Novel anti-human transferrin receptor antibodies that cross the blood-brain barrier

Abstract

The present invention provides a novel anti-human transferrin receptor antibody and analogue that crosses the blood-brain barrier, wherein in the variable region of the heavy chain of the antibody: (a) CDR1 contains an amino acid sequence of sequence number 62 or 63, (b) CDR2 contains an amino acid sequence of sequence number 13 or 14, and (c) CDR3 contains an amino acid sequence of sequence number 15 or 16.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201780071163.0 (International Application No. PCT / JP2017 / 046762), with the Chinese national phase entry date of May 16, 2019 (international application date of December 26, 2017) and the invention title "A Novel Anti-Human Transferrin Receptor Antibody that Crosses the Blood-Brain Barrier". Technical Field

[0002] This invention relates to an anti-human transferrin receptor antibody, its manufacturing method, and its use, in order to enable compounds (proteins, low molecular weight compounds, etc.) that are administered into the blood and are intended to function in the central nervous system (CNS) to cross the blood-brain barrier and bind to these compounds. Background Technology

[0003] Unlike capillaries in muscles and other tissues, the capillaries supplying blood to most brain tissue, except for a few areas including periventricular organs (pineal gland, pituitary gland, and area cerebellopontine), are tightly bound together by strong intercellular connections. This hinders the passive transport of substances from the blood to the brain. While there are exceptions, substances that are highly lipid-soluble or have small molecular weights (below 200–500 Daltons) and are electrically neutral near the physiological pH level rarely migrate from the capillaries to the brain. This mechanism by which the capillary endothelium in the brain restricts the exchange of substances between the blood and brain tissue fluid is called the blood-brain barrier (BBB). Furthermore, the BBB not only restricts the exchange of substances between the brain's tissue fluid and the blood but also between the tissue fluid of the central nervous system, including the brain and spinal cord, and the blood.

[0004] Due to the existence of the blood-brain barrier, most cells of the central nervous system are not affected by changes in the concentration of hormones, lymphokines, and other substances in the blood, thus maintaining their biochemical homeostasis.

[0005] However, the presence of the blood-brain barrier poses challenges in drug development. For example, enzyme replacement therapy with intravenous recombinant α-L-iduronidase is being developed as a treatment for mucopolysaccharidosis type I (Hurler syndrome), a hereditary metabolic disorder caused by α-L-iduronidase deficiency. However, this therapy is ineffective against the significant central nervous system (CNS) abnormalities observed in Hurler syndrome because the enzyme cannot cross the blood-brain barrier.

[0006] Various methods have been developed for enabling macromolecules such as proteins that are intended to function in the central nervous system to cross the blood-brain barrier. For example, in the case of nerve growth factor, attempts have been made to facilitate its passage across the blood-brain barrier by fusing liposomes encapsulating nerve growth factor with the cell membranes of endothelial cells in brain capillaries, but this has not been put into practical use (Non-Patent Literature 1). In the case of α-L-iduronase, attempts have been made to increase its blood concentration by increasing the amount of enzyme administered per dose, thereby enhancing the passive transport of the enzyme across the blood-brain barrier, and the method has been shown to alleviate central nervous system (CNS) abnormalities using an animal model of Hurler syndrome (Non-Patent Literature 2).

[0007] In addition, attempts have been made to directly administer high-molecular-weight substances into the medullary cavity or brain to bypass the blood-brain barrier. For example, methods have been reported such as: intramedullary administration of human α-L-iduronase to patients with Hurler syndrome (mucopolysaccharidosis type I) (Patent Document 1); intraventricular administration of human acid sphingomyelinase to patients with Niemann-Pick disease (Patent Document 2); and intraventricular administration of iduronate 2-sulfatase (I2S) to animal models of Hunter syndrome (Patent Document 3). It is believed that such methods can reliably deliver drugs to the central nervous system, but on the other hand, they present the problem of extremely high invasiveness.

[0008] As a method for enabling macromolecules to cross the blood-brain barrier and reach the brain, various methods have been reported to modify macromolecules in a manner that allows them to have affinity for membrane proteins present on the endothelial cells of brain capillaries. Examples of membrane proteins present on the endothelial cells of brain capillaries include receptors for compounds such as insulin, transferrin, insulin-like growth factor (IGF-I, IGF-II), LDL, and leptin.

