles with a particle size of less than 10 μm. In other words, each surfactant is thought to have a different effect on the stability of aqueous pharmaceutical compositions.

[0164] [Example 7] Stability study of aqueous pharmaceutical composition containing humanized anti-hTfR antibody-hIDUA 3 Using the purified humanized anti-hTfR antibody-hIDUA product obtained in Example 4, five aqueous pharmaceutical compositions were prepared as shown in Table 5, each containing sodium chloride, citrate buffer, sucrose, poloxamer 188, polysorbate 80, and humanized anti-hTfR antibody-hIDUA, differing only in the concentration of polysorbate 80. Each of these five aqueous pharmaceutical compositions (Formulas J-N) was filled into a 2 mL glass vial, sealed, and shaken continuously at room temperature for 24 hours using a shaker (SR-2S, Taitec Co., Ltd.) (shaking speed: 240 reciprocations / min, amplitude: 40 mm). The number of particles per unit volume (particle size: 1-100 μm) contained in the aqueous pharmaceutical composition after shaking was measured using the method described in Example 8. The content of the humanized anti-hTfR antibody-hIDUA polymer contained in the aqueous pharmaceutical composition after shaking was measured using the method described in Example 9. Furthermore, the content of degradation products of humanized anti-hTfR antibody-hIDUA contained in each aqueous pharmaceutical composition was measured using the method described in Example 10.

[0165] [Table 5]

[0166] Figure 7 shows the measurement results of the number of particles contained in the aqueous pharmaceutical composition. It was found that when the concentration of polysorbate 80 was 0.05 mg / mL or higher, the number of particles with a particle size of less than 10 μm contained in the aqueous pharmaceutical composition after shaking was lower compared to when the concentration of polysorbate 80 was 0.025 mg / mL.

[0167] Next, we examine the measurement results for the content of the humanized anti-hTfR antibody-hIDUA polymer contained in the aqueous pharmaceutical composition. The measurement results are shown in Figure 8. Regardless of the concentration of polysorbate 80, almost no polymer was detected in the aqueous pharmaceutical composition.

[0168] Next, we examine the measurement results of the content of degradation products of humanized anti-hTfR antibody-hIDUA contained in the aqueous pharmaceutical composition. The measurement results are shown in Figure 9. Regardless of the concentration of polysorbate 80, almost no degradation products were observed in the aqueous pharmaceutical composition after shaking.

[0169] From the above results, it was found that adding both poloxamer 188 and polysorbate 80 is effective in simultaneously suppressing the generation of polymers of humanized anti-hTfR antibody-hIDUA and the increase in fine particles with a particle size of less than 10 μm in aqueous pharmaceutical compositions, and that adding polysorbate 80 is effective in suppressing the increase in fine particles with a particle size of less than 10 μm. However, since aqueous pharmaceutical compositions are to be administered to humans as drugs, it is preferable to set the concentrations of poloxamer 188 and polysorbate 80 to be low. For example, by setting the concentrations of poloxamer 188 and polysorbate 80 in the aqueous pharmaceutical composition to the ranges of 0.15 to 0.5 mg / mL and 0.025 to 0.125 mg / mL, respectively, a stable aqueous pharmaceutical composition can be obtained.

[0170] [Example 8] Measurement of the number of particles (particle size: 1-100 μm) contained in an aqueous pharmaceutical composition The number of particles contained in an aqueous pharmaceutical composition is measured using a flow imaging particle analyzer called FLOWCAM. TM The measurements were performed using a Fluid Imaging Technologies (Fluid Imaging Technologies) flow imaging particle analyzer. This device draws the sample solution into a flow cell perpendicular to the optical system using a syringe pump, and measures the number of particles in the sample solution by capturing images of the particles passing through the flow cell in real time. The measurements were performed with the particle size to be detected set to 1 to 100 μm.

