A novel anti-human transferrin receptor antibody that crosses the blood-brain barrier

Abstract

To provide an anti-TfR antibody that can be used in combination with compounds (proteins, low molecular weight compounds, etc.) that are to be administered into the blood to function in the central nervous system, so that the compounds can pass through the blood-brain barrier. [Solution] An anti-human transferrin receptor antibody, wherein in the variable region of the heavy chain, (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and (c) CDR3 comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

Description

[Technical Field]

[0001] The present invention relates to anti-human transferrin receptor antibodies that are used in combination with compounds (proteins, low molecular weight compounds, etc.) that are administered into the blood to function in the central nervous system (CNS) so that the compounds can pass through the blood-brain barrier, as well as methods for producing and using the same. [Background technology]

[0002] Capillaries supply blood to most brain tissues, except for a few areas including the circumventricular organs (e.g., the pineal gland, pituitary gland, and area postrema), unlike capillaries in other tissues such as muscle. The endothelial cells that make up the capillaries are tightly connected by strong intercellular junctions. This prevents passive transport of substances from blood to the brain, and, although there are exceptions, only highly lipid-soluble substances or substances with small molecular weights (less than 200-500 daltons) and electrically neutral at near physiological pH are able to pass through capillaries into the brain. This mechanism, which restricts the exchange of substances between blood and brain tissue fluid via the capillary endothelium in the brain, is called the blood-brain barrier (BBB). The BBB also restricts the exchange of substances between the blood and tissue fluid in the central nervous system, including the brain and spinal cord, and not just the brain.

[0003] The existence of the blood-brain barrier allows the majority of cells in the central nervous system to maintain their biochemical homeostasis without being affected by fluctuations in the concentrations of hormones, lymphokines, and other substances in the blood.

[0004] However, the existence of the blood-brain barrier poses a problem in drug development. For example, enzyme replacement therapy using intravenous recombinant α-L-iduronidase is used to treat mucopolysaccharidosis type 1 (Hurler syndrome), a genetic metabolic disorder caused by α-L-iduronidase deficiency. However, this is ineffective for the significant central nervous system (CNS) abnormalities observed in Hurler syndrome because the enzyme cannot cross the blood-brain barrier.

[0005] Various methods have been developed to allow macromolecular substances such as proteins to act in the central nervous system to pass through the blood-brain barrier. For example, in the case of nerve growth factor, attempts have been made to allow it to pass through the blood-brain barrier by fusing liposomes encapsulating it with the cell membrane of endothelial cells in brain capillaries, but this has not yet been put to practical use (Non-Patent Document 1). In the case of α-L-iduronidase, attempts have been made to increase the passive transport of the enzyme across the blood-brain barrier by increasing the amount of enzyme administered per dose and thereby increasing its blood concentration, and this method has been shown to alleviate central nervous system (CNS) abnormalities using an animal model of Hurler syndrome (Non-Patent Document 2).

[0006] Attempts have also been made to bypass the blood-brain barrier and administer high molecular weight substances directly into the spinal cavity or brain. For example, a method of administering human α-L-iduronidase into the spinal cavity of patients with Hurler syndrome (mucopolysaccharidosis type I) (Patent Document 1), a method of administering human acid sphingomyelinase into the ventricles of patients with Niemann-Pick disease (Patent Document 2), and a method of administering iduronate 2-sulfatase (I2S) into the ventricles of an animal model of Hunter syndrome (Patent Document 3) have been reported. While these methods are thought to ensure that drugs can act on the central nervous system, they have the problem of being extremely invasive.

[0007] As a method for delivering high molecular weight substances through the blood-brain barrier to the brain, various methods have been reported in which high molecular weight substances are modified so that they have affinity for membrane proteins present on the endothelial cells of brain capillaries.Membrane proteins present on the endothelial cells of brain capillaries include receptors for compounds such as insulin, transferrin, insulin-like growth factors (IGF-I, IGF-II), LDL, and leptin.

[0008] For example, a technique has been reported in which nerve growth factor (NGF) is synthesized in the form of a fusion protein with insulin, and this fusion protein is allowed to pass through the blood-brain barrier via binding to the insulin receptor (Patent Documents 4 to 6). Another technique has been reported in which nerve growth factor (NGF) is synthesized in the form of a fusion protein with an anti-insulin receptor antibody, and this fusion protein is allowed to pass through the blood-brain barrier via binding to the insulin receptor (Patent Documents 4 and 7). Another technique has been reported in which nerve growth factor (NGF) is synthesized in the form of a fusion protein with transferrin, and this fusion protein is allowed to pass through the blood-brain barrier via binding to the transferrin receptor (TfR) (Patent Document 8). Another technique has been reported in which nerve growth factor (NGF) is synthesized in the form of a fusion protein with an anti-transferrin receptor antibody (anti-TfR antibody), and this fusion protein is allowed to pass through the blood-brain barrier via binding to the TfR (Patent Documents 4 and 9).

[0009] Looking further at technologies using anti-TfR antibodies, it has been reported that single-chain antibodies can be used in technologies that allow drugs to pass through the blood-brain barrier by binding to anti-TfR antibodies (Non-Patent Document 3). It has also been reported that anti-hTfR antibodies with a relatively large dissociation constant with hTfR (low-affinity anti-hTfR antibodies) can be suitably used in technologies that allow drugs to pass through the blood-brain barrier (Patent Documents 10 and 11, Non-Patent Document 4). Furthermore, it has been reported that anti-TfR antibodies whose affinity for hTfR changes depending on pH can be used as carriers for drugs to pass through the blood-brain barrier (Patent Document 12, Non-Patent Document 5). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Special Publication No. 2007-504166 [Patent Document 2] Special Publication No. 2009-525963 [Patent Document 3] Japanese Patent Application Laid-Open No. 2012-62312 [Patent Document 4] U.S. Patent No. 5,154,924 [Patent Document 5] Japanese Patent Application Laid-Open No. 2011-144178 [Patent Document 6] US Patent Publication No. 2004 / 0101904 [Patent Document 7] Special Publication No. 2006-511516 [Patent Document 8] Japanese Patent Application Laid-Open No. H06-228199 [Patent Document 9] U.S. Patent No. 5,977,307 [Patent Document 10] WO2012 / 075037 [Patent Document 11] WO2013 / 177062 [Patent Document 12] WO2012 / 143379 [Non-patent literature]

[0011] [Non-Patent Document 1] Xie Y. et al., J Control Release. 105. 106-19(2005) [Non-patent document 2] Ou L. et al., Mol Genet Metab. 111. 116-22(2014) [Non-patent document 3] Li JY. Protein Engineering. 12. 787-96 (1999) [Non-patent document 4] Bien-Ly N. et al., J Exp Med. 211. 233-44 (2014) [Non-patent document 5] Sada H. PLoS ONE. 9. E96340 (2014) Summary of the Invention [Problem to be solved by the invention]

[0012] Against the above background, the object of the present invention is to provide an anti-TfR antibody that can be used in combination with compounds (proteins, low molecular weight compounds, etc.) that are to be administered into the blood to function in the central nervous system, so that the compounds can pass through the blood-brain barrier, as well as methods for producing and using the same. [Means for solving the problem]

[0013] In research aimed at the above-mentioned object, the present inventors have conducted extensive research and have found that an anti-human transferrin receptor antibody (anti-hTfR antibody) that recognizes the extracellular domain of hTfR, obtained by the antibody production method described in detail in this specification, efficiently crosses the blood-brain barrier when administered in vivo, thereby completing the present invention. That is, the present invention provides the following.

[0014] 1. An anti-human transferrin receptor antibody, wherein the antibody comprises, in the variable region of the heavy chain: (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and (c) An antibody in which CDR3 comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. 2. The antibody of 1 above, wherein framework region 3 of the heavy chain comprises the amino acid sequence of SEQ ID NO: 64. 3. The antibody according to 1 or 2 above, wherein in the variable region of the heavy chain: In place of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 of CDR2, an amino acid sequence having 80% or more homology thereto is included; and An antibody comprising, instead of the amino acid sequence of CDR3 of SEQ ID NO: 15 or SEQ ID NO: 16, an amino acid sequence having 80% or more homology thereto. 4. The antibody according to 1 or 2 above, wherein in the variable region of the heavy chain: In place of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 of CDR2, an amino acid sequence having 90% or more homology thereto is included; and An antibody comprising, instead of the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 of CDR3, an amino acid sequence having 90% or more homology thereto. 5. The antibody according to 1 or 2 above, wherein in the variable region of the heavy chain: (a) SEQ ID NO: 62 or SEQ ID NO: 63 for CDR1, (b) SEQ ID NO: 13 or SEQ ID NO: 14 for CDR2, (c) SEQ ID NO: 15 or SEQ ID NO: 16 for CDR3, and (d) SEQ ID NO: 64 for framework region 3; and wherein at least one of the amino acid sequences is replaced by an amino acid sequence modified by substitution, deletion, or addition of 1 to 5 amino acids, Here, for CDR1, the methionine located at the fifth position from the N-terminus of the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63 is located at the same position in the modified amino acid sequence, and An antibody in which, for framework region 3, the leucine located at the 17th position from the N-terminus of SEQ ID NO: 64 is in the same position in the modified amino acid sequence. 6. The antibody according to 1 or 2 above, wherein in the variable region of the heavy chain: (a) SEQ ID NO: 62 or SEQ ID NO: 63 for CDR1, (b) SEQ ID NO: 13 or SEQ ID NO: 14 for CDR2, (c) SEQ ID NO: 15 or SEQ ID NO: 16 for CDR3, and (d) SEQ ID NO: 64 for framework region 3; and wherein at least one of the amino acid sequences is replaced by an amino acid sequence modified by substitution, deletion, or addition of 1 to 3 amino acids, Here, for CDR1, the methionine located at the fifth position from the N-terminus of the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63 is located at the same position in the modified amino acid sequence, and An antibody in which, for framework region 3, the leucine located at the 17th position from the N-terminus of SEQ ID NO: 64 is in the same position in the modified amino acid sequence. 7. The antibody according to 2 above, wherein the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 65. 8. An antibody according to 7 above, wherein in the variable region of the heavy chain, in the portions other than the amino acid sequences of SEQ ID NO: 62 or SEQ ID NO: 63 in CDR1 and SEQ ID NO: 64 in framework region 3, an amino acid sequence having 80% or more homology with said portions is included instead of said portions. 9. An antibody according to 7 above, wherein in the variable region of the heavy chain, in the portions other than the amino acid sequences of SEQ ID NO: 62 or SEQ ID NO: 63 in CDR1 and SEQ ID NO: 64 in framework region 3, an amino acid sequence having 90% or more homology with said portions is included instead of said portions. 10. The antibody according to 7 above, which comprises an amino acid sequence modified by substitution, deletion or addition of 1 to 5 amino acids in place of the amino acid sequence constituting the variable region of the heavy chain, Here, for CDR1, the methionine located at the fifth position from the N-terminus of the amino acid sequence of SEQ ID NO: 63 is in the same position in the modified amino acid sequence, and for framework region 3, the leucine located at the 17th position from the N-terminus of SEQ ID NO: 64 is in the same position in the modified amino acid sequence. 11. The antibody according to 7 above, which comprises an amino acid sequence modified by substitution, deletion, or addition of 1 to 3 amino acids in place of the amino acid sequence constituting the variable region of the heavy chain, Here, for CDR1, the methionine located at the fifth position from the N-terminus of the amino acid sequence of SEQ ID NO: 63 is in the same position in the modified amino acid sequence, and for framework region 3, the leucine located at the 17th position from the N-terminus of SEQ ID NO: 64 is in the same position in the modified amino acid sequence. 12. The antibody according to 7 above, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68. 13. An antibody according to claim 12, wherein the heavy chain contains, in place of the portions other than the amino acid sequences of SEQ ID NO: 62 or SEQ ID NO: 63 in CDR1 and SEQ ID NO: 64 in framework region 3, an amino acid sequence having 80% or more homology to said portions. 14. An antibody according to claim 12, wherein the heavy chain contains, in place of the portions other than the amino acid sequences of SEQ ID NO: 62 or SEQ ID NO: 63 in CDR1 and SEQ ID NO: 64 in framework region 3, an amino acid sequence having 90% or more homology to said portions. 15. The antibody according to claim 12, wherein the heavy chain comprises an amino acid sequence modified by substitution, deletion, or addition of 1 to 5 amino acids in place of the amino acid sequence constituting the heavy chain, Here, for CDR1, the methionine located at the fifth position from the N-terminus of the amino acid sequence of SEQ ID NO: 63 is in the same position in the modified amino acid sequence, and for framework region 3, the leucine located at the 17th position from the N-terminus of SEQ ID NO: 64 is in the same position in the modified amino acid sequence. 16. The antibody according to claim 12, wherein the heavy chain comprises an amino acid sequence modified by substitution, deletion, or addition of 1 to 3 amino acids, instead of the amino acid sequence constituting the heavy chain, Here, for CDR1, the methionine located at the fifth position from the N-terminus of the amino acid sequence of SEQ ID NO: 63 is in the same position in the modified amino acid sequence, and for framework region 3, the leucine located at the 17th position from the N-terminus of SEQ ID NO: 64 is in the same position in the modified amino acid sequence. 17. Any one of the antibodies 1 to 16 above, wherein in the light chain variable region of the antibody: (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence Lys-Val-Ser, and (c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10, antibody. 18. The antibody according to 17 above, wherein the variable region of the light chain comprises: (a) comprising an amino acid sequence having 80% or more homology thereto instead of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 of CDR1, (b) comprising an amino acid sequence having 80% or more homology thereto in place of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9 of CDR2, or the amino acid sequence Lys-Val-Ser; and (c) comprising an amino acid sequence having 80% or more homology thereto instead of the amino acid sequence of SEQ ID NO: 10 of CDR3, antibody. 19. The antibody according to claim 17, wherein the variable region of the light chain comprises: (a) comprising an amino acid sequence having 90% or more homology thereto instead of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 of CDR1, (b) comprising an amino acid sequence having 90% or more homology thereto in place of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9 of CDR2, or the amino acid sequence Lys-Val-Ser; and (c) comprising an amino acid sequence having 90% or more homology thereto instead of the amino acid sequence of SEQ ID NO: 10 of CDR3, antibody. 20. The antibody according to claim 17, wherein the variable region of the light chain comprises: (a) SEQ ID NO: 6 or SEQ ID NO: 7 for CDR1, (b) SEQ ID NO: 8 or SEQ ID NO: 9 for CDR2, or the amino acid sequence Lys-Val-Ser, and (c) SEQ ID NO: 10 for CDR3 In place of at least one of the amino acid sequences, an amino acid sequence is modified by substitution, deletion, or addition of 1 to 5 amino acids. antibody. 21. The antibody according to claim 17, wherein the variable region of the light chain comprises: (a) SEQ ID NO: 6 or SEQ ID NO: 7 for CDR1, (b) SEQ ID NO: 8 or SEQ ID NO: 9 for CDR2, or the amino acid sequence Lys-Val-Ser, and (c) SEQ ID NO: 10 for CDR3 In place of at least one of the amino acid sequences, an amino acid sequence is modified by substitution, deletion, or addition of 1 to 3 amino acids. antibody. 22. Any one of the antibodies 1 to 16 above, An antibody, wherein the variable region of the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22. 23. An antibody according to 22 above, which comprises, in place of the amino acid sequence of the variable region of the light chain, an amino acid sequence having 80% or more homology thereto. 24. An antibody according to 22 above, which comprises, in place of the amino acid sequence of the variable region of the light chain, an amino acid sequence having 90% or more homology thereto. 25. An antibody according to claim 22, which comprises an amino acid sequence in which the amino acid sequence of the variable region of the light chain has been modified by substitution, deletion or addition of 1 to 5 amino acids. 26. An antibody according to claim 22, which comprises an amino acid sequence in which the amino acid sequence of the variable region of the light chain has been modified by substitution, deletion or addition of 1 to 3 amino acids. 27. An antibody according to any one of 1 to 16 above, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 29. 28. An antibody according to 27 above, which comprises, in place of the amino acid sequence of the light chain, an amino acid sequence having 80% or more homology thereto. 29. An antibody according to 27 above, which comprises, in place of the amino acid sequence of the light chain, an amino acid sequence having 90% or more homology thereto. 30. An antibody according to 27 above, which comprises an amino acid sequence which has been modified by substitution, deletion or addition of 1 to 5 amino acids in place of the amino acid sequence of the light chain. 31. An antibody according to 27 above, which comprises an amino acid sequence which has been modified by substitution, deletion or addition of 1 to 3 amino acids in place of the amino acid sequence of the light chain. 32. An antibody according to any one of 1 to 31 above, which has affinity for both the extracellular domain of the human transferrin receptor and the extracellular domain of the monkey transferrin receptor. 33. The antibody according to claim 32, wherein the dissociation constant with the extracellular domain of the human transferrin receptor is 1×10 -10 M or less, and the dissociation constant with the extracellular domain of the monkey transferrin receptor is 1X10 -9 M or less, antibodies. 34. Any one of the antibodies 1 to 33 above, which is a Fab antibody, a F(ab')2 antibody, or a F(ab')2 antibody. 35. An anti-human transferrin receptor antibody according to any one of 1 to 33 above, which is a single-chain antibody selected from the group consisting of scFab, scF(ab'), scF(ab')2 and scFv. 36. The antibody according to 35 above, wherein the light chain and heavy chain are linked via a linker sequence. 37. The antibody according to 36 above, wherein the heavy chain is linked to the C-terminus of the light chain via a linker sequence. 38. The antibody according to 35 above, wherein the light chain is linked to the C-terminus of the heavy chain via a linker sequence. 39. An antibody according to any one of 36 to 38 above, wherein the linker sequence consists of 8 to 50 amino acid residues. 40. The antibody of 39 above, wherein the linker sequence is selected from the group consisting of the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, each of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO: 3, and an amino acid sequence consisting of 1 to 10 consecutive amino acids of these amino acid sequences. 41. A fusion protein of an anti-human transferrin receptor antibody and another protein (A), the anti-human transferrin receptor antibody is any one of the antibodies 1 to 40 above, A fusion protein in which the protein (A) is bound to the C-terminal or N-terminal side of the light chain of the antibody. 42. The fusion protein according to 41 above, wherein said protein (A) is bound to the C-terminal or N-terminal side of said light chain directly or via a linker. 43. The fusion protein according to 41 or 42 above, wherein said protein (A) is bound via a linker to the C-terminal or N-terminal side of said light chain. 44. The fusion protein according to 43 above, wherein the linker is a peptide consisting of 1 to 50 amino acid residues. 45. The fusion protein of 44 above, wherein the linker is a peptide comprising an amino acid sequence selected from the group consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and amino acid sequences consisting of 1 to 10 consecutive amino acids of these amino acid sequences. 46. ​​A fusion protein of an anti-human transferrin receptor antibody and another protein (A), the anti-human transferrin receptor antibody is any one of the antibodies 1 to 40 above, A fusion protein in which the protein (A) is bound to the C-terminal or N-terminal side of the heavy chain of the antibody. 47. The fusion protein according to 46 above, wherein said protein (A) is bound to the C-terminal or N-terminal side of said heavy chain directly or via a linker. 48. The fusion protein according to 46 or 47 above, wherein said protein (A) is bound via a linker to the C-terminal or N-terminal side of said heavy chain. 49. The fusion protein according to 48 above, wherein the linker sequence is a peptide consisting of 1 to 50 amino acid residues. 50. The fusion protein of 49 above, wherein the linker is a peptide comprising an amino acid sequence selected from the group consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and amino acid sequences consisting of 1 to 10 consecutive amino acids of these amino acid sequences. 51. A fusion protein according to any one of 41 to 50 above, wherein the protein (A) is a protein of human origin. 52. The fusion protein according to any one of 41 to 51 above, wherein the protein (A) is selected from the group consisting of nerve growth factor (NGF), lysosomal enzyme, ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, granulocyte colony activator (GCM), and erythropoietin. A fusion protein of any one of 39 to 49 above, selected from the group consisting of macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), various cytokines, tumor necrosis factor alpha receptor (TNF-α receptor), PD-1 ligand, PD-L1, PD-L2, an enzyme having the activity of degrading beta-amyloid, anti-beta-amyloid antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-HER2 antibody, anti-TNF-α antibody, anti-CTLA-4 antibody, and other antibody pharmaceuticals. 53. The protein (A) is a lysosomal enzyme, and the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, human acid α-glucosidase, glucocerebrosidase, β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. A fusion protein selected from the group consisting of A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2. 54. A fusion protein according to any one of 41 to 51 above, wherein said protein (A) is any one of human iduronate-2-sulfatase, human acid α-glucosidase, and human α-L-iduronidase. 55. The fusion protein according to 51 above, wherein the protein (A) is human acid α-glucosidase; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The heavy chain of the antibody binds to human acid alpha-glucosidase at its C-terminus via the amino acid sequence Gly-Ser, thereby forming the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58. fusion protein. 56. The fusion protein according to 51 above, wherein the protein (A) is human acid α-glucosidase; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68, and the heavy chain is bound to human acid α-glucosidase having the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 56 at its C-terminus via the amino acid sequence Gly-Ser. fusion protein. 57. The fusion protein according to 51 above, wherein the protein (A) is human acid α-glucosidase and the antibody is a Fab antibody; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The heavy chain of the antibody binds to human acid alpha-glucosidase at its C-terminus via an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO: 3, thereby forming the amino acid sequence of SEQ ID NO: 89. fusion protein. 58. The fusion protein according to 51 above, wherein the protein (A) is human acid α-glucosidase and the antibody is a Fab antibody; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 61, and the heavy chain is bound to human acid α-glucosidase having the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 56 at its C-terminus via an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO: 3. fusion protein. 59. The fusion protein according to 51 above, wherein the protein (A) is human α-L-iduronidase and the antibody is a Fab antibody; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The heavy chain of the antibody binds to human α-L-iduronidase at its C-terminus via an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO: 3, thereby forming the amino acid sequence of SEQ ID NO: 93. fusion protein. 60. The fusion protein according to 51 above, wherein the protein (A) is human α-L-iduronidase and the antibody is a Fab antibody; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 61, and the heavy chain is bound to human α-L-iduronidase having the amino acid sequence of SEQ ID NO: 75 or SEQ ID NO: 76 at its C-terminus via an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO: 3. fusion protein. 61. The fusion protein according to 41 above, wherein a human IgG Fc region or a part thereof is introduced between said protein (A) and said antibody. 62. A fusion protein according to 61 above, in which a human IgG Fc region is bound to the C-terminus of protein (A) directly or via a linker sequence, and the heavy chain or light chain of the antibody is bound to the C-terminus of the human IgG Fc region directly or via a linker sequence. 63. The fusion protein according to 61 or 62 above, wherein the human IgG Fc region comprises the amino acid sequence of SEQ ID NO: 70. 64. A fusion protein according to claim 63, in which a human IgG Fc region is linked via a linker sequence to a Fab heavy chain consisting of the amino acid sequence of SEQ ID NO: 61, thereby forming the amino acid sequence of SEQ ID NO: 71. 65. A DNA fragment encoding the amino acid sequence of any one of the anti-human transferrin receptor antibodies set forth in 1 to 40 above. 66. A DNA fragment encoding the amino acid sequence of any one of the fusion proteins 41 to 64 above. 67. An expression vector incorporating the DNA fragment of 65 or 66 above. 68. A mammalian cell transformed with the expression vector of 67 above. 69. An anti-human transferrin receptor antibody-pharmacologically active substance conjugate in which a low molecular weight pharmacologically active substance that is capable of passing through the blood-brain barrier and exerting its function in the brain is bound to either the light chain and / or the heavy chain of any of the anti-human transferrin receptor antibodies 1 to 40 above. 70. The anti-human transferrin receptor antibody-pharmacologically active substance conjugate of claim 69, wherein the pharmacologically active substance is any one selected from the group consisting of an anticancer agent, an agent for treating Alzheimer's disease, an agent for treating Parkinson's disease, an agent for treating Huntington's disease, an agent for treating schizophrenia, an agent for treating depression, an agent for treating multiple sclerosis, an agent for treating amyotrophic lateral sclerosis, an agent for treating tumors of the central nervous system including brain tumors, an agent for treating lysosomal diseases associated with encephalopathy, an agent for treating glycogen storage disease, an agent for treating muscular dystrophy, an agent for treating cerebral ischemia, an agent for treating prion disease, an agent for treating traumatic central nervous system disorders, an agent for treating viral and bacterial central nervous system diseases, an agent for use in recovery after brain surgery, an agent for recovery after spinal surgery, siRNA, antisense DNA, and a peptide. 71. Use of any of the anti-human transferrin receptor antibodies set forth in 1 to 40 above, for allowing a protein (A) or a low-molecular-weight pharmacologically active substance to pass through the blood-brain barrier and exert its function in the brain. 72. Use of any of the anti-human transferrin receptor antibodies set forth in 1 to 40 above, by binding to a physiologically active protein or pharmacologically active low molecular weight compound molecule for the manufacture of a drug for administration into the bloodstream for the treatment of a disease state of the central nervous system. 73. A method for treating a disease of the central nervous system, comprising administering into the blood of a patient having the disease a therapeutically effective amount of a physiologically active protein or pharmacologically active low molecular weight compound for the disease in the form of a conjugate with an anti-human transferrin receptor antibody molecule of any one of 1 to 40 above. 74. Use of any of the fusion proteins set forth in 52 to 58 above to allow human acid α-glucosidase to pass through the blood-brain barrier and exert its function in the brain. 75. Use of any of the fusion proteins set forth in 52 to 58 above for the manufacture of a drug for administration into the blood for the treatment of a disease state of the central nervous system associated with Pompe disease. 76. A method for treating a central nervous system disorder associated with Pompe disease, comprising administering a therapeutically effective amount of any of the fusion proteins set forth in 52 to 58 above into the blood of a patient having the disease. 77. Use of the fusion protein according to any one of items 52 to 54, 59, or 60 above, to allow human acid α-L-iduronidase to pass through the blood-brain barrier and exert its function in the brain. 78. Use of any of the fusion proteins set forth in 52 to 54, 59, or 60 above for the manufacture of a drug for administration into the blood for the treatment of a disease state of the central nervous system associated with Hurler syndrome or Hurler-Scheie syndrome. 79. A method for treating a central nervous system disease associated with Hurler syndrome or Hurler-Scheie syndrome, comprising administering a therapeutically effective amount of any of the fusion proteins set forth in 52 to 54, 59, or 60 above into the blood of a patient having the disease. [Effects of the Invention]