[0009] For example, the following techniques have been reported: synthesizing nerve growth factor (NGF) in the form of a fusion protein with insulin, and allowing the fusion protein to cross the blood-brain barrier by binding to the insulin receptor (Patent Documents 4-6). Another technique has been reported: synthesizing nerve growth factor (NGF) in the form of a fusion protein with an anti-insulin receptor antibody, and allowing the fusion protein to cross the blood-brain barrier by binding to the insulin receptor (Patent Documents 4 and 7). Furthermore, the following technique has been reported: synthesizing nerve growth factor (NGF) in the form of a fusion protein with transferrin, and allowing the fusion protein to cross the blood-brain barrier by binding to the transferrin receptor (TfR) (Patent Document 8). Finally, the following technique has been reported: synthesizing nerve growth factor (NGF) in the form of a fusion protein with an anti-transferrin receptor antibody (anti-TfR antibody), and allowing the fusion protein to cross the blood-brain barrier by binding to TfR (Patent Documents 4 and 9).

[0010] The report states that further examination of the technology using anti-TfR antibodies has revealed that single-chain antibodies can be used in techniques that allow drugs to cross the blood-brain barrier by binding to anti-TfR antibodies (Non-Patent Document 3). It also reports that anti-hTfR antibodies with a large dissociation constant with hTfR (low-affinity anti-hTfR antibodies) can be formulated into drugs for appropriate use in techniques crossing the blood-brain barrier (Patent Documents 10 and 11, Non-Patent Document 4). Furthermore, it reports that anti-TfR antibodies whose affinity for hTfR varies with pH can be used as carriers for allowing drugs to cross the blood-brain barrier (Patent Document 12, Non-Patent Document 5).

[0011] Existing technical documents

[0012] Patent documents

[0013] Patent Document 1: Japanese Patent Publication No. 2007-504166

[0014] Patent Document 2: Japanese Patent Publication No. 2009-525963

[0015] Patent Document 3: Japanese Patent Application Publication No. 2012-62312

[0016] Patent Document 4: U.S. Patent No. 5,154,924

[0017] Patent Document 5: Japanese Patent Application Publication No. 2011-144178

[0018] Patent Document 6: U.S. Patent No. 2004 / 0101904

[0019] Patent Document 7: Japanese Patent Publication No. 2006-511516

[0020] Patent Document 8: Japanese Patent Application Publication No. H06-228199

[0021] Patent Document 9: U.S. Patent No. 5977307

[0022] Patent Document 10: WO2012 / 075037

[0023] Patent Document 11: WO2013 / 177062

[0024] Patent Document 12: WO2012 / 143379

[0025] Non-patent literature

[0026] Non-patent literature 1: Xie Y. et al., J Control Release. 105. 106-19 (2005)

[0027] Non-patent literature 2: Ou L. et al., Mol Genet Metab. 111.116-22 (2014)

[0028] Non-patent literature 3: Li JY. Protein Engineering. 12. 787-96 (1999)

[0029] Non-patent literature 4: Bien-Ly N. et al., J Exp Med. 211. 233-44 (2014)

[0030] Non-patent literature 5: Sada H. PLoS ONE.9.E96340 (2014) Summary of the Invention

[0031] The problem that the invention aims to solve

[0032] Against the above background, the object of the present invention is to provide an anti-TfR antibody that enables compounds (proteins, low molecular weight compounds, etc.) to act in the central nervous system when administered into the blood to cross the blood-brain barrier, a method for manufacturing the antibody, and a method for using the antibody.

[0033] Methods for solving problems

[0034] In research aimed at achieving the aforementioned objectives, the inventors conducted repeated and in-depth studies, and discovered that the anti-human transferrin receptor antibody (anti-hTfR antibody) that recognizes the extracellular region of hTfR, obtained through the antibody preparation method detailed in this specification, efficiently crosses the blood-brain barrier when administered to a living organism, thus completing this invention. Specifically, this invention provides the following solution.

[0035] 1. An anti-human transferrin receptor antibody, wherein, in the variable region of the heavy chain of the antibody,

[0036] (a) CDR1 contains the amino acid sequence of sequence number 62 or 63.

[0037] (b) CDR2 contains the amino acid sequence of sequence number 13 or 14, and

[0038] (c) CDR3 contains an amino acid sequence of sequence number 15 or sequence number 16.

[0039] 2. According to the antibody in 1 above, the framework region 3 of the heavy chain contains the amino acid sequence of sequence number 64.

[0040] 3. Based on the antibody described in 1 or 2 above, wherein, in the variable region of the heavy chain,

[0041] The amino acid sequence replacing CDR2 at sequence number 13 or 14 contains an amino acid sequence with more than 80% homology to that sequence, and

[0042] The amino acid sequence that replaces CDR3 in sequence number 15 or 16 contains an amino acid sequence that has more than 80% homology to that sequence.