[0171] [Example 9] Measurement of the content of humanized anti-hTfR antibody-hIDUA polymer contained in aqueous pharmaceutical composition The Shimadzu HPLC system LC-20A (Shimadzu Corporation) is equipped with the TSKgel G3000SW size exclusion chromatography column.XL A 5 μm column (7.8 mm diameter × 30 cm length, TOSOH Corporation) was set up. An absorbance spectrophotometer was also installed downstream of the column to allow continuous measurement of the absorbance of the effluent (measurement wavelength 215 nm). After equilibrating the column with 0.2 M sodium phosphate buffer solution, a sample solution containing 10 μg of humanized anti-hTfR antibody-hIDUA was loaded onto the column, and then 0.2 M sodium phosphate buffer solution was flowed at a flow rate of 0.6 mL / min. During this time, the absorbance of the effluent (measurement wavelength 215 nm) was measured to obtain the elution profile. From the obtained elution profile, the peak area of ​​the monomer of humanized anti-hTfR antibody-hIDUA (monomer peak area), the peak area of ​​the polymer of humanized anti-hTfR antibody-hIDUA that appears before this monomer peak (polymer peak area), and the peak area of ​​the degradation products of humanized anti-hTfR antibody-hIDUA that appears after this monomer peak (degradation product peak area) were determined. The polymer content (%) was calculated using the following formula. Polymer content (%) = {polymer peak area / (monomer peak area + polymer peak area + decomposition product peak area)} × 100

[0172] [Example 10] Measurement of the content of degradation products of humanized anti-hTfR antibody-hIDUA contained in aqueous pharmaceutical composition The Shimadzu HPLC system LC-20A (Shimadzu Corporation) is equipped with the TSKgel G3000SW size exclusion chromatography column. XLA 5 μm column (7.8 mm diameter × 30 cm length, TOSOH Corporation) was set up. An absorbance spectrophotometer was also installed downstream of the column to continuously measure the absorbance of the effluent (measurement wavelength 215 nm). After equilibrating the column with 0.2 M sodium phosphate buffer solution, a sample solution containing 10 μg of humanized anti-hTfR antibody-hIDUA was loaded onto the column, and then 0.2 M sodium phosphate buffer solution was flowed at a flow rate of 0.6 mL / min. During this time, the absorbance of the effluent (measurement wavelength 215 nm) was measured to obtain the elution profile. From the obtained elution profile, the peak area of ​​the monomer of humanized anti-hTfR antibody-hIDUA (monomer peak area), the peak area of ​​the polymer of humanized anti-hTfR antibody-hIDUA that appears before this monomer peak (polymer peak area), and the peak area of ​​the degradation products of humanized anti-hTfR antibody-hIDUA that appears after this monomer peak (degradation product peak area) were determined. The content (%) of the degradation products was calculated using the following formula. Decomposition product content (%) = {Decomposition product peak area / (Monomer peak area + Polymer peak area + Decomposition product peak area)} × 100

[0173] [Example 11] Formulation design of aqueous pharmaceutical composition Based on the results of the studies shown in Examples 5-7 above, a formulation example of an aqueous pharmaceutical composition containing humanized anti-hTfR antibody-hIDUA was designed, having the composition shown in Table 6 (Formulation O). This aqueous pharmaceutical composition is filled and sealed in glass or plastic vials, ampoules, or syringes in volumes of 1-10 mL and stored at low temperatures (e.g., 4°C). When filled and sealed in syringes, it becomes a pre-filled syringe type formulation.

[0174] [Table 6]

[0175] [Example 12] Long-term storage test of aqueous pharmaceutical composition Formula O, an aqueous pharmaceutical composition containing humanized anti-hTfR antibody-hIDUA, was filled into a glass vial at a temperature of 2-8 °C in the dark. During storage, the pH of the solution, the number of particles per unit volume (200 μL) (particle size: 1-100 μm), the content of the humanized anti-hTfR antibody-hIDUA polymer, and the content of the humanized anti-hTfR antibody-hIDUA degradation products were measured over time. The number of particles was measured by the method described in Example 8, the content of the humanized anti-hTfR antibody-hIDUA polymer was measured by the method described in Example 9, and the content of the humanized anti-hTfR antibody-hIDUA degradation products was measured by the method described in Example 10.

[0176] Table 7 shows the results of a long-term storage test of aqueous pharmaceutical composition formulation O containing humanized anti-hTfR antibody-hIDUA. At the start of storage, and after 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, and 36 months, the pH of the solution, the number of particles per unit volume (200 μL) (particle size: 1-100 μm), the content of the humanized anti-hTfR antibody-hIDUA polymer, and the content of the humanized anti-hTfR antibody-hIDUA degradation products were measured. During the storage period of the long-term storage test, there was almost no change in pH, particle number, polymer (%), and degradation product (%). These results indicate that aqueous pharmaceutical composition formulation O containing humanized anti-hTfR antibody-hIDUA is stable for at least 36 months at a temperature of 2-8°C in the dark, and is also predicted to remain stable even after 72 months from the start of storage.