[0015] The present invention makes it possible to convert various substances that have physiological or pharmacological activity, such as proteins or low-molecular-weight substances, into a form that can pass through the blood-brain barrier, which has previously been unavailable for administration into the blood, because they are unable to pass through the blood-brain barrier at all, and therefore can be used as new drugs for administration into the blood for the treatment of disease states of the central nervous system. [Brief explanation of the drawings]

[0016] [Figure 1] Photographs showing the results of immunohistochemical staining of the cerebral cortex of cynomolgus monkeys after a single intravenous administration of anti-hTfR antibody: (a) no anti-hTfR antibody administered, (b) anti-hTfR antibody No. 3 administered. The bar at the bottom right of each photograph is a gauge representing 50 μm. [Figure 2] Immunohistochemical staining of the hippocampus of cynomolgus monkeys after a single intravenous administration of anti-hTfR antibody (a) without anti-hTfR antibody administration, and (b) with anti-hTfR antibody No. 3 administration. The bar at the bottom right of each photograph is a gauge representing 50 μm. [Figure 3] Photographs showing the results of immunohistochemical staining of the cerebellum of a cynomolgus monkey after a single intravenous administration of anti-hTfR antibody: (a) no anti-hTfR antibody administered, (b) anti-hTfR antibody No. 3 administered. The bar at the bottom right of each photograph is a gauge representing 50 μm. [Figure 4]This graph shows the accumulation of humanized anti-hTfR antibodies in various organs of cynomolgus monkeys other than the brain after a single intravenous administration. The vertical axis shows the amount of humanized anti-hTfR antibody per wet weight of each organ (μg / g wet weight). The white bars show, from left to right, the accumulation in each organ of monkeys administered humanized anti-hTfR antibody No. 3, humanized anti-hTfR antibody No. 3-2, humanized anti-hTfR antibody No. 3 (IgG4), and humanized anti-hTfR antibody No. 3-2 (IgG4), respectively. The black bars show the accumulation in each organ of monkeys administered trastuzumab (Herceptin™). ND means not determined. [Figure 5] Photographs showing the results of immunohistochemical staining of the cerebral cortex of cynomolgus monkeys for humanized anti-hTfR antibodies after a single intravenous administration: (a) Herceptin, (b) humanized anti-hTfR antibody No. 3, (c) humanized anti-hTfR antibody No. 3-2, (d) humanized anti-hTfR antibody No. 3 (IgG4), and (e) humanized anti-hTfR antibody No. 3-2 (IgG4). The bar at the bottom right of each photograph is a gauge representing 20 μm. [Figure 6] Immunohistochemical staining of the hippocampus of cynomolgus monkeys after a single intravenous administration of humanized anti-hTfR antibodies: (a) Herceptin, (b) humanized anti-hTfR antibody No. 3, (c) humanized anti-hTfR antibody No. 3-2, (d) humanized anti-hTfR antibody No. 3 (IgG4), and (e) humanized anti-hTfR antibody No. 3-2 (IgG4). The bar at the bottom right of each photograph is a gauge representing 20 μm. [Figure 7] Immunohistochemical staining of the cerebellum of cynomolgus monkeys after a single intravenous administration of humanized anti-hTfR antibodies: (a) Herceptin, (b) humanized anti-hTfR antibody No. 3, (c) humanized anti-hTfR antibody No. 3-2, (d) humanized anti-hTfR antibody No. 3 (IgG4), and (e) humanized anti-hTfR antibody No. 3-2 (IgG4). The bar at the bottom right of each photograph is a gauge representing 20 μm. [Figure 8]Immunohistochemical staining of the medulla oblongata of cynomolgus monkeys after a single intravenous administration of (a) Herceptin, (b) humanized anti-hTfR antibody No. 3, (c) humanized anti-hTfR antibody No. 3-2, (d) humanized anti-hTfR antibody No. 3 (IgG4), and (e) humanized anti-hTfR antibody No. 3-2 (IgG4). The bar at the bottom right of each photograph is a gauge representing 20 μm. [Figure 9] Immunohistochemical staining of the cerebellum of cynomolgus monkeys after a single intravenous administration of hGAA (a) hGAA-anti-hTfR antibody 3N (IgG4) and (b) hGAA. The bar at the bottom right of each photograph indicates a gauge of 20 μm. [Figure 10] Figures showing the results of a pharmacological evaluation of hGAA-anti-hTfR antibody 3N (IgG4) using mice. Glycogen concentrations are shown in (a) the right brain, (b) the cervical spinal cord, (c) the heart, (d) the diaphragm, (e) the liver, and (f) the spleen. In each figure, 1 represents the normal control group, 2 represents the pathological control group, 3 represents the group administered 20 mg / kg of hGAA, and 4 to 7 represent the groups administered 2.5 mg / kg, 5.0 mg / kg, 10 mg / kg, and 20 mg / kg of hGAA-anti-hTfR antibody 3N (IgG4), respectively. The vertical axis represents glycogen concentration (mg / g wet weight). Vertical bars indicate SD, # indicates p<0.01 compared with the pathological control group, ♯♯ indicates p<0.05 compared with the pathological control group, $ indicates p<0.01 compared with the hGAA-administered group, and $$ indicates p<0.05 compared with the hGAA-administered group (all by Tukey HSD test). [Figure 11]Figures showing the results of efficacy evaluation of hGAA-anti-hTfR antibody 3N (IgG4) using mice. Glycogen concentrations are shown in (a) quadriceps, (b) gastrocnemius, (c) soleus, (d) tibialis anterior, and (e) extensor digitorum longus muscles. In each figure, 1 represents the normal control group, 2 represents the pathological control group, 3 represents the hGAA-administered group, and 4 to 7 represent the hGAA-anti-hTfR antibody 3N (IgG4) 2.5 mg / kg, 5.0 mg / kg, 10 mg / kg, and 20 mg / kg administration groups, respectively. The vertical axis represents glycogen concentration (mg / g wet weight). Vertical bars indicate SD, # indicates p<0.01 compared with the pathological control group, ♯♯ indicates p<0.05 compared with the pathological control group, $ indicates p<0.01 compared with the hGAA-administered group, and $$ indicates p<0.05 compared with the hGAA-administered group (all by Tukey HSD test). [Figure 12] Figures showing the results of efficacy evaluation of Fab GS-GAA using mice. Glycogen concentrations are shown in (a) the right brain, (b) the heart, (c) the diaphragm, (d) the quadriceps, (e) the soleus, and (f) the tibialis anterior. In each figure, 1 represents the normal control group, 2 represents the pathological control group, 3 represents the hGAA administration group, and 4 and 5 represent the 5.0 mg / kg and 20 mg / kg administration groups of Fab GS-GAA, respectively. The vertical axis represents glycogen concentration (mg / g wet weight). The vertical bars indicate SD. DETAILED DESCRIPTION OF THE INVENTION

[0017] In the present invention, the term "antibody" primarily refers to human antibodies, mouse antibodies, humanized antibodies, chimeric antibodies between human antibodies and antibodies of other mammals, and chimeric antibodies between mouse antibodies and antibodies of other mammals, but is not limited to these, as long as it has the property of specifically binding to a specific antigen, and there are also no particular restrictions on the animal species of the antibody. However, humanized antibodies are preferred.

[0018] In the present invention, the term "human antibody" refers to an antibody whose entire protein is encoded by a gene of human origin. Antibodies encoded by genes in which mutations have been added to the original human gene without changing the original amino acid sequence, for purposes such as increasing gene expression efficiency, are also included in the term "human antibody." Furthermore, antibodies created by combining two or more genes encoding human antibodies and substituting a portion of one human antibody for a portion of another human antibody are also included in the term "human antibody." 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 referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. The three CDRs in the immunoglobulin heavy chain are also referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. Human antibodies also include antibodies in which the antigen specificity, affinity, etc. of a human antibody have been modified by substituting the CDR of one human antibody with the CDR of another human antibody.

[0019] In the present invention, the term "human antibody" also includes antibodies in which mutations such as substitutions, deletions, and additions have been added to the amino acid sequence of the original antibody by modifying the gene of the original human antibody. When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of the original antibody are deleted, the number of deleted amino acids is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, antibodies in which mutations combining these amino acid substitutions and deletions have been added are also human antibodies. When amino acids are added, preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of the original antibody. Antibodies in which mutations combining these amino acid additions, substitutions, and deletions have been added are also human antibodies. The amino acid sequence of the mutated antibody preferably exhibits 80% or more homology with the amino acid sequence of the original antibody, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology. In other words, in the present invention, the term "human-derived gene" includes not only the original human-derived gene, but also genes obtained by modifying it.

[0020] The homology between the amino acid sequence of the original antibody and the amino acid sequence of the mutated antibody can be easily calculated using well-known homology calculation algorithms, such as BLAST (Altschul SF. J Mol. Biol. 215. 403-10, (1990)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), and the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2. 482-9 (1981)).

[0021] In the present invention, the term "mouse antibody" refers to an antibody whose entire protein consists of the same amino acid sequence as an antibody encoded by a mouse-derived gene. Therefore, "mouse antibody" also includes antibodies encoded by genes in which mutations have been added to the original mouse gene without changing the original amino acid sequence, for purposes such as increasing gene expression efficiency. Mouse antibodies also include antibodies in which two or more genes encoding mouse antibodies are combined to replace a portion of one mouse antibody with a portion of another mouse antibody. Mouse 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 referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. The three CDRs in the immunoglobulin heavy chain are referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. For example, antibodies in which the antigen specificity, affinity, etc. of a mouse antibody have been modified by replacing the CDR of a mouse antibody with the CDR of another mouse antibody are also included in the mouse antibody category.

[0022] In the present invention, antibodies in which mutations such as substitution, deletion, and addition have been added to the amino acid sequence of the original mouse antibody by modifying the gene of the original mouse antibody are also included in the "mouse antibody." When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of the original antibody are deleted, the number of deleted amino acids is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, antibodies in which mutations have been added that combine these amino acid substitutions and deletions are also included in the "mouse antibody." When amino acids are added, preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or the N-terminus or C-terminus of the original antibody. Antibodies in which mutations have been added that combine these amino acid additions, substitutions, and deletions are also included in the "mouse antibody." The amino acid sequence of the mutated antibody preferably exhibits 80% or more homology with the amino acid sequence of the original antibody, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology. In other words, in the present invention, the term "mouse-derived gene" includes not only the original mouse-derived gene, but also genes obtained by modifying it.

[0023] In the present invention, the term "humanized antibody" refers to an antibody in which the amino acid sequence of a portion of the variable region (e.g., all or part of the CDRs in particular) is derived from a mammal other than human, and the remaining regions are derived from humans. For example, a humanized antibody may be an antibody produced by replacing three complementarity-determining regions (CDRs) of an immunoglobulin light chain and three complementarity-determining regions (CDRs) of an immunoglobulin heavy chain that constitute a human antibody with CDRs from another mammal. The species of other mammal from which the CDRs to be grafted into appropriate positions in a human antibody are derived is not particularly limited as long as it is a mammal other than human, but is preferably a mouse, rat, rabbit, horse, or non-human primate, more preferably a mouse or rat, and even more preferably a mouse.

[0024] In the present invention, the term "chimeric antibody" refers to an antibody formed by linking fragments of two or more different antibodies derived from two or more different species.

[0025] A chimeric antibody between a human antibody and an antibody from another mammal is an antibody in which parts of a human antibody are replaced with parts of an antibody from a mammal other than a human. The antibody consists of an Fc region, Fab region, and hinge region, as explained below. A specific example of such a chimeric antibody is a chimeric antibody in which the Fc region is derived from a human antibody while the Fab region is derived from an antibody from another mammal. The hinge region is derived from either a human antibody or an antibody from another mammal. Conversely, a chimeric antibody in which the Fc region is derived from another mammal while the Fab region is derived from a human antibody is also included. The hinge region is derived from either a human antibody or an antibody from another mammal.

[0026] Alternatively, an antibody can be said to consist of a variable region and a constant region. Another specific example of a chimeric antibody is a heavy chain constant region (C H ) and the light chain constant region (C L ) is derived from a human antibody, while the variable region of the heavy chain (V H ) and the light chain variable region (V L ) derived from antibodies of other mammals, conversely, the heavy chain constant region (C H ) and the light chain constant region (C L ) are derived from antibodies of other mammals, while the variable region of the heavy chain (V H ) and the light chain variable region (V L ) derived from a human antibody. Here, the other mammalian species is not particularly limited as long as it is a mammal other than a human, but is preferably a mouse, rat, rabbit, horse, or non-human primate, more preferably a mouse.

[0027] A chimeric antibody between a mouse antibody and an antibody from another mammal is an antibody in which a part of the mouse antibody is replaced with a part of the antibody from a mammal other than a mouse. Specific examples of such chimeric antibodies include chimeric antibodies in which the Fc region is derived from a mouse antibody and the Fab region is derived from an antibody from another mammal, and conversely, chimeric antibodies in which the Fc region is derived from another mammal and the Fab region is derived from a mouse antibody. The other mammalian species is not particularly limited as long as it is a mammal other than a mouse, but is preferably a rat, rabbit, horse, or non-human primate, and more preferably a human.

[0028] Chimeric antibodies of human and mouse antibodies are particularly called "human / mouse chimeric antibodies." Examples of 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. Another specific example of a human / mouse chimeric antibody is a chimeric antibody in which the constant region of the heavy chain (C H ) and the light chain constant region (C L ) is derived from a human antibody, while the variable region of the heavy chain (V H ) and the light chain variable region (V L ) is derived from mouse antibodies, while the heavy chain constant region (C H ) and the light chain constant region (C L ) is derived from a mouse antibody, while the variable region of the heavy chain (V H ) and the light chain variable region (V L ) derived from a human antibody.

[0029] An antibody originally has a basic structure consisting of four polypeptide chains: two immunoglobulin light chains and two immunoglobulin heavy chains. However, in the present invention, the term "antibody" refers to an antibody that has this basic structure, as well as: (1) Consists of two polypeptide chains, one immunoglobulin light chain and one immunoglobulin heavy chain, or, as described in more detail below, (2) A single-chain antibody, which is composed of an immunoglobulin light chain with a linker sequence at its C-terminus and an immunoglobulin heavy chain at its C-terminus; (3) Single-chain antibodies are also included, which are composed of a linker sequence attached to the C-terminus of an immunoglobulin heavy chain and an immunoglobulin light chain attached to the C-terminus of the linker sequence. (4) The term "antibody" as used herein also includes antibodies consisting of an Fab region, which is the basic structure of an antibody in the true sense, with the Fc region deleted, and antibodies consisting of an Fab region and all or part of the hinge region (including Fab, F(ab') and F(ab')2).

[0030] Here, Fab is a region consisting of a variable region and a C L One light chain containing the constant region (the light chain constant region) and one variable region and C H Fab is a molecule in which one heavy chain containing one region (the constant region of the heavy chain) is bound by disulfide bonds between the cysteine ​​residues present in each heavy chain. In Fab, the heavy chain consists of a variable region and a C H In addition to the region 1 (part 1 of the heavy chain constant region), 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 an antibody. In Fab, the light chain and the heavy chain are connected by the light chain constant region (C L The cysteine ​​residues in the heavy chain constant region (C H The heavy chains that make up Fab are called Fab heavy chains. Fab lacks the cysteine ​​residues that connect the heavy chains of antibodies in the hinge region, so it consists of one light chain and one heavy chain. The light chains that make up Fab consist of a variable region and a C L The heavy chain that makes up Fab contains the variable region and C H It may consist of one domain, a variable domain, C HIt may also contain a portion of the hinge region in addition to the variable region. In this case, however, the hinge region is selected so as not to contain cysteine ​​residues that connect the heavy chains, so that disulfide bonds are not formed between the two heavy chains at the hinge region. In F(ab'), the heavy chain consists of a variable region and a C H In addition to the first region, the heavy chain includes all or part of the hinge region containing the cysteine ​​residues that link the heavy chains. F(ab')2 refers to a molecule in which two F(ab')s 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 a Fab' heavy chain. Furthermore, polymers such as dimers and trimers formed by multiple antibodies linked directly or via linkers are also antibodies. Furthermore, the term "antibody" as used herein includes any molecule that contains a portion of an immunoglobulin molecule and has the property of specifically binding to an antigen. In other words, the term "immunoglobulin light chain" as used herein includes those derived from an immunoglobulin light chain and having all or part of the amino acid sequence of its variable region. Furthermore, the term "immunoglobulin heavy chain" 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, for example, even one lacking the Fc region is an immunoglobulin heavy chain.

[0031] Also, here, Fc or Fc region refers to the C region in an antibody molecule. H 2 region (part 2 of the heavy chain constant region), and C H It refers to a region containing a fragment consisting of three regions (part 3 of the heavy chain constant region).

[0032] Furthermore, in the present invention, when referring to an "antibody", (5) Also included are scFab, scF(ab'), and scF(ab')2, which are single-chain antibodies formed by linking the light chain and heavy chain constituting the Fab, F(ab'), or F(ab')2 shown in (4) above via a linker sequence. Here, scFab, scF(ab'), and scF(ab')2 may be formed by linking a linker sequence to the C-terminus of the light chain and then linking a heavy chain to that C-terminus, or by linking a linker sequence to the C-terminus of the heavy chain and then linking a light chain to that C-terminus. Furthermore, the antibodies of the present invention also include 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 sequence. In the case of scFv, it may be composed of a linker sequence attached to the C-terminus of the light chain variable region, and then the heavy chain variable region attached to the C-terminus of that, or it may be composed of a linker sequence attached to the C-terminus of the heavy chain variable region, and then the light chain variable region attached to the C-terminus of that.

[0033] Furthermore, the term "antibody" as used herein includes all forms of full-length antibodies, as well as antigen-binding fragments (antibody fragments) that are missing portions of full-length antibodies, which is a broader concept that includes (4) and (5) above, in addition to those shown in (1) to (5) above.

[0034] The term "antigen-binding fragment" refers to a fragment of an antibody that retains at least a portion of the specific binding activity with an antigen. Examples of binding fragments include, in addition to those shown in (4) and (5) above, Fab, Fab', F(ab')2, variable region (Fv), heavy chain variable region (V), and the like. H ) and the light chain variable region (V L ) linked with an appropriate linker, a single-chain antibody (scFv), a heavy chain variable region (V H ) and the light chain variable region (V L ) and scFv heavy chain (H chain) containing a portion of the constant region (C H3) and other minibodies, which are dimers of the antibody fragments bound to the antibody protein, as well as other minibodies. However, they are not limited to these molecules as long as they have the ability to bind to antigens. Furthermore, these binding fragments include not only those obtained by treating full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

[0035] In the present invention, the term "single-chain antibody" refers to a protein that can specifically bind to a specific antigen, comprising an amino acid sequence containing all or part of the variable region of an immunoglobulin light chain, to which a linker sequence is attached at the C-terminus, and to which an amino acid sequence containing all or part of the variable region of an immunoglobulin heavy chain is further attached at the C-terminus of the linker sequence. For example, the above (2), (3), and (5) are included in single-chain antibodies. Furthermore, a protein that can specifically bind to a specific antigen, comprising an amino acid sequence containing all or part of the variable region of an immunoglobulin heavy chain, to which a linker sequence is attached at the C-terminus of the linker sequence, and to which an amino acid sequence containing all or part of the variable region of an immunoglobulin light chain is further attached at the C-terminus of the linker sequence, is also a "single-chain antibody" in the present invention. In a single-chain antibody in which an immunoglobulin light chain is attached to the C-terminus of the immunoglobulin heavy chain via a linker sequence, 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 an immunoglobulin heavy chain also has three CDRs. These CDRs are the main regions that determine the antigen specificity of an antibody. Therefore, a single-chain antibody preferably contains all three CDRs of an immunoglobulin heavy chain and all three CDRs of an immunoglobulin light chain. However, as long as the antigen-specific affinity of the antibody is maintained, a single-chain antibody can also be produced by deleting one or more CDRs.