[0177] [Table 7]

[0178] [Example 13] Production of freeze-dried pharmaceutical composition A 2.4 mL aqueous pharmaceutical composition formulation O containing the humanized anti-hTfR antibody-hIDUA was filled into a glass vial, partially sealed with a chlorinated butyl rubber stopper, and freeze-dried. During the freeze-drying process, the gas phase inside the vial was replaced with nitrogen before the rubber stopper was fully sealed. The resulting freeze-dried product formed a white mass in the vial. The resulting freeze-dried product can be restored to the original aqueous pharmaceutical composition by adding pure water to the vial and shaking to make a 2.4 mL solution.

[0179] [Example 14] Long-term storage test of freeze-dried pharmaceutical composition The lyophilized pharmaceutical composition formulation O containing humanized anti-hTfR antibody-hIDUA obtained in Example 13 was stored in the dark at a temperature of 2 to 8 °C. During the storage period, the pH of the solution obtained by dissolving the lyophilized pharmaceutical composition in pure water, the number of particles per unit volume (200 μL) (particle size: 1 to 100 μm), the content of the humanized anti-hTfR antibody-hIDUA polymer, and the content of the humanized anti-hTfR antibody-hIDUA degradation products were measured over time. The number of particles was measured by the method described in Example 8, the content of the humanized anti-hTfR antibody-hIDUA polymer was measured by the method described in Example 9, and the content of the humanized anti-hTfR antibody-hIDUA degradation products was measured by the method described in Example 10.

[0180] Table 8 shows the results of a long-term storage test of the lyophilized pharmaceutical composition of Formulation O containing humanized anti-hTfR antibody-hIDUA. At the start of storage, and at 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, and 36 months after the start of storage, the pH of the solution, the number of particles per unit volume (200 μL) (particle size: 1-100 μm), the content of the humanized anti-hTfR antibody-hIDUA polymer, and the content of the humanized anti-hTfR antibody-hIDUA degradation products were measured. During the storage period of the long-term storage test, there was almost no change in pH, particle number, polymer (%), and degradation product (%). These results indicate that the lyophilized pharmaceutical composition Formulation O containing humanized anti-hTfR antibody-hIDUA is stable for at least 36 months at a temperature of 2-8°C in the dark, and is also predicted to be stable even after 72 months from the start of storage.

[0181] [Table 8] [Industrial applicability]

[0182] According to the present invention, aqueous pharmaceutical compositions or freeze-dried pharmaceutical compositions containing physiologically active proteins as active ingredients can be supplied to the market. [Sequence Listing Free Text]