[0036] In single-chain antibodies, the linker sequence disposed between the light and heavy chains of immunoglobulin is a peptide chain composed of preferably 2 to 50, more preferably 8 to 50, even more preferably 10 to 30, and even more preferably 12 to 18 or 15 to 25 amino acid residues, for example, 15 or 25. The amino acid sequence of such a linker sequence is not limited as long as the anti-hTfR antibody linking both chains retains affinity for hTfR, but preferably is composed of only glycine or glycine and serine, such as the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence in which any of these amino acid sequences is repeated 2 to 10 times or 2 to 5 times. For example, when linking the variable region of an immunoglobulin light chain to the C-terminus of an amino acid sequence consisting of the entire variable region of an immunoglobulin heavy chain via a linker sequence, a linker sequence containing a total of 15 amino acids corresponding to three consecutive amino acids of the amino acid sequence Gly-Gly-Gly-Gly-Ser (sequence number 3) is preferred.

[0037] In the present invention, the term "human transferrin receptor" or "hTfR" refers to a membrane protein having the amino acid sequence shown in SEQ ID NO: 1. 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: 1 from the 89th cysteine ​​residue from the N-terminus to the phenylalanine at the C-terminus (extracellular domain of hTfR), but is not limited to this. Furthermore, in the present invention, the term "monkey transferrin receptor" or "monkey TfR" particularly refers to a membrane protein derived from cynomolgus monkeys (Macaca fascicularis) and having the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the anti-hTfR antibody of the present invention also binds to the portion of the amino acid sequence shown in SEQ ID NO: 2 from the 89th cysteine ​​residue from the N-terminus to the phenylalanine at the C-terminus (extracellular domain of monkey TfR), but is not limited to this.

[0038] A common method for producing antibodies against hTfR is to produce recombinant human transferrin receptor (rhTfR) using cells transfected with an expression vector incorporating the hTfR gene, and then immunize animals such as mice with this rhTfR.Hybridoma cells capable of producing anti-hTfR antibodies can be produced by extracting antibody-producing cells against hTfR from the immunized animal and fusing these with myeloma cells.

[0039] 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 an in vitro immunization method. When using an in vitro immunization method, there are no particular limitations on the animal species from which the immune system cells are derived, but preferred are mice, rats, rabbits, guinea pigs, dogs, cats, horses, and primates including humans, more preferably mice, rats, and humans, and even more preferably mice and humans. As mouse immune system cells, for example, splenocytes prepared from mouse spleens 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 using an in vitro immunization method, human antibodies against hTfR can be obtained.

[0040] Hybridoma cells capable of producing antibodies can be produced by immunizing immune system cells using in vitro immunization and then fusing the cells with myeloma cells. Alternatively, mRNA can be extracted from the immunized cells, cDNA can be synthesized, and DNA fragments containing genes encoding the light and heavy chains of immunoglobulins can be amplified by PCR using this cDNA as a template, and these can then be used to artificially reconstruct antibody genes.

[0041] The hybridoma cells obtained as they are by the above method also include cells that produce antibodies that recognize proteins other than hTfR as antigens. Furthermore, not all hybridoma cells that produce anti-hTfR antibodies necessarily produce anti-hTfR antibodies that exhibit high affinity for hTfR.

[0042] Similarly, artificially reconstructed antibody genes also include genes encoding antibodies that recognize proteins other than hTfR as antigens. Furthermore, not all genes encoding anti-hTfR antibodies necessarily have the desired properties, such as encoding anti-hTfR antibodies that exhibit high affinity for hTfR.

[0043] Therefore, a step of selecting hybridoma cells that produce antibodies with desired properties (such as high affinity for hTfR) from the hybridoma cells obtained as described above is required. Furthermore, in the case of artificially reconstructed antibody genes, a step of selecting genes encoding antibodies with desired properties (such as high affinity for hTfR) from the antibody genes is required. The method described in detail below is effective as a method for selecting hybridoma cells that produce antibodies that exhibit high affinity for hTfR (high-affinity antibodies), or genes that encode high-affinity antibodies. Note that an antibody that exhibits high affinity for hTfR is one that has a dissociation constant with hTfR (K D ) but preferably 1X10 -8 M or less, and more preferably 1X10 -9 M or less, and more preferably 1X10 -10 M or less, and even more preferably 1X10 -11 For example, a suitable one has a dissociation constant of 1×10 -13 M~1X10 -9 M, 1X10 -13 M~1X10 -10 M can be mentioned.

[0044] For example, when selecting hybridoma cells that produce antibodies with high affinity for anti-hTfR antibodies, recombinant hTfR is added to a plate and allowed to remain thereon, followed by the addition of the hybridoma cell culture supernatant, followed by the removal of antibodies not bound to the recombinant hTfR from the plate, and the measurement of the amount of antibody retained on the plate. According to this method, the higher the affinity for hTfR of the antibodies contained in the hybridoma cell culture supernatant added to the plate, the greater the amount of antibody retained on the plate. Therefore, the amount of antibody retained on the plate can be measured, and hybridoma cells corresponding to plates retaining a greater amount of antibody can be selected as cell lines that produce anti-hTfR antibodies with relatively high affinity for hTfR. From the cell lines selected in this way, mRNA can be extracted to synthesize cDNA, and the cDNA can be used as a template to amplify a DNA fragment containing the gene encoding the anti-hTfR antibody by PCR, thereby isolating the gene encoding the high-affinity antibody.

[0045] When selecting a gene encoding an anti-hTfR antibody with high affinity from the above-mentioned artificially reconstructed antibody genes, the artificially reconstructed antibody gene is first incorporated into an expression vector, and this expression vector is then introduced into host cells. The host cells used in this process are not particularly limited, regardless of whether they are prokaryotic or eukaryotic, as long as they can express the antibody gene upon introduction of an expression vector incorporating the artificially reconstructed antibody gene. However, mammalian cells such as human, mouse, or Chinese hamster are preferred, with Chinese hamster ovary-derived CHO cells or mouse myeloma-derived NS / 0 cells being particularly preferred. Furthermore, the expression vector used to incorporate and express a gene encoding the antibody gene can be used without particular limitation, as long as it expresses the gene when introduced into mammalian cells. The gene incorporated into the expression vector is located downstream of a DNA sequence (gene expression control site) that can regulate the frequency of gene transcription in mammalian cells. Examples of gene expression control sites that can be used in the present invention include promoters derived from cytomegalovirus, SV40 early promoters, human elongation factor-1 alpha (EF-1α) promoters, and human ubiquitin C promoters.

[0046] Mammalian cells into which such an expression vector has been introduced express the above-mentioned artificially reconstructed antibody incorporated into the expression vector. To select cells producing antibodies with high affinity for anti-hTfR antibodies from the cells expressing the artificially reconstructed antibodies obtained in this manner, recombinant hTfR is added to a plate and allowed to remain there, followed by contacting the cell culture supernatant with the recombinant hTfR, and then removing antibodies not bound to the recombinant hTfR from the plate and measuring the amount of antibody retained on the plate. According to this method, the higher the affinity of antibodies contained in the cell culture supernatant for hTfR, the greater the amount of antibody retained on the plate. Therefore, by measuring the amount of antibody retained on the plate, cells corresponding to plates retaining a greater amount of antibody can be selected as cell lines producing anti-hTfR antibodies with relatively high affinity for hTfR, and thus genes encoding anti-hTfR antibodies with high affinity for hTfR can be selected. From the cell lines selected in this manner, genes encoding high-affinity antibodies can be isolated by amplifying DNA fragments containing genes encoding anti-hTfR antibodies using PCR.

[0047] Selection of a gene encoding an anti-hTfR antibody with high affinity from the above-mentioned artificially reconstructed antibody genes can also be achieved by incorporating the artificially reconstructed antibody gene into an expression vector, introducing this expression vector into E. coli, and then using the culture supernatant obtained by culturing the E. coli or a solution containing antibodies obtained by lysing the E. coli to select E. coli strains carrying the desired gene in a manner similar to that used for selecting hybridoma cells described above. The selected E. coli strain expresses a gene encoding an anti-hTfR antibody with a relatively high affinity for hTfR. From this cell line, a gene encoding an anti-hTfR antibody with a relatively high affinity for hTfR can be selected. To secrete the antibody into the E. coli culture supernatant, the antibody gene can be incorporated into an expression vector so that a secretory signal sequence is attached to the N-terminus.

[0048] Another method for selecting genes encoding anti-hTfR antibodies with high affinity is to retain and express antibodies encoded by the above-mentioned artificially reconstructed antibody genes on phage particles. In this case, the antibody genes are reconstructed as genes encoding single-chain antibodies. Methods for retaining antibodies on phage particles are well known and are described in international publications (WO1997 / 09436, WO1995 / 11317, etc.). When selecting phages carrying antibodies with high affinity for anti-hTfR antibodies from phages carrying antibodies encoded by artificially reconstructed antibody genes, a method is used in which recombinant hTfR is added to a plate and retained, followed by contact with phages, followed by removal of phages not bound to the recombinant hTfR from the plate, and the amount of phages retained on the plate is measured. According to this method, the higher the affinity for hTfR of the antibodies retained on the phage particles, the greater the amount of phages retained on the plate. Therefore, the amount of phage retained on the plate can be measured, and phage particles corresponding to plates retaining more phage can be selected as phage particles that produce anti-hTfR antibodies with relatively high affinity for hTfR, and thus genes encoding anti-hTfR antibodies with high affinity for hTfR can be selected. From the phage particles selected in this way, DNA fragments containing the genes encoding the anti-hTfR antibodies can be amplified using PCR, allowing the genes encoding the high-affinity antibodies to be isolated.

[0049] cDNA or phage DNA can be prepared from cells such as hybridoma cells that produce antibodies with high affinity for the above-mentioned anti-hTfR antibodies, or from phage particles that contain antibodies with high affinity for the anti-hTfR antibodies, and using this as a template, DNA fragments containing genes encoding all or part of the light chain of an anti-hTfR antibody, the heavy chain of an anti-hTfR antibody, or a single-chain anti-hTfR antibody can be amplified and isolated by PCR, etc. Similarly, DNA fragments containing genes encoding all or part of the variable region of the light chain of an anti-hTfR antibody, and DNA fragments containing genes encoding all or part of the variable region of the heavy chain of an anti-hTfR antibody can also be amplified and isolated by PCR, etc.

[0050] High-affinity anti-hTfR antibodies can be produced by incorporating all or part of the genes encoding the light and heavy chains of this high-affinity anti-hTfR antibody into an expression vector, transforming host cells such as mammalian cells with this expression vector, and culturing the resulting transformed cells. The amino acid sequence of the anti-hTfR antibody can also be translated from the nucleotide sequence of the gene encoding the isolated anti-hTfR antibody, and a DNA fragment encoding this amino acid sequence can be artificially synthesized. When artificially synthesizing a DNA fragment, the expression level of the anti-hTfR antibody in host cells can be increased by selecting appropriate codons.

[0051] To add mutations such as substitutions, deletions, or additions to the amino acid sequence of the original anti-hTfR antibody, appropriate mutations can be added to the gene encoding the anti-hTfR antibody contained in the isolated DNA fragment. The gene encoding the anti-hTfR antibody after mutation preferably has 80% or more homology with the original gene, more preferably 90% or more homology, but there are no particular restrictions on the homology. Adding mutations to the amino acid sequence can also modify the number and type of sugar chains that bind to the anti-hTfR antibody, thereby increasing the stability of the anti-hTfR antibody in vivo.

[0052] When a mutation is introduced into a gene encoding all or part of the light chain variable region of an anti-hTfR antibody, the gene after the mutation preferably has 80% or more homology with the original gene, more preferably 90% or more homology, but there is no particular limit to the homology. When amino acids in the amino acid sequence of the light chain variable region are substituted 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 amino acids in the amino acid sequence of the light chain variable region are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be introduced. When amino acids are added to the light chain variable region, 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 the light chain variable region or to the N-terminus or C-terminus thereof. Mutations combining these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of the mutated light chain variable region preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original light chain variable region. In particular, when amino acids in the CDR amino acid sequence are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. When amino acids in the CDR amino acid sequence are deleted, the number of deleted amino acids is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. Mutations combining these amino acid substitutions and deletions can also be added. When amino acids are added, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence or to the N-terminal or C-terminal side. Mutations that combine addition, substitution, and deletion of these amino acids can also be added.The amino acid sequence of each mutated CDR preferably has 80% or more homology with the original amino acid sequence of each CDR, more preferably 90% or more homology, and even more preferably 95% or more homology.

[0053] When a mutation is introduced into a gene encoding all or part of the heavy chain variable region of an anti-hTfR antibody, the gene after the mutation preferably has 80% or more homology with the original gene, more preferably 90% or more homology, but there is no particular limit to the homology. When amino acids in the amino acid sequence of the heavy chain variable region are substituted 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 amino acids in the amino acid sequence of the heavy chain variable region are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be introduced. When amino acids are added to the heavy chain variable region, 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 the heavy chain variable region or to the N-terminus or C-terminus thereof. Mutations combining these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of the mutated heavy chain variable region preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original heavy chain variable region. In particular, when amino acids in the CDR amino acid sequence are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. When amino acids in the CDR amino acid sequence are deleted, the number of deleted amino acids is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. Mutations combining these amino acid substitutions and deletions can also be added. When amino acids are added, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence or to the N-terminal or C-terminal side. Mutations that combine addition, substitution, and deletion of these amino acids can also be added.The amino acid sequence of each mutated CDR preferably has 80% or more homology with the original amino acid sequence of each CDR, more preferably 90% or more homology, and even more preferably 95% or more homology.

[0054] Mutations can also be made in both the light chain and heavy chain variable regions of the anti-hTfR antibody by combining the above-mentioned mutations in the light chain variable region of the anti-hTfR antibody with the above-mentioned mutations in the heavy chain variable region of the anti-hTfR antibody.

[0055] Substitutions of amino acids in the light and heavy chain amino acid sequences of the above-mentioned anti-hTfR antibodies with other amino acids include, for example, amino acids classified in the same group, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), and amino acids with hydroxyl groups (Ser, Thr). Substitutions with such similar amino acids are expected not to alter the phenotype of the protein (i.e., conservative amino acid substitutions). However, the amino acid sequence of framework region 3 of hTfR in antibody No. 3 (unpublished at the time of filing this application), previously discovered by the present inventor and confirmed to have the same effects as the present invention, is SEQ ID NO: 83, and its 17th amino acid is Trp. In contrast, the framework region 3 of the heavy chain of anti-hTfR antibody No. 3N in the present invention has the amino acid sequence of SEQ ID NO: 68, with the 17th amino acid from the N-terminus substituted with Leu. Furthermore, in the amino acid sequence of CDR1, the fifth Thr in SEQ ID NO: 12 has been substituted with Met as shown in SEQ ID NO: 66. Trp and Leu are not in the similar relationship described above, and Thr and Met are not in the similar relationship described above. However, as will be described later, antibody No. 3N containing this substitution is unexpectedly similar in effect to antibody No. 3, and exhibits even superior effects.

[0056] In addition, if an amino acid is added to the C-terminus or N-terminus by mutating an anti-hTfR antibody, and the added amino acid is located between the anti-hTfR antibody and another protein (A) when the anti-hTfR antibody is fused with the protein (A), the added amino acid will constitute a part of the linker. The linker placed between the anti-hTfR antibody and protein (A) in the fusion protein of the anti-hTfR antibody and protein (A) will be described in detail later.

[0057] Anti-hTfR antibodies obtained by culturing cells that produce anti-hTfR antibodies with relatively high affinity for hTfR selected by the above-mentioned methods, and anti-hTfR antibodies obtained by expressing genes that encode high-affinity anti-hTfR antibodies, can also be modified to have desired properties by introducing mutations such as substitutions, deletions, and additions into their amino acid sequences. Introduction of mutations into the amino acid sequence of an anti-hTfR antibody is carried out by making mutations in the gene corresponding to that amino acid sequence.

[0058] The affinity of an anti-hTfR antibody for hTfR can be adjusted appropriately by introducing mutations such as substitutions, deletions, and additions into the amino acid sequence of the antibody's variable region. For example, if an antibody has high affinity for an antigen and an extremely low dissociation constant in aqueous solution, the antibody may not dissociate from the antigen when administered to the body, potentially resulting in functional problems. In such cases, by introducing mutations into the antibody's variable region, the dissociation constant can be adjusted in stages, such as 2-5 times, 5-10 times, or 10-100 times that of the original antibody, thereby obtaining the most desirable antibody for the intended purpose. Conversely, the introduction of such mutations can also adjust the dissociation constant in stages, such as 1 / 2-1 / 5 times, 1 / 5-1 / 10 times, or 1 / 10-1 / 100 times that of the original antibody.

[0059] Introduction of mutations such as substitutions, deletions, and additions into the amino acid sequence of an anti-hTfR antibody can be carried out, for example, by using the gene encoding the anti-hTfR antibody as a template and introducing mutations into specific sites in the base sequence of the gene using methods such as PCR, or by introducing mutations randomly.

[0060] Mutations can be introduced into the amino acid sequence of an anti-hTfR antibody for the purpose of adjusting the affinity of the antibody with hTfR by, for example, incorporating a gene encoding a single-chain anti-hTfR antibody into a phagemid, using this phagemid to produce a phage that expresses the single-chain antibody on the capsid surface, and then propagating the phage while introducing mutations into the gene encoding the single-chain antibody by applying a mutagen or the like, and selecting from the propagated phages those phages that express a single-chain antibody with the desired dissociation constant by the method described above, or by purifying using an antigen column under certain conditions.

[0061] The antibody having a relatively high affinity for hTfR obtained by the above-mentioned method for selecting cells producing high-affinity antibodies preferably has a dissociation constant (K) with hTfR measured by the method described in Example 7. D ) but preferably 1X10 -8 M or less, and more preferably 1X10 -9 M or less, and more preferably 1X10 -10 M or less, and even more preferably 1X10 -11 For example, a suitable one has a dissociation constant of 1×10 -13 M~1X10 -9 M, 1X10 -13 M~1X10 -10 Examples of such antibodies include those in which the amino acid sequence is M. The same applies to single-chain antibodies. Once an antibody is obtained, it can be appropriately modified by introducing mutations or the like so that it has desired properties.

[0062] By selecting antibodies with affinity for monkey TfR from the anti-hTfR antibodies obtained as described above, it is possible to obtain antibodies with affinity for both human and monkey TfR. Antibodies with affinity for monkey TfR can be selected, for example, by ELISA using recombinant monkey TfR produced using genetic engineering. In this ELISA, recombinant monkey TfR is added to a plate and allowed to remain on the plate, followed by contact with an anti-hTfR antibody. Antibodies that do not bind to the recombinant monkey TfR are then removed from the plate, and the amount of antibody retained on the plate is measured. Since the higher the affinity for recombinant monkey TfR, the greater the amount of antibody retained on the plate, an antibody corresponding to a plate retaining a larger number of antibodies can be selected as an antibody with affinity for monkey TfR. Note that the "monkey" referred to here is preferably an anthropoid animal excluding humans, more preferably one classified as Cercopithecidae, and even more preferably one classified as Macaca, such as a cynomolgus monkey or a rhesus monkey. Cynomolgus monkeys are particularly convenient for evaluation.

[0063] An antibody that has affinity for both human and monkey hTfR has the advantageous effect that the in vivo kinetics of the antibody when administered can be observed using monkeys. For example, when developing a drug using the anti-hTfR antibody of the present invention, pharmacokinetic testing of the drug can be carried out using monkeys, which can significantly accelerate the development of the drug.

[0064] In the present invention, an antibody having a relatively high affinity for hTfR and also having affinity for monkey TfR has the following dissociation constants with human and monkey TfR, particularly when measured by the method described in Example 7: (a) Dissociation constant with hTfR: preferably 1×10 -10 M or less, preferably 2.5X10 -11 M or less, more preferably 5X10 -12 M or less, and even more preferably 1X10 -12 M or less, (b) Dissociation constant with monkey TfR: preferably 1×10-9 M or less, more preferably 5X10 -10 M or less, more preferably 1X10 -10 M or less, for example 7.5X10 -11 M or less.

[0065] For example, the dissociation constants for human TfR and monkey TfR are 1X10 -10 M or less and 1X10 -9 M or less, 1X10 -11 M or less and 5X10 -10 M or less, 5X10 -12 M or less and 1X10 -10 M or less, 5X10 -12 M or less and 7.5X10 -11 M or less, 1X10 -12 M or less and 1X10 -10 M or less, 1X10 -12 M or less and 7.5X10 -11 There is no particular lower limit to the dissociation constant with human TfR, but for example, -13 M,1X10 -13 M, etc. In addition, there is no particularly clear lower limit for the dissociation constant with monkey TfR, but for example, -11 M,1X10 -12 M, etc. The same applies to the case where the antibody is a single-chain antibody.

[0066] Antibodies with relatively high affinity for hTfR obtained by the above-mentioned method for selecting cells producing high-affinity antibodies can be humanized if they are antibodies of animals other than humans. A humanized antibody is an antibody obtained by using the amino acid sequence of a portion of the variable region of an antibody of a nonhuman animal (e.g., all or part of the CDRs in particular) and replacing appropriate regions of a human antibody with that sequence (transplanting that sequence into a human antibody) while maintaining its specificity for the antigen. For example, a humanized antibody may be an antibody in which the three complementarity-determining regions (CDRs) of the immunoglobulin light chain and the three complementarity-determining regions (CDRs) of the immunoglobulin heavy chain constituting a human antibody are replaced with CDRs from another mammal. The species from which the CDRs incorporated into a human antibody are derived is not particularly limited as long as it is a mammal other than humans, but is preferably a mouse, rat, rabbit, horse, or nonhuman primate, more preferably a mouse or rat, and even more preferably a mouse. However, an antibody in which a portion of a human antibody is replaced with a portion of another human antibody may also be obtained.

[0067] Methods for producing humanized antibodies are well known in the art, and the most common method is based on the method devised by Winter et al., in which the amino acid sequences of the complementarity-determining regions (CDRs) in the variable regions of a human antibody are replaced with the CDRs of an antibody from a non-human mammal (Verhoeyen M. Science. 239, 1534-1536 (1988)). It is also well known that in order to reproduce the original activity of a donor antibody, it may be necessary to replace not only the CDRs of a non-human mammalian antibody but also the amino acid sequences of regions outside the CDRs that are involved in maintaining the CDR structure or binding to the antigen with the corresponding regions of the acceptor human antibody (Queen C. Proc. Natl. Acad. Sci. USA. 86, 10029-10033 (1989)). The regions outside the CDRs are also referred to as framework regions (FRs).

[0068] The variable regions of both the heavy and light chains of an antibody each contain four framework regions 1 to 4 (FR1 to FR4). FR1 is a region adjacent to CDR1 on the N-terminus side, and consists of the amino acid sequence from its N-terminus to the amino acid adjacent to the N-terminus of CDR1 in each peptide constituting the heavy and light chains. FR2 consists of the amino acid sequence between CDR1 and CDR2 in each peptide constituting the heavy and light chains. FR3 consists of the amino acid sequence between CDR2 and CDR3 in each peptide constituting the heavy and light chains. FR4 consists of the amino acid sequence from the amino acid adjacent to the C-terminus of CDR3 to the C-terminus of the variable region. However, without being limited to this, in the present invention, the framework region may also be a region excluding 1 to 5 amino acids on the N-terminus side and / or 1 to 5 amino acids on the C-terminus side of each of the above FR regions.

[0069] The production of humanized antibodies involves transplanting the CDRs (and, where appropriate, the surrounding FRs) of an antibody from a non-human mammal into the CDRs (and, where appropriate, the surrounding FRs) of the variable region of a human antibody. In this process, the framework regions of the variable region of the original human antibody can be obtained from public DNA databases containing germline antibody gene sequences. For example, the germline DNA sequences and amino acid sequences of human heavy and light chain variable region genes can be selected from the "VBase" human germline sequence database (available on the Internet at www.mrccpe.cam.ac.uk / vbase). In addition, DNA sequences and amino acid sequences can be selected from published literature, such as "Kabat EA. Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)," "Tomlinson IM. J. fol. Biol. 227. 776-98 (1992)," and "Cox JPL. Eur. J Immunol. 24:827-836 (1994)."

[0070] As described above, in humanized antibodies, the region of the antibody of a non-human mammal that is grafted onto the variable region of the original human antibody generally includes the CDR itself or the FR surrounding the CDR. However, since the FR grafted together with the CDR is also considered to be involved in maintaining the CDR structure or binding with an antigen and to have a function that essentially determines the complementarity of the antibody, the term "CDR" in the present invention refers to the region that is grafted or can be grafted from an antibody of a non-human mammal into a humanized antibody when producing a humanized antibody. In other words, even if a region is generally considered to be a FR, it is included in the CDR in the present invention as long as it is involved in maintaining the CDR structure or binding with an antigen and is considered to have a function that essentially determines the complementarity of the antibody.

[0071] When administered into the body by intravenous injection or other means, the anti-hTfR antibodies of the present invention can efficiently bind to hTfR present on endothelial cells of brain capillaries. The antibodies bound to hTfR are taken up into the brain by passing through the blood-brain barrier via mechanisms such as endocytosis and transcytosis. Therefore, by binding proteins, low-molecular-weight compounds, etc. that should function in the brain to the anti-hTfR antibodies of the present invention, these substances can efficiently pass through the blood-brain barrier and reach the brain. Furthermore, after passing through the blood-brain barrier, the anti-hTfR antibodies of the present invention can reach the cerebral parenchyma, hippocampal neuron-like cells, cerebellar Purkinje cells, etc., or at least any of these. They are also expected to reach neuron-like cells in the cerebral striatum and the substantia nigra of the midbrain. Therefore, proteins, low-molecular-weight compounds, etc. that act on these tissues or cells can be delivered to these tissues or cells by binding them to the anti-hTfR antibodies of the present invention.

[0072] The anti-hTfR antibodies of the present invention can be an effective means for transporting substances (proteins, low-molecular-weight compounds, etc.) that normally cannot cross the blood-brain barrier and therefore have little or no physiological or pharmacological effect in the brain when administered intravenously from the blood to the brain, where they can exert their effects. In particular, the anti-hTfR antibodies of the present invention reach the cerebral parenchyma, hippocampal neuron-like cells, cerebellar Purkinje cells, etc., or at least some of these, after passing through the blood-brain barrier. They are also expected to reach neuron-like cells in the cerebral striatum and the substantia nigra of the midbrain. Therefore, by administering these substances into the blood via intravenous administration or other means in a form bound to the anti-hTfR antibody molecules of the present invention, it becomes possible to exert or enhance their effects in these brain tissues or cells.

[0073] Methods for linking anti-hTfR antibodies to such substances (proteins, low-molecular-weight compounds, etc.) include linking via a non-peptide linker or a peptide linker. Examples of non-peptide linkers that can be used include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, as well as derivatives or combinations of these. A peptide linker is a peptide chain or derivative thereof consisting of 1 to 50 peptide-bonded amino acids, whose N-terminus and C-terminus form covalent bonds with either the anti-hTfR antibody, protein, low-molecular-weight compound, etc., thereby linking the anti-hTfR antibody to the protein, low-molecular-weight compound, etc.

[0074] The anti-hTfR antibody of the present invention conjugated to a desired other protein (A) using PEG as a non-peptide linker is specifically referred to as an anti-hTfR antibody-PEG-protein. The anti-hTfR antibody-PEG-protein can be produced by conjugating the anti-hTfR antibody to PEG to prepare an anti-hTfR antibody-PEG, and then conjugating the anti-hTfR antibody-PEG to the other protein (A). Alternatively, the anti-hTfR antibody-PEG-protein can be produced by conjugating the other protein (A) to PEG to prepare a protein-PEG, and then conjugating the protein-PEG to the anti-hTfR antibody. When conjugating PEG to an anti-hTfR antibody and the other protein (A), PEG modified with a functional group such as carbonate, carbonylimidazole, active ester of carboxylic acid, azlactone, cyclic imidothione, isocyanate, isothiocyanate, imidate, or aldehyde is used. The functional groups introduced into these PEGs react primarily with amino groups in the anti-hTfR antibody and other protein (A) molecules, thereby covalently bonding the PEG to the hTfR antibody and other protein (A). While there are no particular limitations on the molecular weight or shape of the PEG used, its average molecular weight (MW) is preferably 500 to 60,000, and more preferably 500 to 20,000. For example, PEGs with average molecular weights of about 300, about 500, about 1,000, about 2,000, about 4,000, about 10,000, or about 20,000 can be suitably used as non-peptide linkers. The same applies when linking an anti-hTfR antibody to a desired low-molecular-weight compound.

[0075] For example, anti-hTfR antibody-PEG can be obtained by mixing an anti-hTfR 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 11, 12.5, 15, 110, 120, etc., and then adding a reducing agent such as NaCNBH3 to allow the mixture to react. The anti-hTfR antibody-PEG is then reacted with another protein (A) in the presence of a reducing agent such as NaCNBH3 to obtain an anti-hTfR antibody-PEG-protein. Conversely, anti-hTfR antibody-PEG-protein can also be obtained by first conjugating another protein (A) with ALD-PEG-ALD to prepare a protein-PEG, and then conjugating the protein-PEG to an anti-hTfR antibody.

[0076] The anti-hTfR antibody and the other protein (A) can also be linked by peptide bonding the N-terminus or C-terminus of the other protein (A) to the C-terminus or N-terminus of the heavy chain or light chain of the anti-hTfR antibody, either directly or via a linker sequence. A fusion protein formed by linking an anti-hTfR antibody and the other protein (A) in this way can be obtained by incorporating a DNA fragment in which the cDNA encoding the other protein (A) is placed in-frame at the 3'-terminus or 5'-terminus of the cDNA encoding the heavy chain or light chain of the anti-hTfR antibody, either directly or via a DNA fragment encoding a linker sequence, into an expression vector for mammalian cells, and culturing mammalian cells into which this expression vector has been introduced. When a DNA fragment encoding another protein (A) is to be linked to the heavy chain, an expression vector for mammalian cells incorporating a cDNA fragment encoding the light chain of an anti-hTfR antibody is also introduced into the same host cell. When a DNA fragment encoding another protein (A) is to be linked to the light chain, an expression vector for mammalian cells incorporating a cDNA fragment encoding the heavy chain of an anti-hTfR antibody is also introduced into the same host cell. When the anti-hTfR antibody is a single-chain antibody, a fusion protein combining the anti-hTfR antibody and the other protein (A) can be obtained by incorporating a DNA fragment in which a cDNA encoding a single-chain anti-hTfR antibody is linked to the 5'- or 3'-end of the cDNA encoding the other protein (A) directly or via a DNA fragment encoding a linker sequence into an expression vector (for mammalian cells, eukaryotes such as yeast, or prokaryote cells such as Escherichia coli), and expressing the DNA fragment in the cells into which the expression vector has been introduced.

[0077] A fusion protein of the type in which another protein (A) is bound to the C-terminus of the light chain of an anti-hTfR antibody is one in which the anti-human transferrin receptor antibody contains an amino acid sequence including all or part of the variable region of the light chain and an amino acid sequence including all or part of the variable region of the heavy chain, and the other protein (A) is bound to the C-terminus of the light chain of this anti-human transferrin receptor antibody. Here, the light chain of the anti-hTfR antibody and the other protein (A) may be bound directly or via a linker.

[0078] A fusion protein of the type in which another protein (A) is bound to the C-terminus of the heavy chain of an anti-hTfR antibody is one in which the anti-human transferrin receptor antibody contains an amino acid sequence including all or part of the variable region of the light chain and an amino acid sequence including all or part of the variable region of the heavy chain, and the other protein (A) is bound to the C-terminus of the heavy chain of this anti-human transferrin receptor antibody. Here, the heavy chain of the anti-hTfR antibody and the other protein (A) may be bound directly or via a linker.

[0079] A fusion protein of the type in which another protein (A) is bound to the N-terminus of the light chain of an anti-hTfR antibody is one in which the anti-human transferrin receptor antibody contains an amino acid sequence containing all or part of the variable region of the light chain and an amino acid sequence containing all or part of the variable region of the heavy chain, and the other protein (A) is bound to the N-terminus of the light chain of this anti-human transferrin receptor antibody. Here, the light chain of the anti-hTfR antibody and the other protein (A) may be bound directly or via a linker.

[0080] A fusion protein of the type in which another protein (A) is bound to the N-terminus of the heavy chain of an anti-hTfR antibody is one in which the anti-human transferrin receptor antibody contains an amino acid sequence including all or part of the variable region of the light chain and an amino acid sequence including all or part of the variable region of the heavy chain, and the other protein (A) is bound to the N-terminus of the heavy chain of this anti-human transferrin receptor antibody. Here, the heavy chain of the anti-hTfR antibody and the other protein (A) may be bound directly or via a linker.

[0081] In this case, the linker sequence placed between the anti-hTfR antibody and the other protein (A) is a peptide chain composed of preferably 1 to 50, more preferably 1 to 17, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids, but the number of amino acids constituting the linker sequence 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, 27, etc., depending on the other protein (A) to be bound to the anti-hTfR antibody. There are no limitations on the amino acid sequence of such a linker sequence, as long as the anti-hTfR antibody linked thereto retains its affinity for hTfR and the other protein (A) linked via the linker sequence can exert the physiological activity of the protein under physiological conditions, but preferably the linker sequence is composed of glycine and serine. Examples include those consisting of a single amino acid, either glycine or serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or those containing 1 to 10 or 2 to 5 consecutive amino acids of these amino acid sequences. Also included are sequences consisting of 1 to 50 amino acids, or sequences consisting of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, or 27 amino acids. For example, those containing the amino acid sequence Gly-Ser can be suitably used as a linker sequence. In addition, a linker sequence containing a total of 27 amino acids consisting of the amino acid sequence Gly-Ser followed by five consecutive amino acid sequences Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3) can be suitably used.Furthermore, a linker sequence containing a total of 25 amino acids consisting of five consecutive amino acid sequences Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3) can also be suitably used.

[0082] In a fusion protein of an anti-hTfR antibody and another protein (A), if the anti-hTfR antibody is a single-chain antibody, the amino acid sequence containing all or part of the variable region of the immunoglobulin light chain and the amino acid sequence containing all or part of the variable region of the immunoglobulin heavy chain are generally linked via a linker sequence. In this case, as long as the affinity of the anti-hTfR antibody for hTfR is maintained, the linker sequence may be linked to the C-terminus of the amino acid sequence derived from the light chain, and the amino acid sequence derived from the heavy chain may be further linked to the C-terminus of that, or conversely, the linker sequence may be linked to the C-terminus of the amino acid sequence derived from the heavy chain, and the amino acid sequence derived from the light chain may be further linked to the C-terminus of that.

[0083] The linker sequence placed between the light and heavy chains of an immunoglobulin is a peptide chain composed of preferably 2 to 50, more preferably 8 to 50, even more preferably 10 to 30, and even more preferably 12 to 18 or 15 to 25, for example, 15 or 25 amino acids. The amino acid sequence of such a linker sequence is not limited, as long as the anti-hTfR antibody formed by linking both chains therethrough retains affinity for hTfR and the other protein (A) bound to the antibody can exert its physiological activity under physiological conditions, but preferably it is composed of glycine or glycine and serine, and includes, for example, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence consisting of 2 to 10 or 2 to 5 consecutive amino acids of these amino acid sequences. A preferred embodiment of the linker sequence includes a sequence consisting of 15 amino acids, each consisting of three consecutive amino acids of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

[0084] In a specific example of a fusion protein of the humanized anti-hTfR antibody of the present invention with another protein (A) when the anti-hTfR antibody is a single-chain antibody, the single-chain antibody is linked to the C-terminus of the other protein (A) via a first linker sequence consisting of a total of 27 amino acids, consisting of the amino acid sequence Gly-Ser followed by five consecutive amino acid sequences of Gly-Gly-Gly-Gly-Ser (sequence number 3). Preferred embodiments of the single-chain antibodies used herein include those having the amino acid sequence of SEQ ID NO: 60, in which the variable region of the heavy chain of humanized anti-hTfR antibody No. 3N, having the amino acid sequence of SEQ ID NO: 65, is linked to the C-terminus of the variable region, via a first linker sequence consisting of 15 amino acids in total, consisting of three consecutive amino acid sequences of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3); and those having the amino acid sequence of SEQ ID NO: 98, in which the light chain of anti-hTfR antibody, having the amino acid sequence of SEQ ID NO: 23, is linked to the C-terminus of the Fab heavy chain of humanized anti-hTfR antibody No. 3N, having the amino acid sequence of SEQ ID NO: 61, via a linker consisting of 32 amino acids, consisting of an amino acid sequence of six consecutive amino acids of the amino acid sequence of SEQ ID NO: 3 followed by the amino acid sequence Gly-Gly.

[0085] When the anti-hTfR antibody is a single-chain antibody, such a fusion protein can be produced, for example, by transforming a host cell such as a mammalian cell with an expression vector incorporating a DNA fragment having a base sequence encoding the fusion protein, and culturing the host cell.

[0086] In the present invention, when one peptide chain contains multiple linker sequences, for convenience, the linker sequences are named in order from the N-terminus side as the first linker sequence, the second linker sequence, and so on.

[0087] As an example of a specific embodiment of a fusion protein of the humanized anti-hTfR antibody of the present invention and another protein (A) when the anti-hTfR antibody is Fab, the variable region of the anti-hTfR antibody heavy chain and the C-terminal end of the other protein (A) are linked via a linker sequence consisting of 27 amino acids in total, consisting of Gly-Ser followed by five consecutive amino acid sequences of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3). H In this case, C H In addition to the region, a part of the hinge region may be included, but the hinge region does not contain cysteine ​​residues that form disulfide bonds between heavy chains.

[0088] An example of a suitable heavy chain when the anti-hTfR antibody is a Fab is one having the amino acid sequence set forth in SEQ ID NO: 61. The amino acid sequence of SEQ ID NO: 61 is the Fab of the heavy chain of humanized anti-hTfR antibody No. 3N, which has the amino acid sequence set forth in SEQ ID NO: 66, and corresponds to the portion from the 1st to the 226th amino acid sequence from the N-terminus of SEQ ID NO: 66. The portion from the 1st to the 118th amino acid sequence from the N-terminus of SEQ ID NO: 61 corresponds to the variable region (SEQ ID NO: 65), and the portion from the 119th to the 216th amino acid sequence corresponds to the C H The region from 217th to 226th corresponds to the hinge region.

[0089] When the anti-hTfR antibody is a Fab, the Fc region of another IgG can also be introduced into the fusion protein. Introducing an Fc region into the fusion protein can increase the stability of the fusion protein in the blood or other living organisms. Examples of such fusion proteins incorporating an Fc region include those in which a human IgG Fc region is linked to the C-terminus of another protein (A) directly or via a linker sequence, and in which the Fab heavy chain of an anti-human transferrin receptor antibody is linked to the C-terminus of the human IgG Fc region directly or via a linker sequence.

[0090] The linker sequence between the other protein (A) and the human IgG Fc region preferably consists of 1 to 50 amino acids. Here, the number of amino acids is appropriately adjusted to 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, 27, etc. The amino acid sequence of such a linker sequence is not limited, but is preferably composed of glycine and serine. Examples include those consisting of a single amino acid, either glycine or serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or sequences consisting of 50 or less amino acids, formed by linking 1 to 10 or 2 to 5 of these amino acid sequences, or sequences consisting of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, or 25 amino acids. For example, a sequence consisting of 25 consecutive amino acids, formed by five consecutive amino acids of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), can be suitably used as a linker sequence. The same applies to linker sequences between the human IgG Fc region and the Fab heavy chain.

[0091] When the human IgG Fc region is introduced, the human IgG Fc region may be bound to either the heavy chain or the light chain of the anti-human transferrin receptor antibody. The antibody may also be other antigen-binding fragments, including F(ab')2, F(ab'), and single-chain antibodies.

[0092] The type of IgG for the human IgG Fc region to be introduced is not particularly limited, and may be any of IgG1 to IgG5. Furthermore, the human IgG Fc region to be introduced may be the entire Fc region or a portion thereof. A preferred embodiment of such a human IgG Fc region includes one having the amino acid sequence shown in SEQ ID NO: 70, which is the entire Fc region of human IgG1. An example of the amino acid sequence of the Fab heavy chain into which the human IgG Fc region has been introduced is one having the amino acid sequence shown in SEQ ID NO: 71, in which the human IgG Fc region having the amino acid sequence shown in SEQ ID NO: 70 is linked to the N-terminus of the amino acid sequence of the Fab heavy chain of humanized anti-hTfR antibody 3N (SEQ ID NO: 61) via a linker sequence comprising a total of 25 amino acids consisting of five consecutive amino acids in the sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

[0093] Fusion proteins of anti-hTfR antibodies and other proteins (A) can also be modified to have affinity for albumin. Affinity for albumin can be achieved by conjugating the fusion protein with a compound, peptide, protein, or the like that has affinity for albumin. Fusion proteins into which affinity for albumin has been introduced circulate in the blood, at least a portion of which binds to albumin. Albumin has the function of stabilizing proteins bound to it. Therefore, by introducing affinity for albumin, the half-life of fusion proteins administered in vivo can be prolonged in the blood, thereby enhancing their efficacy. Introducing affinity for albumin is more effective when the anti-hTfR antibody lacks the Fc region that contributes to antibody stability, such as Fab.

[0094] Furthermore, introducing affinity for albumin is also effective when a fusion protein of an anti-hTfR antibody and another protein (A) exhibits immunogenicity when administered in vivo. The immunogenicity of the fusion protein is reduced because the immunogenic site of the fusion protein is prevented from being presented to immune cells by binding to albumin.

[0095] When affinity for albumin is introduced into a fusion protein of an anti-hTfR antibody and another protein (A), the part into which such affinity is introduced may be any of the light chain of the anti-hTfR antibody, the heavy chain of the anti-hTfR antibody, the other protein (A), or the linker part, or it may be introduced into two or more of these parts.

[0096] Examples of peptides or proteins with affinity for albumin include, but are not limited to, peptides obtained by modifying the albumin-binding domain of a protein derived from Streptococcus strain G418, which has the amino acid sequence set forth in SEQ ID NO: 74 (Alm T. Biotechnol J. 5. 605-17 (2010)), to exhibit alkali resistance. Methods for linking a peptide or protein with affinity for albumin (albumin affinity peptide) to a fusion protein of an anti-hTfR antibody and another protein (A) (anti-hTfR antibody-protein (A) fusion protein) include linking via a non-peptide linker or a peptide linker. Examples of non-peptide linkers that can be used include polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, or derivatives or combinations thereof. A peptide linker is a peptide chain or derivative thereof consisting of 1 to 50 peptide-bonded amino acids, and its N-terminus and C-terminus form covalent bonds with either the albumin affinity peptide or the fusion protein, respectively, thereby linking the albumin affinity peptide and the fusion protein.

[0097] A fusion protein of an albumin affinity peptide, an anti-hTfR antibody, and another protein (A) linked using PEG as a non-peptide linker is specifically referred to as an albumin affinity peptide-PEG-protein (A) fusion protein. The albumin affinity peptide-anti-hTfR antibody-protein (A) fusion protein can be produced by linking the albumin affinity peptide to PEG to prepare an albumin affinity peptide-PEG, and then linking the albumin affinity peptide-PEG to the anti-hTfR antibody-protein (A) fusion protein. Alternatively, the albumin affinity peptide-anti-hTfR antibody-protein (A) fusion protein can also be produced by linking the anti-hTfR antibody-protein (A) fusion protein to PEG to prepare an anti-hTfR antibody-protein (A) fusion protein, and then linking the anti-hTfR antibody-protein (A) fusion protein-PEG to the albumin affinity peptide. When binding PEG to the albumin affinity peptide and the anti-hTfR antibody-protein (A) fusion protein, PEG modified with a functional group such as carbonate, carbonylimidazole, activated ester of carboxylic acid, azlactone, cyclic imidothione, isocyanate, isothiocyanate, imidate, or aldehyde is used. These functional groups introduced into PEG react primarily with amino groups in the albumin affinity peptide and anti-hTfR antibody-protein (A) fusion protein molecules, thereby covalently binding PEG to the albumin affinity peptide and anti-hTfR antibody-protein (A) fusion protein. The molecular weight and shape of the PEG used are not particularly limited, but its average molecular weight (MW) is preferably 500 to 60,000, more preferably 500 to 20,000. For example, PEG having an average molecular weight of about 300, about 500, about 1000, about 2000, about 4000, about 10000, about 20000, etc. can be suitably used as a non-peptide linker.

[0098] For example, albumin affinity peptide-PEG can be obtained by mixing an albumin affinity peptide with aldehyde group-modified PEG (ALD-PEG-ALD) so that the molar ratio of the modified PEG to the albumin affinity peptide is 11, 12.5, 15, 110, 120, etc., and then adding a reducing agent such as NaCNBH3 to allow the mixture to react. The albumin affinity peptide-PEG is then reacted with an anti-hTfR antibody-protein (A) fusion protein in the presence of a reducing agent such as NaCNBH3 to obtain the albumin affinity peptide-PEG-anti-hTfR antibody-protein (A) fusion protein. Conversely, the albumin affinity peptide-PEG-anti-hTfR antibody-protein (A) fusion protein can also be obtained by first conjugating the anti-hTfR antibody-protein (A) fusion protein with ALD-PEG-ALD to prepare the anti-hTfR antibody-protein (A) fusion protein-PEG, and then conjugating the fusion protein-PEG to the albumin affinity peptide.