[0183] 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 of linker example 4 Sequence ID 5: Amino acid sequence of the human transferrin receptor Sequence ID 6: Amino acid sequence 1 of human IDUA Sequence ID 7: Amino acid sequence 2 of human IDUA SEQ ID NO: 8: Amino acid sequence 1 of the light chain CDR1 of the anti-hTfR antibody SEQ ID NO: 9: Amino acid sequence 2 of the light chain CDR1 of the anti-hTfR antibody SEQ ID NO: 10: Amino acid sequence 1 of the light chain CDR2 of the anti-hTfR antibody SEQ ID NO: 11: Amino acid sequence 2 of the light chain CDR2 of the anti-hTfR antibody SEQ ID NO: 12: Amino acid sequence 1 of the light chain CDR3 of the anti-hTfR antibody SEQ ID NO: 13: Amino acid sequence 1 of the heavy chain CDR1 of the anti-hTfR antibody SEQ ID NO: 14: Amino acid sequence 2 of the heavy chain CDR1 of the anti-hTfR antibody SEQ ID NO: 15: Amino acid sequence 1 of the heavy chain CDR2 of the anti-hTfR antibody SEQ ID NO: 16: Amino acid sequence 2 of the heavy chain CDR2 of the anti-hTfR antibody SEQ ID NO: 17: Amino acid sequence 1 of the heavy chain CDR3 of the anti-hTfR antibody SEQ ID NO: 18: Amino acid sequence 2 of the heavy chain CDR3 of the anti-hTfR antibody SEQ ID NO: 19: Amino acid sequence of heavy chain framework region 3 of anti-hTfR antibody SEQ ID NO: 20: Amino acid sequence of the variable region of the light chain of an anti-hTfR antibody SEQ ID NO: 21: Amino acid sequence of the variable region of the heavy chain of an anti-hTfR antibody Sequence ID 22: Amino acid sequence of the light chain of the anti-hTfR antibody SEQ ID NO: 23: Amino acid sequence of the Fab heavy chain of the anti-hTfR antibody Sequence ID 24: Primer Hyg-Sfi5', synthetic sequence Sequence ID 25: Primer Hyg-BstX3', synthetic sequence Sequence ID 26: Base sequence and synthetic sequence encoding the amino acid sequence of the light chain of the anti-hTfR antibody. Sequence ID 27: Amino acid sequence of the fusion protein of the Fab heavy chain of the humanized anti-hTfR antibody and human IDUA. Sequence ID 28: Base sequence and synthetic sequence containing the gene encoding the amino acid sequence of the fusion protein of the Fab heavy chain of the humanized anti-hTfR antibody and human IDUA. Sequence ID 29: Primer IRES5', synthetic sequence Sequence ID 30: Primer IRES3', synthetic sequence Sequence ID 31: Primer mPGKP5', synthetic sequence Sequence ID 32: Primer mPGKP3', synthetic sequence Sequence ID 33: Primer GS5', synthetic sequence Sequence ID 34: Primer GS3', synthetic sequence Sequence ID 35: Primer puro5', synthetic sequence Sequence ID 36: Primer puro3', synthetic sequence Sequence ID 37: Primer SV40polyA5', synthetic sequence Sequence ID 38: Primer SV40polyA3', synthetic sequence Sequence ID 39: Primer mIRES-GS5', synthetic sequence Sequence ID 40: Primer mIRES-GS3', synthetic sequence Sequence ID 41: CMVE-EF-1αp-IFNβMAR, synthetic sequence Sequence ID 42: IRES-HygroR-mPGKpA, synthetic sequence

Claims

1. A fusion protein of an antibody and a lysosomal enzyme, a neutral salt, a disaccharide, a buffer, and two types of nonionic surfactants, polysorbate and poloxamer, The antibody is a humanized anti-human transferrin receptor (hTfR) antibody which is a Fab antibody, the human lysosomal enzyme is human α-L-iduronidase, and the fusion protein is represented by the following (a) or (b): (a) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:22; and The heavy chain of the antibody is bound to human α-L-iduronidase at its C-terminus via the amino acid sequence shown in SEQ ID NO:4, thereby forming the amino acid sequence shown in SEQ ID NO:27; (b) the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:22; and The heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:23, and the heavy chain is bound to human α-L-iduronidase having the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6 at its C-terminus via the amino acid sequence shown in SEQ ID NO:

4.

2. 2. The aqueous pharmaceutical composition or lyophilized pharmaceutical composition of claim 1, wherein the polysorbate is polysorbate 80 and the poloxamer is polyoxyethylene (160) polyoxypropylene (30) glycol.

3. The aqueous pharmaceutical composition of claim 1, 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 0.105 to 0.6 mg / mL.

4. The aqueous pharmaceutical composition of claim 1, 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 0.1 to 0.5 mg / mL.

5. The aqueous pharmaceutical composition of claim 1, 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 0.15 to 0.45 mg / mL.

6. The aqueous pharmaceutical composition of claim 3, wherein the neutral salt is sodium chloride, the disaccharide is sucrose, the buffer is a citrate buffer, 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 3 to 30 mM, and the pH is 4.5 to 6.

5.

7. The aqueous pharmaceutical composition of claim 3, wherein the neutral salt is sodium chloride, the disaccharide is sucrose, the buffer is a citrate buffer, the concentration of the neutral salt is 0.5 to 1.0 mg / mL, the concentration of the disaccharide is 55 to 95 mg / mL, the concentration of the buffer is 10 to 30 mM, and the pH is 5.0 to 6.

0.

8. The aqueous pharmaceutical composition of claim 3, wherein the neutral salt is sodium chloride, the disaccharide is sucrose, the buffer is a citrate buffer, 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, and the pH is 5.2 to 5.

8.

9. 7. The aqueous pharmaceutical composition according to claim 6, wherein the polymer content and the decomposition product content are 0.5% or less and 1% or less, respectively, after storage for 36 months in a dark place at 2 to 8°C.

10. A freeze-dried pharmaceutical composition, which is obtained by freeze-drying the aqueous pharmaceutical composition described in any one of claims 3 to 9.

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