[0099] The anti-hTfR antibody-protein (A) fusion protein can also be fused with an albumin affinity peptide. Such a fusion protein (anti-hTfR antibody-protein (A) fusion protein-albumin affinity peptide) can be obtained by incorporating a DNA fragment in which a cDNA encoding an albumin affinity peptide is located in frame at the 3'-end or 5'-end of the cDNA encoding the heavy chain (including a fusion protein of the heavy chain and protein (A)) or light chain (including a fusion protein of the light chain and protein (A)) of the anti-hTfR antibody-protein (A) fusion protein, either directly or via a DNA fragment encoding a linker sequence, into an expression vector for mammalian cells, and then culturing mammalian cells into which this expression vector has been introduced. When a DNA fragment encoding an albumin affinity peptide is bound to a heavy chain (or a fusion protein of a heavy chain and protein (A)), an expression vector for mammalian cells incorporating a cDNA fragment encoding a fusion protein (or light chain) of the light chain constituting the anti-hTfR antibody and protein (A) is also introduced into the same host cells; when a DNA fragment encoding an albumin affinity peptide is bound to a light chain (or a fusion protein of a light chain and protein (A)), an expression vector for mammalian cells incorporating a cDNA fragment encoding a fusion protein (or heavy chain) of the heavy chain of the anti-hTfR antibody and protein (A) is also introduced into the same host cells. In other words, the albumin affinity peptide may be bound to either the N-terminus or C-terminus of the heavy chain (including a fusion protein of a heavy chain and protein (A)) or light chain (including a fusion protein of a light chain and protein (A)) of the anti-hTfR antibody-protein (A) fusion protein, but when protein (A) is bound to the N-terminus of the heavy chain of the anti-hTfR antibody, it is preferable to bind it to the C-terminus of the anti-hTfR antibody, and it is particularly preferable to bind it to the C-terminus of the heavy chain.

[0100] When the anti-hTfR antibody-protein (A) fusion protein is fused to an albumin affinity peptide, the fusion can be performed directly or via a linker sequence. The linker sequence preferably consists of 1 to 50 amino acids. The number of amino acids is adjusted appropriately to 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, 27, etc. The amino acid sequence of such a linker sequence is not limited, but is preferably composed of glycine and serine. Examples include those consisting of a single amino acid of either glycine or serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or sequences consisting of 50 or less amino acids formed by linking 1 to 10 or 2 to 5 of these amino acid sequences, or sequences consisting of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, or 25 amino acids, etc. For example, a sequence consisting of a total of 15 amino acids formed by three consecutive amino acid sequences of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3) can be suitably used as a linker sequence.

[0101] The binding affinity of the anti-hTfR antibody-protein (A) fusion protein to which the albumin-affinity peptide has been introduced is preferably 1×10 -7 M or less, more preferably 5X10 -7 M or less, more preferably 1X10 -8 M or less, and even more preferably 1X10 -9 It is below M.

[0102] Fab antibodies can also be stabilized in the blood by methods other than the introduction of an Fc region or an albumin affinity peptide. For example, Fab antibodies can be stabilized by PEG-modifying the Fab antibody itself or a fusion of a Fab antibody with another protein. This method is commonly used in the field of protein medicine, and PEGylated erythropoietin, interferon, and the like have been put to practical use as pharmaceuticals. Fab antibodies can also be stabilized by introducing mutations into them. For example, Fab antibodies can be stabilized by substituting leucine for the fourth methionine from the N-terminus of the light chain. However, the method for introducing mutations is not limited to this, and mutations can also be introduced into the heavy chain. Furthermore, methods for stabilizing Fab antibodies are not limited to these, and all well-known methods can be used.

[0103] There are no particular limitations on the other protein (A) to be bound to the anti-hTfR antibody, but it must be a protein that can exert physiological activity in the body, and in particular, a protein that should reach the brain to exert its function, but cannot pass through the blood-brain barrier as is, and therefore cannot be expected to function in the brain when administered intravenously. Such proteins include, for example, nerve growth factor (NGF), α-L-iduronidase (IDUA), iduronate-2-sulfatase (IDS), glucocerebrosidase (GBA), β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase (LAMAN), β-mannosidase, galactosylceramidase (GALC), saposin C, arylsulfatase A (ARSA), α-L-fucosidase (FUCA1), aspartylglucosaminidase, α-N-acetylgalactosaminidase, and acid sphingomyelinase. These include lysosomal enzymes such as lysosomal enzymes such as acetylcholinesterase (ASM), α-galactosidase A, β-glucuronidase (GUSB), heparan N-sulfatase (SGSH), α-N-acetylglucosaminidase (NAGLU), acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase (AC), amylo-1,6-glucosidase, sialidase, aspartylglucosaminidase, palmitoyl protein thioesterase-1 (PPT-1), tripeptidyl peptidase-1 (TPP-1), hyaluronidase-1, acid α-glucosidase (GAA), CLN1, and CLN2.

[0104] Nerve growth factor (NGF) conjugated with anti-hTfR antibody is a therapeutic agent for dementia in Alzheimer's disease, α-L-iduronidase (IDUA) fused with anti-hTfR antibody is a therapeutic agent for central nervous system disorders in Hurler syndrome or Hurler-Scheie syndrome, iduronate-2-sulfatase (IDS) fused with anti-hTfR antibody is a therapeutic agent for central nervous system disorders in Hunter syndrome, glucocerebrosidase (GBA) is a therapeutic agent for central nervous system disorders in Gaucher disease, and β-galactosidase is a therapeutic agent for GM1-gangliosidosis. GM2 activator protein is a therapeutic agent for central nervous system disorders in GM2-gangliosidosis types 1-3, GM2-gangliosidosis AB variant is a therapeutic agent for central nervous system disorders in Sandhoff disease and Tisachs disease, β-hexosaminidase B is a therapeutic agent for central nervous system disorders in Sandhoff disease, N-acetylglucosamine-1-phosphotransferase is a therapeutic agent for central nervous system disorders in I-cell disease, and α-mannosidase (LAMAN) is a therapeutic agent for central nervous system disorders in α-mannosidosis. As therapeutic agents, β-mannosidase is a therapeutic agent for central nervous system disorders in β-mannosidosis, galactosylceramidase (GALC) is a therapeutic agent for central nervous system disorders in Krabbe disease, saposin C is a therapeutic agent for central nervous system disorders in Gaucher-like storage diseases, arylsulfatase A (ARSA) is a therapeutic agent for central nervous system disorders in metachromatic leukodystrophy, α-L-fucosidase (FUCA1) is a therapeutic agent for central nervous system disorders in fucosidosis, and aspartylglucosaminidase is a therapeutic agent for aspartylglucosaminidase. α-N-acetylgalactosaminidase is used as a treatment for central nervous system disorders in glucosaminuria, α-N-acetylgalactosaminidase is used as a treatment for central nervous system disorders in Schindler disease and Kawasaki disease, acid sphingomyelinase (ASM) is used as a treatment for central nervous system disorders in Niemann-Pick disease, α-galactosidase A is used as a treatment for central nervous system disorders in Fabry disease, β-glucuronidase (GUSB) is used as a treatment for central nervous system disorders in Sly syndrome, heparan N-sulfatase (SGSH), α-N-acetylglucosaminidase (NAGLU),Acetyl-CoA α-glucosaminide N-acetyltransferase and N-acetylglucosamine-6-sulfate sulfatase are potential therapeutic agents for central nervous system disorders in Sanfilippo syndrome, acid ceramidase (AC) is a potential therapeutic agent for central nervous system disorders in Farber disease, amylo-1,6-glucosidase is a potential therapeutic agent for central nervous system disorders in Cori's disease (Forbes-Coli disease), sialidase is a potential therapeutic agent for central nervous system disorders in sialidase deficiency, and palmitoyl protein thioesterase-1 (PPT-1) is a potential therapeutic agent for central nervous system disorders in neuronal cell Tripeptidyl peptidase-1 (TPP-1) can be used as a therapeutic agent for central nervous system disorders in idiolipofuscinosis or Santavuori-Haltia disease; hyaluronidase-1 can be used as a therapeutic agent for central nervous system disorders in hyaluronidase deficiency; acid α-glucosidase (GAA) can be used as a therapeutic agent for central nervous system disorders in Pompe disease; and CLN1 and 2 can be used as therapeutic agents for central nervous system disorders in Batten disease. In particular, the anti-hTfR antibodies of the present invention are expected to reach the cerebral parenchyma, hippocampal neuron-like cells, and cerebellar Purkinje cells after passing through the blood-brain barrier, as well as neuron-like cells in the cerebral striatum and the substantia nigra of the midbrain, and therefore the medicinal efficacy of the proteins can be enhanced by fusing them with proteins that should exert their medicinal effects in these tissues or cells. However, its medicinal use is not limited to these diseases.

[0105] In addition, the therapeutic agents used in the present invention can also be used to prevent the onset of diseases.

[0106] Other proteins that can exert their therapeutic effects when bound to anti-hTfR antibodies include lysosomal enzymes, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, and interferon These include Ikin-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), various cytokines, tumor necrosis factor alpha receptors (TNF-α receptors), PD-1 ligands, PD-L1, PD-L2, enzymes with the activity of degrading beta-amyloid, anti-beta-amyloid antibodies, anti-BACE antibodies, anti-EGFR antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-HER2 antibodies, anti-TNF-α antibodies, anti-CTLA-4 antibodies, and other antibody drugs.

[0107] Lysosomal enzymes conjugated with anti-hTfR antibodies can be used as therapeutic agents for central nervous system disorders in lysosomal diseases; CNTF can be used as a therapeutic agent for amyotrophic lateral sclerosis; GDNF, neurotrophin 3, and neurotrophin 4 / 5 can be used as therapeutic agents for cerebral ischemia; GDNF can be used as a therapeutic agent for Parkinson's disease; neuregulin 1 can be used as a therapeutic agent for schizophrenia; erythropoietin and darbepoietin can be used as therapeutic agents for cerebral ischemia; bFGF and FGF2 can be used as therapeutic agents for traumatic central nervous system injuries and for recovery after brain surgery and spinal surgery; enzymes with beta-amyloid degrading activity, anti-beta-amyloid antibodies, and anti-BACE antibodies can be used as therapeutic agents for Alzheimer's disease; anti-EGFR antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-HER2 antibodies, and anti-CTLA-4 antibodies can be used as therapeutic agents for central nervous system tumors including brain tumors; and TNFαR-anti-hTfR antibodies can be used as a therapeutic agent for cerebral ischemia and cerebral inflammatory diseases.

[0108] Therapeutic agents for diseases such as neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease; psychiatric disorders such as schizophrenia and depression; multiple sclerosis, amyotrophic lateral sclerosis, central nervous system tumors including brain tumors; lysosomal diseases associated with encephalopathy; glycogen storage diseases, muscular dystrophies, cerebral ischemia, cerebral inflammatory diseases, prion diseases, and traumatic central nervous system disorders can generally be considered as other proteins (A) to be fused to anti-hTfR antibodies. Furthermore, therapeutic agents for viral and bacterial central nervous system diseases can also generally be considered as other proteins (A) to be fused to anti-hTfR antibodies. Furthermore, drugs that can be used for recovery after brain surgery or spinal surgery can also generally be considered as other proteins (A) to be fused to anti-hTfR antibodies.

[0109] As other proteins (A) to be bound to anti-hTfR antibodies, in addition to the natural (wild-type) proteins described above, analogs in which one or more amino acids of the natural (wild-type) proteins have been substituted with other amino acids or deleted, etc., are included in these proteins, as long as they fully or partially function as these proteins. When amino acids are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, the desired analogs can be obtained by combining these amino acid substitutions and deletions. Furthermore, proteins in which one or more amino acids have been added to the amino acid sequence or to the N-terminus or C-terminus of the natural (wild-type) proteins or their analogs are also included, as long as they fully or partially function as these proteins. In this case, the number of amino acids added is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. The addition, substitution, and deletion of these amino acids can be combined to produce a desired analogue of these proteins.

[0110] Furthermore, if a mutation is made into these other proteins (A) to add an amino acid to the C-terminus or N-terminus, and the added amino acid is located between these proteins and the anti-hTfR antibody when these proteins are fused with the anti-hTfR antibody, the added amino acid will form part of the linker.

[0111] Native human acid α-glucosidase (hGAA) is a type of lysosomal enzyme consisting of an 883 amino acid sequence shown in SEQ ID NO: 55. However, native hGAA also includes a sequence consisting of 896 amino acids shown in SEQ ID NO: 56, which is obtained by adding 13 more amino acids to the N-terminus of the amino acid sequence shown in SEQ ID NO: 55.

[0112] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) of the present invention is one in which natural hGAA is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hGAA bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. An antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and one having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody. An example of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 66 is SEQ ID NO: 67, and an example of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 68 is SEQ ID NO: 69.

[0113] Human acid α-glucosidase (hGAA), also known as α-1,4-glucosidase or acid maltase, has the activity of hydrolyzing α-1,4- and α-1,6-glycosidic bonds in lysosomal glycogen, thereby degrading glycogen. Pompe disease, also known as glycogen storage disease type II (GSD II), is a disease caused by intracellular glycogen accumulation due to a deficiency of intralysosomal acid α-glucosidase (acid maltase) activity. Patients with Pompe disease may also suffer from central nervous system disorders. hGAA conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with Pompe disease.

[0114] In the present invention, the term "human GAA" or "hGAA" particularly refers to hGAA having the same amino acid sequence as native hGAA, but is not limited to this and also includes hGAA obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hGAA, as long as it has hGAA activity. When amino acids in the amino acid sequence of hGAA are substituted 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 amino acids in the amino acid sequence of hGAA are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hGAA, 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 hGAA or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated hGAA preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hGAA.

[0115] Here, when hGAA is said to have hGAA activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hGAA. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hGAA. The same applies when the hGAA fused with an anti-hTfR antibody has been mutated.

[0116] A fusion protein of an anti-hTfR antibody and hGAA can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment having the nucleotide sequence shown in SEQ ID NO: 59, which encodes the amino acid sequence shown in SEQ ID NO: 58, and an expression vector incorporating a DNA fragment having the nucleotide sequence shown in SEQ ID NO: 24, which encodes the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The produced fusion protein can be used as a therapeutic agent for Pompe disease, particularly as a therapeutic agent for central nervous system disorders associated with Pompe disease. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hGAA.

[0117] In addition, when an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or hGAA, if the added amino acid is positioned between the anti-hTfR antibody and hGAA, the added amino acid will form part of the linker.

[0118] Native human I2S (hI2S) is a type of lysosomal enzyme consisting of a 525-amino acid sequence shown in SEQ ID NO: 50. A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) of the present invention is one in which native human I2S is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence shown in SEQ ID NO: 23, and human I2S is bound to the C-terminus of the heavy chain via a linker consisting of the amino acid sequence Gly-Ser, forming the amino acid sequence shown in SEQ ID NO: 53. The protein having the amino acid sequence shown in SEQ ID NO: 53 is a fusion of the heavy chain of an IgG1-type anti-hTfR antibody with hI2S, and the heavy chain portion has the amino acid sequence shown in SEQ ID NO: 66. A fusion of an IgG4-type anti-hTfR antibody with hI2S can also be obtained by replacing this heavy chain portion with the amino acid sequence shown in SEQ ID NO: 68.

[0119] Human I2S (hI2S) has the activity of hydrolyzing sulfate ester bonds in heparan sulfate and dermatan sulfate, which belong to the glycosaminoglycans. Patients with Hunter syndrome, who have a genetic abnormality in this enzyme, have abnormal metabolism of heparan sulfate and dermatan sulfate, resulting in the accumulation of these partial degradation products in tissues such as the liver and spleen, leading to symptoms such as skeletal abnormalities. Patients with Hunter syndrome may also suffer from central nervous system disorders. hI2S conjugated with an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with Hunter syndrome.

[0120] In the present invention, the terms "human I2S" or "hI2S" particularly refer to hI2S having the same amino acid sequence as native hI2S. However, this is not limited to this, and includes hI2S obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hI2S, as long as the mutations have I2S activity. When amino acids in the amino acid sequence of hI2S are substituted 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 amino acids in the amino acid sequence of hI2S are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hI2S, 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 hI2S or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated hI2S preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hI2S.

[0121] Here, when hI2S is said to have I2S activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hI2S. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hI2S. The same applies when the hI2S fused with an anti-hTfR antibody has been mutated.

[0122] A fusion protein of an anti-hTfR antibody and hGAA can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment having the nucleotide sequence of SEQ ID NO: 54, which encodes the amino acid sequence of SEQ ID NO: 53, and an expression vector incorporating a DNA fragment having the nucleotide sequence of SEQ ID NO: 24, which encodes the amino acid sequence of SEQ ID NO: 23 (anti-hTfR antibody light chain), and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for Pompe disease, particularly for central nervous system disorders associated with Pompe disease. The fusion protein obtained in this manner is a fusion protein of an IgG1-type humanized anti-hTfR antibody and hGAA.

[0123] In addition, if an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or human I2S, and the added amino acid is positioned between the anti-hTfR antibody and human I2S, the added amino acid will form part of the linker.

[0124] Native human α-L-iduronidase (hIDUA) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO: 75 or 76. The hIDUA shown in SEQ ID NO: 76 is a type in which Ala-Pro has been added to the N-terminus of the hIDUA shown in SEQ ID NO: 75.

[0125] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which natural hIDUA is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hIDUA bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0126] Human α-L-iduronidase (hIDUA) is a lysosomal enzyme that hydrolyzes iduronic acid bonds present in dermatan sulfate and heparan sulfate molecules. Hurler syndrome, also known as mucopolysaccharidosis type I, is a disease caused by the accumulation of dermatan sulfate and other substances in the cells due to a deficiency of α-L-iduronidase activity in the lysosomes. Patients with Hurler syndrome may suffer from central nervous system disorders. hIDUA conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with Hurler syndrome.

[0127] In the present invention, the term "human IDUA" or "hIDUA" particularly refers to hIDUA having the same amino acid sequence as native hIDUA. However, this is not limited to this, and includes hIDUA obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hIDUA, as long as it has hIDUA activity. When amino acids in the amino acid sequence of hIDUA are substituted 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 amino acids in the amino acid sequence of hIDUA are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added 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 that combine these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of the mutated hIDUA preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hIDUA.

[0128] Here, when hIDUA is said to have hIDUA activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hIDUA. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hIDUA. The same applies when the hIDUA fused with an anti-hTfR antibody has been mutated.

[0129] A fusion protein of an anti-hTfR antibody and hIDUA can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 90 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for Hurler syndrome, particularly as a therapeutic agent for central nervous system disorders associated with Hurler syndrome. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hIDUA.

[0130] In addition, if an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or hIDUA, and the added amino acid is positioned between the anti-hTfR antibody and hIDUA, the added amino acid will form part of the linker.

[0131] Native human palmitoyl protein thioesterase-1 (hPPT-1) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:77.

[0132] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which native hPPT-1 is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hPPT-1 bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0133] Neuronal ceroid lipofuscinosis (including Santavuori-Haltia disease) is a disease caused by the accumulation of intracellular ceroid lipofuscin due to a deficiency of palmitoyl protein thioesterase-1 activity in lysosomes. Patients with neuronal ceroid lipofuscinosis may suffer from central nervous system disorders. hPPT-1 conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with neuronal ceroid lipofuscinosis.

[0134] In the present invention, the terms "human PPT-1" or "hPPT-1" particularly refer to hPPT-1 having the same amino acid sequence as native hPPT-1. However, this is not limiting, and hPPT-1 also includes mutations such as substitutions, deletions, and additions to the amino acid sequence of native hPPT-1, so long as they have hPPT-1 activity. When amino acids in the amino acid sequence of hPPT-1 are substituted 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 amino acids in the amino acid sequence of hPPT-1 are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hPPT-1, 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 hPPT-1 or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of mutated hPPT-1 preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hPPT-1.

[0135] Here, when hPPT-1 is said to have hPPT-1 activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hPPT-1. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hPPT-1. The same applies when the hPPT-1 fused with an anti-hTfR antibody has been mutated.

[0136] A fusion protein of an anti-hTfR antibody and hPPT-1 can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 100 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for neuronal ceroid lipofuscinosis, particularly as a therapeutic agent for central nervous system disorders associated with neuronal ceroid lipofuscinosis. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hPPT-1.

[0137] In addition, if an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or hPPT-1, and the added amino acid is positioned between the anti-hTfR antibody and hPPT-1, the added amino acid will form part of the linker.

[0138] Native human acid sphingomyelinase (hASM) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:78.

[0139] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which natural hASM is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hASM bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0140] Human acid sphingomyelinase (hASM) is a lysosomal enzyme that hydrolyzes sphingomyelin into choline phosphate and ceramide. Niemann-Pick disease is classified into types A through F based on differences in etiology and symptoms. Types A and B are caused by a genetic deficiency of acid sphingomyelinase (ASM). Niemann-Pick disease is caused by intracellular accumulation of sphingomyelin due to a deficiency of intralysosomal acid sphingomyelinase activity. Patients with Niemann-Pick disease may also experience central nervous system disorders. hASM conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with Niemann-Pick disease.

[0141] In the present invention, the terms "human ASM" or "hASM" particularly refer to hASM having the same amino acid sequence as native hASM. However, this is not limited to this, and includes hASM obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hASM, as long as it has hASM activity. When amino acids in the amino acid sequence of hASM are substituted 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 amino acids in the amino acid sequence of hASM are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hASM, 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 within the amino acid sequence of hASM or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of mutated hASM preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hASM.

[0142] Here, when hASM is said to have hASM activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hASM. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hASM. The same applies when the hASM fused with an anti-hTfR antibody has been mutated.

[0143] A fusion protein of an anti-hTfR antibody and hASM can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 106 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and then culturing the host cells. The fusion protein produced can be used as a therapeutic agent for Niemann-Pick disease, particularly as a therapeutic agent for central nervous system disorders associated with Niemann-Pick disease. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hASM.

[0144] In addition, when an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or hASM, if the added amino acid is positioned between the anti-hTfR antibody and hASM, the added amino acid will form part of the linker.

[0145] Native human arylsulfatase A (hARSA) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:79.

[0146] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which natural hARSA is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hARSA bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0147] Metachromatic leukodystrophy (MED) is a disease caused by the accumulation of intracellular sulfatides due to a deficiency in lysosomal arylsulfatase A activity. Patients with MED may experience central nervous system disorders. hARSA conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with MED.

[0148] In the present invention, the terms "human ARSA" or "hARSA" particularly refer to hARSA having the same amino acid sequence as native hARSA, but are not limited to this and also include hARSA obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hARSA, as long as it has hARSA activity. When amino acids in the amino acid sequence of hARSA are substituted 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 amino acids in the amino acid sequence of hARSA are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hARSA, 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 hARSA or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of mutated hARSA preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hARSA.

[0149] Here, when hARSA is said to have hARSA activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hARSA. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hARSA. The same applies when the hARSA fused with an anti-hTfR antibody has been mutated.

[0150] A fusion protein of an anti-hTfR antibody and hARSA can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 115 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for metachromatic leukodystrophy, particularly as a therapeutic agent for central nervous system disorders associated with metachromatic leukodystrophy. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hARSA.

[0151] In addition, when an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or hARSA, if the added amino acid is positioned between the anti-hTfR antibody and hARSA, the added amino acid will form part of the linker.

[0152] Native human heparan N-sulfatase (hSGSH) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:80.

[0153] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which natural hSGSH is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hSGSH bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0154] Sanfilippo syndrome, also known as mucopolysaccharidosis type II (MPS type III), is a disease caused by the accumulation of intracellular heparan sulfate due to a deficiency in intralysosomal heparan N-sulfatase activity. However, deficiencies of other enzymes, such as α-N-acetylglucosaminidase, can also be a cause of the disease. Patients with Sanfilippo syndrome may also suffer from central nervous system disorders. hSGSH conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with Sanfilippo syndrome.

[0155] In the present invention, the terms "human SGSH" or "hSGSH" particularly refer to hSGSH having the same amino acid sequence as native hSGSH, but are not limited to this and also include hSGSH obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hSGSH, as long as the resulting hSGSH has hSGSH activity. When amino acids in the amino acid sequence of hSGSH are substituted 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 amino acids in the amino acid sequence of hSGSH are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hSGSH, 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 hSGSH or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of mutated hSGSH preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hSGSH.

[0156] Here, when hSGSH is said to have hSGSH activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hSGSH. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hSGSH. The same applies when the hSGSH fused with an anti-hTfR antibody has been mutated.

[0157] A fusion protein of an anti-hTfR antibody and hSGSH can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 124 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for Sanfilippo syndrome, particularly as a therapeutic agent for central nervous system disorders associated with Sanfilippo syndrome. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hSGSH.

[0158] In addition, when an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or hSGSH, if the added amino acid is positioned between the anti-hTfR antibody and hSGSH, the added amino acid will form part of the linker.

[0159] Native human glucocerebrosidase (hGBA) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:81.

[0160] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which natural hGBA is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hGBA bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0161] Human glucocerebrosidase (hGBA) is a lysosomal enzyme that hydrolyzes the glycolipid glucocerebroside (or glucosylceramide). Gaucher disease is a disease caused by the accumulation of glucocerebroside in cells due to a deficiency of glucocerebrosidase activity in lysosomes. Patients with Gaucher disease may suffer from central nervous system disorders. hGBA conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with Gaucher disease.

[0162] In the present invention, the terms "human GBA" or "hGBA" particularly refer to hGBA having the same amino acid sequence as native hGBA, but are not limited to this and also include hGBA obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hGBA, as long as they have hGBA activity. When amino acids in the amino acid sequence of hGBA are substituted 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 amino acids in the amino acid sequence of hGBA are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hGBA, 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 hGBA or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated hGBA preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hGBA.

[0163] Here, when hGBA is said to have hGBA activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hGBA. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hGBA. The same applies when the hGBA fused with an anti-hTfR antibody has been mutated.

[0164] A fusion protein of an anti-hTfR antibody and hGBA can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 130 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for Gaucher disease, particularly as a therapeutic agent for central nervous system disorders associated with Gaucher disease. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hGBA.

[0165] In addition, when an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or hGBA, if the added amino acid is positioned between the anti-hTfR antibody and hGBA, the added amino acid will form part of the linker.

[0166] Naturally occurring human tripeptidyl peptidase-1 (hTPP-1) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:82.

[0167] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which natural hTPP-1 is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hTPP-1 bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0168] Neuronal ceroid lipofuscinosis (including Santavuori-Haltia disease) is a disease caused by the accumulation of intracellular lipofuscin due to a deficiency in intralysosomal tripeptidyl peptidase-1 activity. Patients with neuronal ceroid lipofuscinosis may suffer from central nervous system disorders. hTPP-1 conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with neuronal ceroid lipofuscinosis.

[0169] In the present invention, the terms "human TPP-1" or "hTPP-1" particularly refer to hTPP-1 having the same amino acid sequence as native hTPP-1. However, this is not limited to this, and hTPP-1 also includes mutations such as substitutions, deletions, and additions to the amino acid sequence of native hTPP-1, as long as they have hTPP-1 activity. When amino acids in the amino acid sequence of hTPP-1 are substituted 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 amino acids in the amino acid sequence of hTPP-1 are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hTPP-1, 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 hTPP-1 or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of mutated hTPP-1 preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hTPP-1.

[0170] Here, when hTPP-1 is said to have hTPP-1 activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hTPP-1. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hTPP-1. The same applies when the hTPP-1 fused with an anti-hTfR antibody has been mutated.

[0171] A fusion protein of an anti-hTfR antibody and hTPP-1 can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 136 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for neuronal ceroid lipofuscinosis, particularly as a therapeutic agent for central nervous system disorders associated with neuronal ceroid lipofuscinosis. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hTPP-1.

[0172] In addition, if an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or hTPP-1, and the added amino acid is positioned between the anti-hTfR antibody and hTPP-1, the added amino acid will form part of the linker.

[0173] Native human α-N-acetylglucosaminidase (hNAGLU) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:83.

[0174] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) of the present invention is one in which natural hNAGLU is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hNAGLU bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0175] Sanfilippo syndrome is a disease caused by the accumulation of intracellular heparan sulfate due to a deficiency of α-N-acetylglucosaminidase activity in lysosomes. Patients with Sanfilippo syndrome may suffer from central nervous system disorders. hNAGLU conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with Sanfilippo syndrome.

[0176] In the present invention, the term "human NAGLU" or "hNAGLU" particularly refers to hNAGLU having the same amino acid sequence as native hNAGLU, but is not limited to this. hNAGLU also includes mutations such as substitutions, deletions, and additions to the amino acid sequence of native hNAGLU, as long as they have hNAGLU activity. When amino acids in the amino acid sequence of hNAGLU are substituted 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 amino acids in the amino acid sequence of hNAGLU are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hNAGLU, 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 hNAGLU or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of mutated hNAGLU preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hNAGLU.

[0177] Here, when hNAGLU is said to have hNAGLU activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hNAGLU. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hNAGLU. The same applies when the hNAGLU fused with an anti-hTfR antibody has been mutated.

[0178] A fusion protein of an anti-hTfR antibody and hNAGLU can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 142 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for Sanfilippo syndrome, particularly as a therapeutic agent for central nervous system disorders associated with Sanfilippo syndrome. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hNAGLU.

[0179] In addition, if a mutation is made into an anti-hTfR antibody or hNAGLU to add an amino acid to the C-terminus or N-terminus, and the added amino acid is positioned between the anti-hTfR antibody and hNAGLU, the added amino acid will form part of the linker.

[0180] Native human β-glucuronidase (hGUSB) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:84.

[0181] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) in the present invention is one in which a native hGUSB is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hGUSB bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0182] Sly syndrome, also known as mucopolysaccharidosis type VII (MPS VII), is a disease caused by the accumulation of intracellular mucopolysaccharides due to a deficiency of intralysosomal β-glucuronidase activity. Patients with Sly syndrome may experience central nervous system disorders. hGUSB conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with Sly syndrome.

[0183] In the present invention, the terms "human GUSB" or "hGUSB" particularly refer to an hGUSB having the same amino acid sequence as native hGUSB, but are not limited to this. hGUSBs also include those obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hGUSB, as long as they have hGUSB activity. When amino acids in the amino acid sequence of hGUSB are replaced with other amino acids, the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. When amino acids in the amino acid sequence of hGUSB are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hGUSB, 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 within the amino acid sequence of hGUSB or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated hGUSB preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hGUSB.

[0184] Here, when hGUSB is said to have hGUSB activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherently possessed by natural hGUSB. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherently possessed by natural hGUSB. The same applies when the hGUSB fused with an anti-hTfR antibody has been mutated.

[0185] A fusion protein of an anti-hTfR antibody and hGUSB can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 150 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hGUSB. The produced fusion protein can be used as a therapeutic agent for Sly syndrome, particularly as a therapeutic agent for central nervous system disorders associated with Sly syndrome.

[0186] In addition, when a mutation is made into an anti-hTfR antibody or hGUSB to add an amino acid to the C-terminus or N-terminus, if the added amino acid is positioned between the anti-hTfR antibody and the hGUSB, the added amino acid will form part of the linker.

[0187] Native human galactosylceramidase (hGALC) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:85.

[0188] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which native hGALC is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hGALC bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0189] Krabbe disease is a disease caused by a deficiency of galactosylceramidase activity. Patients with Krabbe disease may suffer from central nervous system disorders. hGALC conjugated to anti-hTfR antibodies can be used as a therapeutic agent for central nervous system disorders associated with Krabbe disease.

[0190] In the present invention, the terms "human GALC" or "hGALC" particularly refer to hGALC having the same amino acid sequence as native hGALC, but are not limited thereto, and include hGALCs obtained by adding mutations such as substitutions, deletions, and additions to the native hGALC amino acid sequence, as long as they have hGALC activity. When amino acids in the hGALC amino acid sequence are substituted 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 amino acids in the hGALC amino acid sequence are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hGALC, 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 hGALC or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of mutated hGALC preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hGALC.

[0191] Here, when hGALC is said to have hGALC activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hGALC. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hGALC. The same applies when the hGALC fused with an anti-hTfR antibody has been mutated.

[0192] A fusion protein of an anti-hTfR antibody and hGALC can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 158 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for Krabbe disease, particularly for central nervous system disorders associated with Krabbe disease. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hGALC.

[0193] In addition, if a mutation is made in an anti-hTfR antibody or hGALC to add an amino acid to the C-terminus or N-terminus, and the added amino acid is positioned between the anti-hTfR antibody and hGALC, the added amino acid will form part of the linker.

[0194] Naturally occurring human acid ceramidase (hAC) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:86.

[0195] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which natural hAC is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hAC bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0196] Farber disease is a disease caused by the accumulation of intracellular ceramide due to a deficiency in lysosomal acid ceramidase activity. Patients with Farber disease may also suffer from central nervous system disorders. hAC conjugated to an anti-hTfR antibody can be used as a therapeutic agent for the central nervous system disorders associated with Farber disease.

[0197] In the present invention, the term "human AC" or "hAC" particularly refers to hAC having the same amino acid sequence as native hAC. However, this is not limited to this, and includes hACs obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hAC, as long as they have hAC activity. When amino acids in the amino acid sequence of hAC are substituted 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 amino acids in the amino acid sequence of hAC are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hAC, 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 or the N-terminus or C-terminus of hAC. Mutations that combine these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of the mutated hAC has a homology of preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more with the amino acid sequence of the original hAC.

[0198] Here, when hAC is said to have hAC activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hAC. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hAC. The same applies when the hAC fused with an anti-hTfR antibody has been mutated.

[0199] A fusion protein of an anti-hTfR antibody and hAC can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 166 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for Farber disease, particularly as a therapeutic agent for central nervous system disorders associated with Farber disease. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hAC.

[0200] In addition, when an amino acid is added to the C-terminus or N-terminus by making a mutation in an anti-hTfR antibody or hAC, if the added amino acid is positioned between the anti-hTfR antibody and the hAC, the added amino acid will form part of the linker.

[0201] Native human α-L-fucosidase (hFUCA1) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:87.

[0202] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) in the present invention is one in which native hFUCA1 is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hFUCA1 bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0203] Fucosidosis is a disease caused by the accumulation of intracellular fucose-containing oligosaccharides due to a deficiency of α-L-fucosidase activity in lysosomes. Patients with fucosidosis may suffer from central nervous system disorders. hFUCA1 conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with fucosidosis.

[0204] In the present invention, the terms "human FUCA1" or "hFUCA1" particularly refer to hFUCA1 having the same amino acid sequence as native hFUCA1, but are not limited to this. hFUCA1 also includes mutations such as substitutions, deletions, and additions to the amino acid sequence of native hFUCA1, as long as they have hFUCA1 activity. When amino acids in the amino acid sequence of hFUCA1 are substituted 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 amino acids in the amino acid sequence of hFUCA1 are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hFUCA1, 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 hFUCA1 or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of mutated hFUCA1 preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hFUCA1.

[0205] Here, when hFUCA1 is said to have hFUCA1 activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hFUCA1. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hFUCA1. The same applies when the hFUCA1 fused with an anti-hTfR antibody has been mutated.

[0206] A fusion protein of an anti-hTfR antibody and hFUCA1 can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 174 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for fucosidosis, particularly as a therapeutic agent for central nervous system disorders associated with fucosidosis. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hFUCA1.

[0207] In addition, if a mutation is made in an anti-hTfR antibody or hFUCA1 to add an amino acid to the C-terminus or N-terminus, and the added amino acid is positioned between the anti-hTfR antibody and hFUCA1, the added amino acid will form part of the linker.

[0208] Native human α-mannosidase (hLAMAN) is a type of lysosomal enzyme composed of the amino acid sequence shown in SEQ ID NO:88.

[0209] A specific example of a fusion protein of an anti-hTfR antibody and another protein (A) according to the present invention is one in which natural hLAMAN is fused to the C-terminus of the anti-hTfR antibody heavy chain via the amino acid sequence Gly-Ser as a linker sequence. An example of such a fusion protein is one in which the light chain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 66 or 68, with hLAMAN bound to the C-terminus via a peptide bond via the linker sequence Gly-Ser. The antibody having the amino acid sequence set forth in SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the antibody having the amino acid sequence set forth in SEQ ID NO: 68 is an IgG4-type anti-hTfR antibody.

[0210] Human α-mannosidase (hLAMAN) is a lysosomal enzyme that hydrolyzes α-mannose. α-Mannosidosis is a disease caused by the accumulation of mannose-containing oligosaccharides in cells due to a deficiency of α-mannosidase activity in lysosomes. Patients with α-mannosidosis may suffer from central nervous system disorders. hLAMAN conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with α-mannosidosis.

[0211] In the present invention, the terms "human LAMAN" or "hLAMAN" particularly refer to hLAMAN having the same amino acid sequence as native hLAMAN. However, this is not limited to this, and includes hLAMANs obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of native hLAMAN, so long as they have hLAMAN activity. When amino acids in the amino acid sequence of hLAMAN are substituted 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 amino acids in the amino acid sequence of hLAMAN are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to hLAMAN, 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 hLAMAN or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of mutated hLAMAN preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original hLAMAN.

[0212] Here, when hLAMAN is said to have hLAMAN activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in natural hLAMAN. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hLAMAN. The same applies when the hLAMAN fused with an anti-hTfR antibody has been mutated.

[0213] A fusion protein of an anti-hTfR antibody and hLAMAN can be produced, for example, by transforming host cells such as mammalian cells with an expression vector incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 182 and an expression vector incorporating a DNA fragment encoding the anti-hTfR antibody light chain having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The fusion protein produced can be used as a therapeutic agent for α-mannosidosis, particularly as a therapeutic agent for central nervous system disorders associated with α-mannosidosis. The fusion protein obtained in this manner is a fusion protein of an IgG4-type humanized anti-hTfR antibody and hLAMAN.

[0214] In addition, if a mutation is made into an anti-hTfR antibody or hLAMAN to add an amino acid to the C-terminus or N-terminus, and the added amino acid is positioned between the anti-hTfR antibody and the hLAMAN, the added amino acid will form part of the linker.

[0215] As described above, examples of fusion proteins of anti-hTfR antibodies and other proteins (A) have been given in which the other proteins (A) are hGAA, hI2S, hIDUA, hPPT-1, hASM, hARSA, hSGSH, hGBA, hTTP-1, hNAGLU, hGUSB, hAC, hFUca1, and hLAMAN. However, in preferred embodiments of fusion proteins of anti-hTfR antibodies and other proteins (A) including these, there are no particular restrictions on the amino acid sequences of the CDRs of the heavy and light chains of the anti-hTfR antibodies, as long as the antibody has specific affinity for hTfR. However, the anti-TfR antibody used herein, particularly when measured by the method described in Example 7, The dissociation constant with human TfR is preferably 1X10 -10 M or less, more preferably 1X10 -11 M or less, more preferably 5X10 -12 M or less, and even more preferably 1X10 -12 M or less, The dissociation constant with monkey TfR is preferably 1X10 -9 M or less, more preferably 5X10 -10 M or less, more preferably 1X10 -10 M or less, for example 7.5X10 -11 It is M or less. For example, the dissociation constants for human TfR and monkey TfR are 1X10 -10 M or less and 1X10 -9 M or less, 1X10 -11 M or less and 5X10 -10 M or less, 5X10 -12 M or less and 1X10 -10 M or less, 5X10 -12 M or less and 7.5X10 -11 M or less, 1X10 -12 M or less and 1X10 -10 M or less, 1X10 -12 M or less and 7.5X10 -11 There is no particular lower limit to the dissociation constant with human TfR, but for example, -13 M,1X10 -13M, etc. In addition, there is no particularly clear lower limit for the dissociation constant with monkey TfR, but for example, -11 M,1X10 -12 M, etc. The same applies to the case where the antibody is a single-chain antibody.

[0216] A relatively short peptide chain can also be bound to an anti-hTfR antibody using the same technique as for binding other proteins (A) to an anti-hTfR antibody. There are no limitations on the peptide chain to be bound to an anti-hTfR antibody, as long as the peptide chain has the desired physiological activity, and examples include peptide chains having the amino acid sequence of a region that exerts the physiological activity of various proteins. There are no particular limitations on the length of the peptide chain, but it is preferably composed of 2 to 200 amino acids, for example, 5 to 50 amino acids.

[0217] When binding a low molecular weight substance to an anti-hTfR antibody, there are no particular limitations on the low molecular weight substances that can be used as candidates, but they are low molecular weight substances that cannot be expected to function in the brain when administered intravenously because they cannot pass through the blood-brain barrier as they are, even though they should reach the brain and exert their function there. For example, such low molecular weight substances include cyclophosphamide, ifosfamide, melphalan, busulfan, thioteva, nimustine, ranimustine, dacarbazine, procarbazine, temozolomide, carmustine, streptozotocin, pendamustine, cisplatin, carboplatin, oxaliplatin, nedaplatin, 5-fluorouracil, sulfadiazine, sulfamethoxazole, methotrexate, Examples of anticancer drugs include trimethoprim, pyrimethamine, fluorouracil, flucytosine, azathioprine, pentostatin, hydroxyurea, fludarabine, cytarabine, gemcitabine, irinotecan, doxorubicin, etoposide, levofloxacin, ciprofloxacin, vinblastine, vincristine, paclitaxel, dodetaxel, mitomycin C, doxorubicin, and epirubicin. Other low-molecular-weight substances that can be bound to anti-hTfR antibodies include siRNA, antisense DNA, and short peptides.

[0218] When an anti-hTfR antibody is bound to a low molecular weight substance, the low molecular weight substance may be bound to either the light chain or the heavy chain alone, or to both the light chain and the heavy chain. Furthermore, as long as the anti-hTfR antibody has affinity for hTfR, it may contain an amino acid sequence containing all or part of the variable region of the light chain and / or an amino acid sequence containing all or part of the variable region of the heavy chain.

[0219] Therapeutic agents for diseases such as neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease; psychiatric disorders such as schizophrenia and depression; multiple sclerosis, amyotrophic lateral sclerosis, central nervous system tumors including brain tumors; lysosomal diseases associated with encephalopathy; glycogen storage diseases, muscular dystrophies, cerebral ischemia, cerebral inflammatory diseases, prion diseases, and traumatic central nervous system disorders are generally potential candidates for small molecule substances to be fused to anti-hTfR antibodies. Additionally, therapeutic agents for viral and bacterial central nervous system diseases are also generally potential candidates for small molecule substances to be fused to anti-hTfR antibodies. Furthermore, drugs that can be used for recovery after brain surgery or spinal surgery are also generally potential candidates for small molecule substances to be fused to anti-hTfR antibodies.

[0220] If an anti-hTfR antibody is derived from a non-human animal, there is a high risk that its administration to humans will trigger an antigen-antibody reaction, resulting in undesirable side effects. By humanizing non-human animal antibodies, such antigenicity can be reduced, thereby suppressing the occurrence of side effects caused by antigen-antibody reactions when administered to humans. Furthermore, experiments using monkeys have reported that humanized antibodies are more stable in the blood than mouse antibodies, which is thought to enable the therapeutic effect to be sustained for a longer period of time. The occurrence of side effects caused by antigen-antibody reactions can also be suppressed by using a human antibody as the anti-hTfR antibody.

[0221] The case where the anti-hTfR antibody is a humanized antibody or a human antibody will be explained in more detail below. Human antibody light chains include λ chains and κ chains. The light chain constituting an anti-hTfR antibody may be either a λ chain or a κ chain. Human heavy chains include γ chains, μ chains, α chains, σ chains, and ε chains, which correspond to IgG, IgM, IgA, IgD, and IgE, respectively. The heavy chain constituting an anti-hTfR antibody may be any of γ chains, μ chains, α chains, σ chains, and ε chains, but is preferably a γ chain. Furthermore, the γ chain of human heavy chains includes γ1 chains, γ2 chains, γ3 chains, and γ4 chains, which correspond to IgG1, IgG2, IgG3, and IgG4, respectively. When the heavy chain constituting an anti-hTfR antibody is a γ chain, the γ chain may be any of γ1 chains, γ2 chains, γ3 chains, and γ4 chains, but is preferably a γ1 chain or γ4 chain. When the anti-hTfR antibody is a humanized antibody or a human antibody and is an IgG, the light chain of the human antibody may be either a λ chain or a κ chain, and the heavy chain of the human antibody may be either a γ1 chain, a γ2 chain, a γ3 chain, or a γ4 chain, but is preferably a γ1 chain or a γ4 chain. For example, one preferred embodiment of an anti-hTfR antibody is one in which the light chain is a λ chain and the heavy chain is a γ1 chain.

[0222] When the anti-hTfR antibody is a humanized or human antibody, the anti-hTfR antibody and the other protein (A) can be linked by a peptide bond such that the C-terminus (or N-terminus) of the other protein (A) is linked to the N-terminus (or C-terminus) of the heavy or light chain of the anti-hTfR antibody via a linker sequence or directly. When the other protein (A) is linked to the N-terminus (or C-terminus) of the heavy chain of the anti-hTfR antibody, the C-terminus (or N-terminus) of the other protein (A) is linked to the N-terminus (or C-terminus) of the γ-chain, μ-chain, α-chain, σ-chain, or ε-chain of the anti-hTfR antibody via a linker sequence or directly. When another protein (A) is bound to the N-terminus (or C-terminus) of the light chain of an anti-hTfR antibody, the C-terminus (or N-terminus) of the other protein (A) is bound to the N-terminus (or C-terminus) of the λ chain or κ chain of the anti-hTfR antibody by a peptide bond, either directly or via a linker sequence. However, when the anti-hTfR antibody consists of a Fab region or consists of a Fab region and all or part of the hinge region (Fab, F(ab')2, and F(ab')), the C-terminus (or N-terminus) of the other protein (A) can be bound to the N-terminus (or C-terminus) of the heavy or light chain constituting the Fab, F(ab')2, and F(ab'), either via a linker sequence or directly, by a peptide bond.

[0223] In a fusion protein in which another protein (A) is bound to the C-terminus or N-terminus of the light chain of an anti-hTfR antibody, which is a humanized or human antibody, the anti-human transferrin receptor antibody comprises an amino acid sequence containing all or part of the variable region of the light chain and an amino acid sequence containing all or part of the variable region of the heavy chain. Here, the light chain of the anti-hTfR antibody and the other protein (A) may be bound directly or via a linker.

[0224] In a fusion protein in which another protein (A) is bound to the C-terminus or N-terminus of the heavy chain of an anti-hTfR antibody, which is a humanized or human antibody, the anti-human transferrin receptor antibody comprises an amino acid sequence containing all or part of the variable region of the light chain and an amino acid sequence containing all or part of the variable region of the heavy chain. Here, the heavy chain of the anti-hTfR antibody and the other protein (A) may be bound directly or via a linker.

[0225] When a linker sequence is placed between an anti-hTfR antibody or human antibody and another protein (A), the linker sequence placed between the anti-hTfR antibody and another protein (A) is preferably a peptide chain consisting of 1 to 50 amino acids. However, depending on the other protein (A) to be bound to the anti-hTfR antibody, the number of amino acids constituting the linker sequence is appropriately adjusted to 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, etc. Such a linker sequence is not limited to any particular amino acid sequence, as long as the anti-hTfR antibody and the other protein (A) linked by the linker sequence retain their respective functions (affinity for hTfR and activity or function under physiological conditions), but is preferably composed of glycine and serine, such as a sequence consisting of a single amino acid, either glycine or serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence consisting of 1 to 10 or 2 to 5 consecutive amino acids of these amino acid sequences. For example, a linker sequence consisting of a total of 27 amino acids, consisting of the amino acid sequence Gly-Ser followed by five consecutive amino acids of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), can be suitably used. Furthermore, a linker sequence containing a total of 25 amino acids consisting of five consecutive amino acids of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3) can also be suitably used.

[0226] Here, when another protein (A) fused to an anti-hTfR antibody or human antibody is said to retain the activity or function of the other protein (A) under physiological conditions, or simply to have activity, it means that the other protein (A) retains 3% or more of the activity that the natural form of the other protein (A) originally possesses. However, the activity or function is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity that the natural form of the other protein (A) originally possesses. The same applies when the other protein (A) fused to an anti-hTfR antibody is a mutated protein.

[0227] A further specific example of a fusion protein of a humanized anti-hTfR antibody or human antibody with another protein (A) in the present invention is one in which the other protein (A) is fused to the C-terminus of the anti-hTfR antibody heavy chain via a linker sequence consisting of 27 amino acids in total, which consists of the amino acid sequence Gly-Ser followed by five consecutive amino acid sequences of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

[0228] As an example of a specific embodiment of the fusion protein of the humanized anti-hTfR antibody or human antibody of the present invention with another protein (A) when the anti-hTfR antibody is Fab, the other protein (A) is connected to the C-terminal side of the other protein (A) via a linker sequence consisting of 25 amino acids in total, consisting of five consecutive amino acid sequences of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), which connects the variable region of the anti-hTfR antibody heavy chain to the C H In this case, C H In addition to the region, a part of the hinge region may be included, but the hinge region does not contain cysteine ​​residues that form disulfide bonds between heavy chains.

[0229] The specific affinity of an anti-hTfR antibody for hTfR mainly depends on the amino acid sequences of the CDRs of the heavy and light chains of the anti-hTfR antibody. There are no particular limitations on the amino acid sequences of these CDRs, as long as the anti-hTfR antibody has specific affinity for monkey hTfR in addition to hTfR. However, in the present invention, a humanized anti-hTfR antibody or a human antibody that has a relatively high affinity for hTfR and has affinity for both human and monkey TfR, when measured by the method described in Example 7, The dissociation constant with human TfR is preferably 1X10 -10 M or less, preferably 2.5X10 -11 M or less, more preferably 5X10 -12 M or less, and even more preferably 1X10 -12 M or less, The dissociation constant with monkey TfR is preferably 1X10 -9 M or less, more preferably 5X10 -10 M or less, more preferably 1X10 -10 M or less, for example 7.5X10 -11 It is M or less. For example, the dissociation constants for human TfR and monkey TfR are 1X10 -10 M or less and 1X10 -9 M or less, 1X10 -11 M or less and 5X10 -10 M or less, 5X10 -12 M or less and 1X10 -10 M or less, 5X10 -12 M or less and 7.5X10 -11 M or less, 1X10 -12 M or less and 1X10 -10 M or less, 1X10 -12 M or less and 7.5X10 -11 There is no particular lower limit to the dissociation constant with human TfR, but for example, -13 M,1X10 -13 M, etc. In addition, there is no particularly clear lower limit for the dissociation constant with monkey TfR, but for example, -11 M,1X10 -12 M, etc. The same applies to the case where the antibody is a single-chain antibody.

[0230] A preferred embodiment of an antibody having affinity for hTfR comprises, in the variable region of the heavy chain: (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and (c) An example is an antibody in which CDR3 comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0231] In a more specific embodiment of the antibody having affinity for hTfR, the heavy chain variable region contains: (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 62, (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 13, and (c) An example is an antibody in which CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0232] In a preferred embodiment of the above-mentioned antibody having affinity for hTfR, and in a more specific embodiment of the antibody having affinity for hTfR, a suitable amino acid sequence for framework region 3 of the heavy chain of the antibody is one comprising the amino acid sequence of SEQ ID NO: 64.

[0233] A preferred combination of light and heavy chains of an antibody having affinity for hTfR is, for example, one having the following amino acid sequence in the variable region: (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence Lys-Val-Ser, and (c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10 The light chains, (d) CDR1 comprises the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, (e) CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and (f) CDR3 comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. Combination with heavy chain.

[0234] Specific examples of the combination of light and heavy chains of an antibody having affinity for hTfR include those having the following amino acid sequences in the variable regions: A combination of a light chain comprising the amino acid sequences of SEQ ID NO: 6 for CDR1, SEQ ID NO: 8 for CDR2, and SEQ ID NO: 10 for CDR3, and a heavy chain comprising the amino acid sequences of SEQ ID NO: 62 for CDR1, SEQ ID NO: 13 for CDR2, and SEQ ID NO: 15 for CDR3.

[0235] In the above-mentioned preferred light chain and heavy chain combinations of antibodies with affinity for hTfR, and specific embodiments of the light chain and heavy chain combinations of antibodies with affinity for hTfR, a suitable amino acid sequence for framework region 3 of the antibody heavy chain is one having the amino acid sequence of SEQ ID NO: 64.

[0236] A preferred embodiment of a humanized antibody having affinity for hTfR is one having the amino acid sequence shown below: An anti-hTfR antibody, wherein the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, and the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 65.

[0237] The amino acid sequences of the light chain variable regions shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22 comprise CDR1 with the amino acid sequence of SEQ ID NO: 6 or 7, CDR2 with the amino acid sequence of SEQ ID NO: 8 or 9, and CDR3 with the amino acid sequence of SEQ ID NO: 10. However, when referring to CDRs in the amino acid sequences of the light chain variable regions shown in SEQ ID NOs: 17 to 22, the CDR sequences are not limited to these, and regions containing these CDR amino acid sequences, and amino acid sequences containing any three or more consecutive amino acids of these CDR amino acid sequences can also be CDRs.

[0238] The amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 65 is (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 62 or 63, (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or 14, and (c) CDR3 comprises the amino acid sequence of SEQ ID NO: 15 or 16, and further comprises the amino acid sequence of SEQ ID NO: 64 as framework region 3. However, when referring to the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 65, the CDR sequence is not limited to these, and regions containing these CDR amino acid sequences, and amino acid sequences containing any three or more consecutive amino acids of these CDR amino acid sequences can also be considered CDRs. The same applies to framework regions.

[0239] More specific embodiments of humanized antibodies having affinity for hTfR include: wherein the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 18 and the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 65; the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 20 and the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 65; The variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 21 and the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 65, and The variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 22 and the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 65 Examples include:

[0240] More specific embodiments of humanized antibodies having affinity for hTfR include: wherein the light chain comprises the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 66; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 25 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 66; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 66; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 66; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 68; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 25 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 68; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 68; and The light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 68 In the specific embodiment described above, a humanized anti-hTfR antibody whose heavy chain comprises the amino acid sequence of SEQ ID NO: 66 is an IgG1 type antibody, and one whose heavy chain comprises the amino acid sequence of SEQ ID NO: 68 is an IgG4 type antibody. Both antibodies contain the amino acid sequence of SEQ ID NO: 65 as the variable region.

[0241] Furthermore, a more specific embodiment of the humanized antibody that is a Fab having affinity for hTfR is: wherein the light chain comprises the amino acid sequence of SEQ ID NO: 23 and the Fab heavy chain comprises the amino acid sequence of SEQ ID NO: 61; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 25 and the Fab heavy chain comprises the amino acid sequence of SEQ ID NO: 61; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the Fab heavy chain comprises the amino acid sequence of SEQ ID NO: 61; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the Fab heavy chain comprises the amino acid sequence of SEQ ID NO: 61; Examples include:

[0242] When the anti-hTfR antibody is a Fab, specific examples of antibodies in which a separate Fc region has been introduced into the Fab heavy chain include: A light chain comprising the amino acid sequence of SEQ ID NO: 23 and a Fab heavy chain incorporating an Fc region comprising the amino acid sequence of SEQ ID NO: 71, A light chain comprising the amino acid sequence of SEQ ID NO: 25 and a Fab heavy chain incorporating an Fc region comprising the amino acid sequence of SEQ ID NO: 71, A light chain comprising the amino acid sequence of SEQ ID NO: 27 and a Fab heavy chain incorporating an Fc region comprising the amino acid sequence of SEQ ID NO: 71, An example is one in which the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the Fab heavy chain incorporating the Fc region comprises the amino acid sequence of SEQ ID NO: 71.

[0243] When introducing another Fc region into a Fab heavy chain, for example, the Fab heavy chain into which the Fc region has been introduced can be linked directly or via a linker sequence to the C-terminus of another protein (A). The one having the amino acid sequence shown in SEQ ID NO: 71 is obtained by linking a human IgG Fc region having the amino acid sequence shown in SEQ ID NO: 70 to the N-terminus of the amino acid sequence of the Fab heavy chain of humanized anti-hTfR antibody 3N (amino acid sequence 61) via a linker sequence containing a total of 25 amino acids consisting of five consecutive amino acids of the sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

[0244] Preferred embodiments of antibodies with affinity for hTfR are exemplified above. The light and heavy chains of these anti-hTfR antibodies can be mutated, such as by substitution, deletion, or addition, to the amino acid sequences of their variable regions, for the purpose of adjusting the affinity between the anti-hTfR antibody and hTfR to a desired level.

[0245] When amino acids in the amino acid sequence of the light chain variable region are substituted 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 amino acids in the amino acid sequence of the light chain variable region are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added.

[0246] When amino acids are added to the light chain variable region, 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 the light chain variable region or to the N-terminal or C-terminal side. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated light chain variable region preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original light chain variable region.

[0247] In particular, when amino acids in the amino acid sequence of each CDR or each framework region of the light chain are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and even more preferably 1. When amino acids in the amino acid sequence of each CDR or each framework region are deleted, the number of amino acids to be deleted is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and even more preferably 1. Mutations that combine these amino acid substitutions and deletions can also be added.

[0248] When amino acids are added to the amino acid sequence of each CDR or framework region of the light chain, preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and even more preferably 1 amino acid is added to the amino acid sequence or to the N-terminus or C-terminus. Mutations that combine these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of each mutated CDR or framework region preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original CDR.

[0249] When amino acids in the amino acid sequence of SEQ ID NO: 65, which is the heavy chain variable region, are substituted 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 amino acids in the amino acid sequence of the heavy chain variable region are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be added.

[0250] When amino acids are added to the amino acid sequence of SEQ ID NO: 65, which is the heavy chain variable region, preferably 1 to 10 amino acids are added to the amino acid sequence of the heavy chain variable region or to the N-terminal or C-terminal side, more preferably 1 to 5 amino acids, even more preferably 1 to 3 amino acids, and even more preferably 1 to 2 amino acids. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated heavy chain variable region preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original heavy chain variable region.

[0251] In particular, when amino acids in the amino acid sequence of each CDR or each framework region in the amino acid sequence shown in SEQ ID NO: 65 are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and even more preferably 1. When amino acids in the amino acid sequence of each CDR are deleted, the number of amino acids to be deleted is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and even more preferably 1. Mutations that combine these amino acid substitutions and deletions can also be added.

[0252] When amino acids are added to the amino acid sequence of each CDR or each framework region in the amino acid sequence shown in SEQ ID NO: 65, preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and even more preferably 1 amino acid is added to the amino acid sequence or to the N-terminus or C-terminus. Mutations that combine these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of each mutated CDR preferably exhibits 80% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology with the amino acid sequence of the original CDR.

[0253] When mutations such as substitutions, deletions, and additions are made to the amino acid sequence shown in SEQ ID NO: 65, which is the variable region of the heavy chain of an anti-hTfR antibody, as described above, it is preferable that the methionine, which is the fifth amino acid from the N-terminus of the original CDR1 shown in SEQ ID NO: 62 or 63, and the leucine, which is the 17th amino acid from the N-terminus of framework region 3 shown in SEQ ID NO: 64, be preserved in the same positions as originally. It is also preferable that the amino acid sequences of CDR1 and framework region 3 of the heavy chain be preserved in the same positions as originally.

[0254] Mutations can also be made in both the light chain and heavy chain variable regions of the anti-hTfR antibody by combining the above-mentioned mutations in the light chain variable region of the anti-hTfR antibody with the above-mentioned mutations in the heavy chain variable region of the anti-hTfR antibody.

[0255] Examples of substitutions of amino acids in the amino acid sequences of the variable regions of the heavy and light chains of the above-mentioned anti-hTfR antibodies with other amino acids include amino acids classified in the same group, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), and amino acids with hydroxyl groups (Ser, Thr).

[0256] In addition, if an amino acid is added to the C-terminus or N-terminus by mutating an anti-hTfR antibody, and the added amino acid is located between the anti-hTfR antibody and another protein (A) when the anti-hTfR antibody is fused with the other protein (A), the added amino acid will form part of the linker.

[0257] In a preferred embodiment of the antibody, including the humanized antibody having affinity for hTfR exemplified above, the amino acid sequences of the CDRs of the heavy and light chains of the anti-hTfR antibody are not particularly limited, as long as the antibody has specific affinity for hTfR and monkey TfR. However, in the present invention, a humanized antibody that has a relatively high affinity for hTfR and has affinity for both human and monkey TfR, particularly when measured by the method described in Example 7, The dissociation constant with human TfR is preferably 1X10 -10 M or less, preferably 2.5X10 -11 M or less, more preferably 5X10 -12 M or less, and even more preferably 1X10 -12 M or less, The dissociation constant with monkey TfR is preferably 1X10 -9 M or less, more preferably 5X10 -10 M or less, more preferably 1X10 -10 M or less, for example 7.5X10 -11 It is M or less. For example, the dissociation constants for human TfR and monkey TfR are 1X10 -10 M or less and 1X10 -9 M or less, 1X10 -11 M or less and 5X10 -10 M or less, 5X10 -12 M or less and 1X10 -10 M or less, 5X10 -12 M or less and 7.5X10 -11 M or less, 1X10 -12 M or less and 1X10 -10 M or less, 1X10 -12 M or less and 7.5X10 -11There is no particular lower limit to the dissociation constant with human TfR, but for example, -13 M,1X10 -13 M, etc. Although there is no particularly clear lower limit for the dissociation constant with monkey TfR, for example, -11 M,1X10 -11 M,1X10 -12 M, etc. The same applies to the case where the antibody is a single-chain antibody.

[0258] Specific embodiments of the fusion protein of the humanized antibody having affinity for hTfR shown as the specific embodiment above with another protein (A) include those in which the other protein (A) is human acid α-glucosidase (hGAA), human iduronate-2-sulfatase (hI2S), human α-L-iduronidase (hIDUA), human palmitoyl protein thioesterase-1 (hPPT-1), human acid sphingomyelinase (hASM), human allylsulfonyl sphingomyelinase (hALS), human α-L-iduronidase (hILD), human α-L-glucosidase (hAL ... These include human heparan N-sulfatase A (hARSA), human heparan N-sulfatase (hSGSH), human glucocerebrosidase (hGBA), human tripeptidyl peptidase-1 (hTPP-1), human α-N-acetylglucosaminidase (hNAGLU), human β-glucuronidase (hGUSB), human acid ceramidase (hAC), human α-L-fucosidase (hFUCA1), and α-mannosidase (hLAMAN).

[0259] Specific examples of fusion proteins in which the other protein (A) is human acid alpha-glucosidase (hGAA) include: (1) A compound comprising hGAA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound consisting of hGAA linked to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hGAA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hGAA is bound via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, the heavy chain having an amino acid sequence shown in SEQ ID NO: 66 or 68, and the light chain having an amino acid sequence shown in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids of these amino acid sequences. Here, the antibody is of IgG1 type when the heavy chain has SEQ ID NO: 66, and of IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, hGAA has the amino acid sequence of SEQ ID NO: 55 or 56, or a variant thereof.

[0260] A more specific example of a fusion protein in which the other protein (A) is human acid alpha-glucosidase (hGAA) is: (1) A compound comprising hGAA bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 57 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (2) An example is one in which hGAA is bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 58, and the amino acid sequence of the latter being shown in SEQ ID NO: 23. (1) is an IgG1 type antibody, and (2) is an IgG4 type antibody. Furthermore, hGAA has the amino acid sequence of SEQ ID NO: 55.

[0261] Here, the amino acid sequence of the hTfR heavy chain contained in SEQ ID NO: 57 in (1) above is that shown in SEQ ID NO: 66. That is, the fusion protein in (1) above is a humanized antibody in which the light chain contains the amino acid sequence of SEQ ID NO: 23 and the heavy chain contains the amino acid sequence of SEQ ID NO: 66. Furthermore, the amino acid sequence of the hTfR heavy chain contained in SEQ ID NO: 58 in (2) above is that shown in SEQ ID NO: 68. That is, the fusion protein in (2) above is a humanized antibody in which the light chain contains the amino acid sequence of SEQ ID NO: 23 and the heavy chain contains the amino acid sequence of SEQ ID NO: 68.

[0262] A specific example of a fusion protein in which the other protein (A) is human acid alpha-glucosidase (hGAA) and the humanized antibody is a Fab antibody is: (1) A compound comprising hGAA linked via a linker sequence at the C-terminus or N-terminus of the Fab heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the Fab heavy chain is represented by SEQ ID NO: 61 and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hGAA linked via a linker sequence at the C-terminus or N-terminus of the Fab heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the Fab heavy chain is represented by SEQ ID NO: 61 and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hGAA linked via a linker sequence at the C-terminus or N-terminus of the Fab heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the Fab heavy chain is represented by SEQ ID NO: 61 and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hGAA is bound via a linker sequence to the C-terminus or N-terminus of the Fab heavy chain of hTfR, and those comprising an hTfR light chain, wherein the amino acid sequence of the Fab heavy chain is shown in SEQ ID NO: 61, and the amino acid sequence of the light chain is shown in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids of these amino acid sequences.

[0263] A more specific example of a fusion protein in which the other protein (A) is human acid alpha-glucosidase (hGAA) and the humanized antibody is a Fab antibody is one in which hGAA is linked to the C-terminus of the heavy chain (Fab) of hTfR via a linker consisting of three consecutive amino acid sequences shown in SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 89 and the amino acid sequence of the latter being shown in SEQ ID NO: 23.

[0264] The fusion protein in which the other protein (A) is human acid alpha-glucosidase (hGAA) has affinity for both human and monkey TfR, and when measured by the method described in Example 7, The dissociation constant with monkey TfR is preferably 1X10 -10 M or less, more preferably 5X10 -11 M or less, The dissociation constant with human TfR is preferably 1X10 -10 M or less, more preferably 5X10 -11 M or less, more preferably 1X10 -11 M or less, and even more preferably 1X10 -12 It is M or less. For example, the dissociation constants of monkey TfR and human TfR are 1X10 -10 M or less and 1X10 -10 M or less, 1X10 -10 M or less and 1X10 -11M or less, 1X10 -10 M or less and 1X10 -12 M or less, 5X10 -11 M or less and 1X10 -11 M or less, 5X10 -11 M or less and 1X10 -11 M or less, 5X10 -11 M or less and 1X10 -12 There is no particular lower limit to the dissociation constant with monkey TfR, but for example, -11 M,1X10 -12 M,1X10 -13 There is no particular lower limit to the dissociation constant with human TfR, but for example, 1X10 -12 M,5X10 -13 M,1X10 -13 M, etc. The same applies to the case where the antibody is a single-chain antibody.

[0265] Specific examples of fusion proteins in which the other protein (A) is human iduronate-2-sulfatase (hI2S) include: (1) A compound comprising an hTfR light chain and hI2S linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hI2S linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hI2S linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hI2S is bound via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, wherein the heavy chain has an amino acid sequence shown in SEQ ID NO: 66 or 68, and the light chain has an amino acid sequence shown in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids of these amino acid sequences. Here, if the heavy chain has SEQ ID NO: 66, the antibody is of the IgG1 type, and if it has SEQ ID NO: 68, the antibody is of the IgG4 type.

[0266] A more specific example of the fusion protein in which the other protein (A) is human iduronate-2-sulfatase (hI2S) is: An example of such a fusion protein is one in which hI2S is bound to the C-terminus of the heavy chain of hTfR via the amino acid sequence Gly-Ser, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 53, and the amino acid sequence of the latter being shown in SEQ ID NO: 23. In this case, the antibody is of the IgG1 type. The amino acid sequence of the heavy chain of hTfR contained in SEQ ID NO: 53 is shown in SEQ ID NO: 66. In other words, this fusion protein is a humanized antibody in which the light chain contains the amino acid sequence of SEQ ID NO: 23 and the heavy chain contains the amino acid sequence of SEQ ID NO: 66. The antibody can also be of the IgG4 type, in which case the heavy chain containing the amino acid sequence of SEQ ID NO: 66 is replaced with one containing the amino acid sequence of SEQ ID NO: 68.

[0267] A specific example of a fusion protein in which the other protein (A) is human iduronate-2-sulfatase (hI2S) and the humanized antibody is a Fab antibody is: (1) A compound comprising hI2S linked via a linker sequence at the C-terminus or N-terminus of the Fab heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the Fab heavy chain is represented by SEQ ID NO: 61 and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hI2S linked via a linker sequence at the C-terminus or N-terminus of the Fab heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the Fab heavy chain is represented by SEQ ID NO: 61 and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hI2S linked via a linker sequence at the C-terminus or N-terminus of the Fab heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the Fab heavy chain is represented by SEQ ID NO: 61 and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hI2S is bound via a linker sequence at the C-terminus or N-terminus of the Fab heavy chain of hTfR and an hTfR light chain, wherein the Fab heavy chain has the amino acid sequence shown in SEQ ID NO: 61 and the light chain has the amino acid sequence shown in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids of these amino acid sequences.

[0268] Specific examples of fusion proteins in which the other protein (A) is (hIDUA) include: (1) A compound comprising hIDUA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hIDUA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hIDUA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hIDUA is bound via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, wherein the heavy chain has an amino acid sequence shown in SEQ ID NO: 66 or 68, and the light chain has an amino acid sequence shown in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids or 1 to 20 consecutive amino acids of these amino acid sequences. Here, the antibody is of IgG1 type when the heavy chain has SEQ ID NO: 66, and of IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, hIDUA has the amino acid sequence of SEQ ID NO: 75 or 76, or a variant thereof.

[0269] A more specific example of a fusion protein in which the other protein (A) is (hIDUA) is: (1) A compound comprising hIDUA bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 90 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (2) A compound comprising hIDUA linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 42 amino acids in total, consisting of the amino acid sequence Gly-Ser followed by eight consecutive occurrences of the amino acid sequence shown in SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 91, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (3) A compound comprising hIDUA linked at the C-terminal end of the heavy chain of hTfR via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 92, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (4) A compound comprising hIDUA linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of three consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 93, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (5) A compound comprising hIDUA linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of five consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 94, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (6) A compound comprising hIDUA linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 95, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (7) A compound comprising hIDUA linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of 20 consecutive amino acid sequences of SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 96, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (8) A heavy chain (Fab) of hTfR having a linker consisting of six consecutive amino acid sequences shown in SEQ ID NO: 3 at the C-terminus, to which hIDUA is bound at the C-terminus of two consecutive amino acid sequences, and a light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 97 and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (9) An example is the antibody shown in SEQ ID NO: 99, in which hIDUA is linked to the C-terminus of single-chain humanized anti-hTfR antibody No. 3N(2) shown in SEQ ID NO: 98 via a linker consisting of seven amino acids, the amino acid sequence of which is Gly-Gly, followed by the amino acid sequence of SEQ ID NO: 3. (1) to (3) are IgG1 type antibodies, and (4) to (9) are Fab type antibodies.

[0270] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (3) above is that shown in SEQ ID NO: 68. That is, the fusion proteins in (1) to (3) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprising the amino acid sequence of SEQ ID NO: 68. Furthermore, the amino acid sequence of the heavy chain of hTfR in (4) to (9) above is that shown in SEQ ID NO: 61. That is, the fusion proteins in (4) to (9) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain (Fab) comprising the amino acid sequence of SEQ ID NO: 61.

[0271] Specific examples of fusion proteins in which the other protein (A) is (hPPT-1) include: (1) A compound comprising hPPT-1 linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hPPT-1 linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hPPT-1 linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hPPT-1 is bound via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and which are composed of an hTfR light chain, wherein the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids or 1 to 20 consecutive amino acids of these amino acid sequences. Here, the antibody is of the IgG1 type when the heavy chain has SEQ ID NO: 66, and of the IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, hPPT-1 has the amino acid sequence of SEQ ID NO: 77, or a variant thereof.

[0272] A more specific example of a fusion protein in which the other protein (A) is (hPPT-1) is: (1) A compound comprising hPPT-1 bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 100 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (2) hPPT-1 is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of five consecutive amino acid sequences shown in SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 101, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (3) hPPT-1 is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 102, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (4) hPPT-1 is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 103, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (5) A compound comprising hPPT-1 linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of three consecutive amino acid sequences shown in SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 104, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (6) A heavy chain (Fab) of hTfR having a linker consisting of six consecutive amino acid sequences of SEQ ID NO: 3 at the C-terminus, to which hPPT-1 is bound at the C-terminus of a linker consisting of two consecutive amino acid sequences, and a light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 105 and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (1) to (4) are IgG1 type antibodies, and (5) to (6) are Fab type antibodies.

[0273] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is that shown in SEQ ID NO: 68. That is, the fusion proteins in (1) to (4) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprising the amino acid sequence of SEQ ID NO: 68. Furthermore, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is that shown in SEQ ID NO: 61. That is, the fusion proteins in (5) and (6) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain (Fab) comprising the amino acid sequence of SEQ ID NO: 61.

[0274] Specific examples of fusion proteins in which the other protein (A) is (hASM) include: (1) A compound comprising hASM linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hASM linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hASM linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hASM is bound via a linker sequence to the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, wherein the heavy chain has an amino acid sequence shown in SEQ ID NO: 66 or 68, and the light chain has an amino acid sequence shown in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids or 1 to 20 consecutive amino acids of these amino acid sequences. Here, the antibody is of the IgG1 type when the heavy chain has SEQ ID NO: 66, and is of the IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, the hASM has the amino acid sequence of SEQ ID NO: 78, or a variant thereof.

[0275] A more specific example of a fusion protein in which the other protein (A) is (hASM) is: (1) A compound comprising hASM bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 106 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (2) hASM is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of five consecutive amino acid sequences shown in SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 107, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (3) hASM is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 108, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (4) A compound comprising hASM linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, wherein the amino acid sequence of the former is represented by SEQ ID NO: 109, and the amino acid sequence of the latter is represented by SEQ ID NO: 23; (5) A hTfR light chain comprising hASM linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of three consecutive amino acid sequences represented by SEQ ID NO: 3, wherein the amino acid sequence of the former is represented by SEQ ID NO: 110 and the amino acid sequence of the latter is represented by SEQ ID NO: 23; (6) A compound comprising hASM linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of five consecutive amino acid sequences of SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 111, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (7) A compound comprising hASM linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 112, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (8) A hASM consisting of a heavy chain (Fab) of hTfR linked at the C-terminus via a linker consisting of 20 consecutive amino acid sequences of SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being SEQ ID NO: 113, and the amino acid sequence of the latter being SEQ ID NO: 23; (9) A heavy chain (Fab) of hTfR having a linker consisting of six consecutive amino acid sequences of SEQ ID NO: 3 at the C-terminus, to which hASM is bound at the C-terminus of two consecutive amino acid sequences, and a light chain of hTfR, the amino acid sequence of the former being SEQ ID NO: 114 and the amino acid sequence of the latter being SEQ ID NO: 23; (1) to (4) are IgG1 type antibodies, and (5) to (9) are Fab type antibodies.

[0276] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is that shown in SEQ ID NO: 68. That is, the fusion proteins in (1) to (4) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprising the amino acid sequence of SEQ ID NO: 68. Furthermore, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is that shown in SEQ ID NO: 61. That is, the fusion proteins in (5) and (6) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain (Fab) comprising the amino acid sequence of SEQ ID NO: 61.

[0277] Specific examples of fusion proteins in which the other protein (A) is (hARSA) include: (1) A compound comprising hARSA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hARSA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hARSA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hARSA is bound via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, wherein the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids or 1 to 20 consecutive amino acids of these amino acid sequences. Here, the antibody is of the IgG1 type when the heavy chain has SEQ ID NO: 66, and of the IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, hARSA has the amino acid sequence of SEQ ID NO: 79, or a variant thereof.

[0278] A more specific example of a fusion protein in which the other protein (A) is (hARSA) is: (1) A compound comprising hARSA bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 115 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (2) hARSA is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of five consecutive amino acid sequences shown in SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 116, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (3) hARSA is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 117, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (4) hARSA is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 118, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (5) hARSA is linked to the C-terminal side of the heavy chain (Fab) of hTfR via a linker consisting of three consecutive amino acid sequences shown in SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 119, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (6) hARSA is linked to the C-terminal side of the heavy chain (Fab) of hTfR via a linker consisting of five consecutive amino acid sequences of SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 120, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (7) hARSA is linked to the C-terminal side of the heavy chain (Fab) of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 121, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (8) hARSA is linked to the C-terminal side of the heavy chain (Fab) of hTfR via a linker consisting of 20 consecutive amino acid sequences of SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 122, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (9) A heavy chain (Fab) of hTfR having a linker consisting of six consecutive amino acid sequences of SEQ ID NO: 3 at the C-terminus, to which hARSA is bound at the C-terminus of a linker consisting of two consecutive amino acid sequences, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 123 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (1) to (4) are IgG1 type antibodies, and (5) to (9) are Fab type antibodies.

[0279] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is that shown in SEQ ID NO: 68. That is, the fusion proteins in (1) to (4) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprising the amino acid sequence of SEQ ID NO: 68. Furthermore, the amino acid sequence of the heavy chain of hTfR in (5) to (9) above is that shown in SEQ ID NO: 61. That is, the fusion proteins in (5) to (9) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain (Fab) comprising the amino acid sequence of SEQ ID NO: 61.

[0280] Specific examples of fusion proteins in which the other protein (A) is (hSGSH) include: (1) A compound comprising hSGSH linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hSGSH linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hSGSH linked to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hSGSH is bound via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, wherein the heavy chain has an amino acid sequence shown in SEQ ID NO: 66 or 68, and the light chain has an amino acid sequence shown in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids or 1 to 20 consecutive amino acids of these amino acid sequences. Here, the antibody is of the IgG1 type when the heavy chain has SEQ ID NO: 66, and of the IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, hSGSH has the amino acid sequence of SEQ ID NO: 80, or a variant thereof.

[0281] A more specific example of a fusion protein in which the other protein (A) is (hSGSH) is: (1) A compound comprising hSGSH bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 124 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (2) hSGSH is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of five consecutive amino acid sequences shown in SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 125, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (3) hSGSH is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 126, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (4) hSGSH is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 127, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (5) A compound comprising hSGSH linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of three consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 128, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (6) A heavy chain (Fab) of hTfR having a linker consisting of six consecutive amino acid sequences of SEQ ID NO: 3 at the C-terminus, to which hSGSH is bound at the C-terminus of a linker consisting of two consecutive amino acid sequences, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 129 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (1) to (4) are antibodies of the IgG1 type, and (5) and (6) are antibodies of the Fab type.

[0282] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is that shown in SEQ ID NO: 68. That is, the fusion proteins in (1) to (4) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprising the amino acid sequence of SEQ ID NO: 68. Furthermore, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is that shown in SEQ ID NO: 61. That is, the fusion proteins in (5) and (6) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain (Fab) comprising the amino acid sequence of SEQ ID NO: 61.

[0283] Specific examples of fusion proteins in which the other protein (A) is (hGBA) include: (1) A compound comprising hGBA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hGBA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hGBA linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hGBA is bound via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, wherein the heavy chain has an amino acid sequence shown in SEQ ID NO: 66 or 68, and the light chain has an amino acid sequence shown in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids or 1 to 20 consecutive amino acids of these amino acid sequences. Here, the antibody is of the IgG1 type when the heavy chain has SEQ ID NO: 66, and is of the IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, hGBA has the amino acid sequence of SEQ ID NO: 81, or a variant thereof.

[0284] A more specific example of a fusion protein in which the other protein (A) is (hGBA) is: (1) A compound comprising hGBA bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 130 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (2) A compound comprising hGBA linked at the C-terminal end of the heavy chain of hTfR via a linker consisting of five consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 131, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (3) A compound comprising hGBA linked at the C-terminal end of the heavy chain of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 132, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (4) A compound comprising hGBA linked at the C-terminal end of the heavy chain of hTfR via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 133, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (5) A compound comprising hGBA linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of three consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 134, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (6) A heavy chain (Fab) of hTfR having a linker consisting of six consecutive amino acid sequences of SEQ ID NO: 3 at the C-terminus, to which hGBA is bound at the C-terminus of a linker consisting of two consecutive amino acid sequences, and a light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 135 and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (1) to (4) are antibodies of the IgG1 type, and (5) and (6) are antibodies of the Fab type.

[0285] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is that shown in SEQ ID NO: 68. That is, the fusion proteins in (1) to (4) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprising the amino acid sequence of SEQ ID NO: 68. Furthermore, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is that shown in SEQ ID NO: 61. That is, the fusion proteins in (5) and (6) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain (Fab) comprising the amino acid sequence of SEQ ID NO: 61.

[0286] Specific examples of fusion proteins in which the other protein (A) is (hTPP-1) include: (1) A compound comprising hTPP-1 linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hTPP-1 linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hTPP-1 linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hTPP-1 is bound via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, the heavy chain having an amino acid sequence shown in SEQ ID NO: 66 or 68, and the light chain having an amino acid sequence shown in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids or 1 to 20 consecutive amino acids of these amino acid sequences. Here, the antibody is of IgG1 type when the heavy chain has SEQ ID NO: 66, and of IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, hTPP-1 has the amino acid sequence of SEQ ID NO: 82, or a variant thereof.

[0287] A more specific example of a fusion protein in which the other protein (A) is (hTPP-1) is: (1) A compound comprising hTPP-1 bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 136 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (2) hTPP-1 is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of five consecutive amino acid sequences shown in SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 137, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (3) hTPP-1 is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 10 consecutive amino acid sequences shown in SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 138, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (4) hTPP-1 is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3, and the light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 139, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (5) A compound comprising hTPP-1 linked at the C-terminal side of the heavy chain (Fab) of hTfR via a linker consisting of three consecutive amino acid sequences shown in SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 140, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (6) A heavy chain (Fab) of hTfR having a linker consisting of six consecutive amino acid sequences shown in SEQ ID NO: 3 at the C-terminus, to which hTPP-1 is bound at the C-terminus of a linker consisting of two consecutive amino acid sequences, and a light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 141 and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (1) to (4) are antibodies of the IgG1 type, and (5) and (6) are antibodies of the Fab type.

[0288] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is that shown in SEQ ID NO: 68. That is, the fusion proteins in (1) to (4) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprising the amino acid sequence of SEQ ID NO: 68. Furthermore, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is that shown in SEQ ID NO: 61. That is, the fusion proteins in (5) and (6) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain (Fab) comprising the amino acid sequence of SEQ ID NO: 61.

[0289] Specific examples of fusion proteins in which the other protein (A) is (hNAGLU) include: (1) A compound comprising hNAGLU linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising hNAGLU linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising hNAGLU linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and a light chain of hTfR, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hNAGLU is bound via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, wherein the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids or 1 to 20 consecutive amino acids of these amino acid sequences. Here, the antibody is of the IgG1 type when the heavy chain has SEQ ID NO: 66, and of the IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, hNAGLU has the amino acid sequence of SEQ ID NO: 83, or a variant thereof.

[0290] A more specific example of a fusion protein in which the other protein (A) is (hNAGLU) is: (1) A compound comprising hNAGLU bound to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 142 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (2) A light chain of hTfR in which hNAGLU is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of five consecutive amino acid sequences represented by SEQ ID NO: 3, wherein the amino acid sequence of the former is represented by SEQ ID NO: 143 and the amino acid sequence of the latter is represented by SEQ ID NO: 23; (3) A light chain of hTfR in which hNAGLU is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, wherein the amino acid sequence of the former is represented by SEQ ID NO: 144 and the amino acid sequence of the latter is represented by SEQ ID NO: 23; (4) A light chain of hTfR, in which hNAGLU is linked to the C-terminal side of the heavy chain of hTfR via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3, wherein the amino acid sequence of the former is represented by SEQ ID NO: 145 and the amino acid sequence of the latter is represented by SEQ ID NO: 23; (5) A compound comprising hNAGLU linked to the C-terminal side of the heavy chain (Fab) of hTfR via a linker consisting of three consecutive amino acid sequences shown in SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being shown in SEQ ID NO: 146, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (6) A compound comprising hNAGLU linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of five consecutive amino acid sequences represented by SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 147, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (7) A light chain of hTfR in which hNAGLU is linked to the C-terminal side of the heavy chain (Fab) of hTfR via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, wherein the amino acid sequence of the former is represented by SEQ ID NO: 148 and the amino acid sequence of the latter is represented by SEQ ID NO: 23; (8) A compound comprising hNAGLU linked at the C-terminal end of the heavy chain (Fab) of hTfR via a linker consisting of 20 consecutive amino acid sequences of SEQ ID NO: 3, and a light chain of hTfR, the amino acid sequence of the former being represented by SEQ ID NO: 149, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (1) to (4) are IgG1 type antibodies, and (5) to (8) are Fab type antibodies.

[0291] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is that shown in SEQ ID NO: 68. That is, the fusion proteins in (1) to (4) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprising the amino acid sequence of SEQ ID NO: 68. Furthermore, the amino acid sequence of the heavy chain of hTfR in (5) to (8) above is that shown in SEQ ID NO: 61. That is, the fusion proteins in (5) to (8) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain (Fab) comprising the amino acid sequence of SEQ ID NO: 61.

[0292] Specific examples of fusion proteins in which the other protein (A) is (hGUSB) include: (1) A compound comprising an hGUSB linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and an hTfR light chain, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 23; (2) A compound comprising an hGUSB linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and an hTfR light chain, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 25; (3) A compound comprising an hGUSB linked via a linker sequence at the C-terminal or N-terminal side of the heavy chain of hTfR and an hTfR light chain, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is represented by SEQ ID NO: 27; (4) Examples include those in which hGUSB is linked via a linker sequence at the C-terminus or N-terminus of the heavy chain of hTfR and the light chain of hTfR, wherein the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 29. Here, the linker sequence is preferably one consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence consisting of 1 to 10 consecutive amino acids or 1 to 20 consecutive amino acids of these amino acid sequences. Here, the antibody is of the IgG1 type when the heavy chain has SEQ ID NO: 66, and is of the IgG4 type when the heavy chain has SEQ ID NO: 68. Furthermore, the hGUSB has the amino acid sequence of SEQ ID NO: 84, or a variant thereof.

[0293] A more specific example of a fusion protein in which the other protein (A) is (hGUSB) is: (1) A compound comprising an hGUSB linked to the C-terminal side of the heavy chain of hTfR via the amino acid sequence Gly-Ser and an hTfR light chain, the amino acid sequence of which is shown in SEQ ID NO: 150 and the amino acid sequence of which is shown in SEQ ID NO: 23; (2) hGUSB is composed of an hTfR heavy chain linked at the C-terminal side thereof via a linker consisting of five consecutive amino acid sequences shown in SEQ ID NO: 3, and an hTfR light chain, the amino acid sequence of the former being shown in SEQ ID NO: 151, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (3) hGUSB is composed of an hTfR heavy chain linked at the C-terminal side thereof via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and an hTfR light chain, the amino acid sequence of the former being represented by SEQ ID NO: 152, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (4) An hGUSB consisting of an hTfR heavy chain linked at the C-terminus thereof via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3 and an hTfR light chain, the amino acid sequence of the former being represented by SEQ ID NO: 153 and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (5) hGUSB is composed of an hTfR heavy chain (Fab) linked at the C-terminus via a linker consisting of three consecutive amino acid sequences shown in SEQ ID NO: 3, and an hTfR light chain, the amino acid sequence of the former being shown in SEQ ID NO: 154, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (6) hGUSB is composed of an hTfR heavy chain (Fab) linked at the C-terminus via a linker consisting of five consecutive amino acid sequences shown in SEQ ID NO: 3, and an hTfR light chain, the amino acid sequence of the former being shown in SEQ ID NO: 155, and the amino acid sequence of the latter being shown in SEQ ID NO: 23; (7) hGUSB is composed of an hTfR heavy chain (Fab) linked at the C-terminus via a linker consisting of 10 consecutive amino acid sequences represented by SEQ ID NO: 3, and an hTfR light chain, the amino acid sequence of the former being represented by SEQ ID NO: 156, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (8) hGUSB is composed of an hTfR heavy chain (Fab) linked at the C-terminus via a linker consisting of 20 consecutive amino acid sequences represented by SEQ ID NO: 3, and an hTfR light chain, the amino acid sequence of the former being represented by SEQ ID NO: 157, and the amino acid sequence of the latter being represented by SEQ ID NO: 23; (1) to (4) are IgG1 type antibodies, and (5) to (8) are Fab type antibodies.

[0294] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is that shown in SEQ ID NO: 68. That is, the fusion proteins in (1) to (4) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain comprising the amino acid sequence of SEQ ID NO: 68. Furthermore, the amino acid sequence of the heavy chain of hTfR in (5) to (8) above is that shown in SEQ ID NO: 61. That is, the fusion proteins in (5) to (8) above are humanized antibodies, with the light chain comprising the amino acid sequence of SEQ ID NO: 23 and the heavy chain (Fab) comprising the amino acid sequence of SEQ ID NO: 61. ...

Claims

1. An anti-human transferrin receptor antibody, wherein the variable region of the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 65, and the variable region of the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:

18.

2. 2. The antibody of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 61 and the light chain comprises the amino acid sequence of SEQ ID NO:

23.

3. 3. The antibody according to claim 1 or 2, which has affinity for both the extracellular domain of the human transferrin receptor and the extracellular domain of the monkey transferrin receptor.

4. The dissociation constant with the extracellular domain of the human transferrin receptor is 1 x 10 －１０ M or less, and has a dissociation constant with the extracellular domain of monkey transferrin receptor of 1 x 10 －９ The antibody of claim 3, having a molecular weight of M or less.

5. Fab antibody, F(ab') ２ The antibody according to any one of claims 1 to 4, which is an antibody or an F(ab') antibody.

6. scFab, scF(ab'), scF(ab') ２ The antibody according to any one of claims 1 to 4, which is a single-chain antibody selected from the group consisting of scFv and scFv.

7. A fusion protein of an anti-human transferrin receptor antibody and another protein (A), The anti-human transferrin receptor antibody is the antibody according to any one of claims 1 to 6, A fusion protein in which the protein (A) is bound to the C-terminal or N-terminal side of the heavy chain or light chain of the antibody.

8. 8. The fusion protein according to claim 7, wherein the protein (A) is bound to the C-terminal or N-terminal side of the heavy chain directly or via a linker.

9. 9. The fusion protein according to claim 7 or 8, wherein the protein (A) is a protein of human origin.

10. The protein (A) is selected from the group consisting of nerve growth factor (NGF), lysosomal enzymes, ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, granulocyte macrophage colony-stimulating factor ( 10. The fusion protein according to any one of claims 7 to 9, wherein the fusion protein is selected from the group consisting of: erythrocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), various cytokines, tumor necrosis factor α receptor (TNF-α receptor), PD-1 ligand, PD-L1, PD-L2, an enzyme having an activity of degrading beta-amyloid, anti-beta-amyloid antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-HER2 antibody, anti-TNF-α antibody, anti-CTLA-4 antibody, and other antibody pharmaceuticals.

11. The protein (A) is a lysosomal enzyme, and the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, human acid α-glucosidase, glucocerebrosidase, β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. The fusion protein according to any one of claims 7 to 9, wherein the fusion protein is selected from the group consisting of A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2.

12. the protein (A) is human α-L-iduronidase, and the antibody is a Fab antibody; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The fusion protein according to claim 7, wherein the heavy chain of the antibody binds to human α-L-iduronidase at its C-terminus via an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO: 3, thereby forming the amino acid sequence of SEQ ID NO:

93.

13. the protein (A) is human α-L-iduronidase, and the antibody is a Fab antibody; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The fusion protein according to claim 7, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 61, and the heavy chain is bound to human α-L-iduronidase having the amino acid sequence of SEQ ID NO: 75 or SEQ ID NO: 76 at its C-terminus via an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO:

3.

14. A drug for treating a disease state of the central nervous system associated with Hurler syndrome or Hurler-Scheie syndrome, comprising the fusion protein according to claim 12 or 13 as an active ingredient.

15. the protein (A) is human heparan N-sulfatase, and the antibody is a Fab antibody; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The fusion protein according to claim 7, wherein the heavy chain of the antibody binds to human heparan N-sulfatase at its C-terminus via an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO: 3, thereby forming the amino acid sequence of SEQ ID NO:

128.

16. the protein (A) is human heparan N-sulfatase, and the antibody is a Fab antibody; (1) the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23; and (2) The fusion protein according to claim 7, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 61, and the heavy chain is bound to human heparan N-sulfatase having the amino acid sequence of SEQ ID NO: 80 at its C-terminus via an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO:

3.

17. A drug for treating a disease state of the central nervous system associated with Sanfilippo syndrome, comprising the fusion protein according to claim 15 or 16 as an active ingredient.

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