[0043] 4. Based on the antibody described in 1 or 2 above, wherein, in the variable region of the heavy chain,

[0044] The amino acid sequence replacing CDR2 at sequence number 13 or 14 contains an amino acid sequence with more than 90% homology to that sequence, and

[0045] The amino acid sequence that replaces CDR3 at sequence number 15 or 16 contains an amino acid sequence that has more than 90% homology to that sequence.

[0046] 5. Based on antibody 1 or 2 above, wherein, in the variable region of the heavy chain,

[0047] Replacing at least one amino acid sequence in (a) sequence number 62 or 63 of CDR1, (b) sequence number 13 or 14 of CDR2, (c) sequence number 15 or 16 of CDR3, and (d) sequence number 64 of frame region 3, the amino acid sequence contains an amino acid sequence modified by substitution, deletion, or addition of 1 to 5 amino acids relative to that sequence.

[0048] Here, regarding CDR1, the methionine at position 5 from the N-terminus of amino acid sequence 62 or 63 is also located at the same position in the modified amino acid sequence, and

[0049] Regarding frame region 3, the leucine at position 17, located on the N-terminus side of sequence number 64, also occupies the same position in the modified amino acid sequence.

[0050] 6. The antibody according to 1 or 2 above, wherein, in the variable region of the heavy chain,

[0051] Replacing at least one amino acid sequence in (a) sequence number 62 or 63 of CDR1, (b) sequence number 13 or 14 of CDR2, (c) sequence number 15 or 16 of CDR3, and (d) sequence number 64 of frame region 3, the amino acid sequence contains an amino acid sequence modified by substitution, deletion, or addition of 1 to 3 amino acids relative to that sequence.

[0052] Here, regarding CDR1, the methionine at position 5 from the N-terminus of amino acid sequence 62 or 63 is also located at the same position in the modified amino acid sequence, and

[0053] Regarding frame region 3, the leucine at position 17, located on the N-terminus side of sequence number 64, also occupies the same position in the modified amino acid sequence.

[0054] 7. The antibody according to 2 above, wherein the variable region of the heavy chain contains the amino acid sequence of sequence number 65.

[0055] 8. The antibody according to 7 above, wherein, in the variable region of the heavy chain, in the portion other than the amino acid sequences of sequence number 62 or 63 of CDR1 and sequence number 64 of frame region 3, the portion contains an amino acid sequence having more than 80% homology with the portion.

[0056] 9. The antibody according to 7 above, wherein, in the variable region of the heavy chain, in the portion other than the amino acid sequences of sequence number 62 or 63 of CDR1 and sequence number 64 of frame region 3, the portion contains an amino acid sequence having more than 90% homology with the portion.

[0057] 10. The antibody according to 7 above, wherein, instead of the amino acid sequence constituting the variable region of the heavy chain, contains an amino acid sequence modified by substitution, deletion, or addition of 1 to 5 amino acids relative to that sequence.

[0058] Here, regarding CDR1, the methionine at position 5 from the N-terminus of amino acid sequence 63 is also located at the same position in the modified amino acid sequence, and regarding frame region 3, the leucine at position 17 from the N-terminus of sequence 64 is also located at the same position in the modified amino acid sequence.

[0059] 11. The antibody according to 7 above, wherein, instead of the amino acid sequence constituting the variable region of the heavy chain, contains an amino acid sequence modified by substitution, deletion, or addition of 1 to 3 amino acids relative to that sequence.

[0060] Here, regarding CDR1, the methionine at position 5 from the N-terminus of amino acid sequence 63 is also located at the same position in the modified amino acid sequence, and regarding frame region 3, the leucine at position 17 from the N-terminus of sequence 64 is also located at the same position in the modified amino acid sequence.

[0061] 12. The antibody according to 7 above, wherein the heavy chain contains the amino acid sequence of sequence number 66 or sequence number 68.

[0062] 13. The antibody according to 12 above, wherein, in the heavy chain, the portion other than each amino acid sequence of sequence number 62 or 63 of CDR1 and sequence number 64 of frame region 3 contains an amino acid sequence having more than 80% homology with respect to that portion.

[0063] 14. The antibody according to 12 above, wherein, in the heavy chain, the portion other than each amino acid sequence of sequence number 62 or 63 of CDR1 and sequence number 64 of frame region 3 contains an amino acid sequence having more than 90% homology with respect to that portion.

[0064] 15. The antibody according to 12 above, wherein, instead of the amino acid sequence constituting the heavy chain, contains an amino acid sequence modified by substitution, deletion, or addition of 1 to 5 amino acids relative to that sequence.

[0065] Here, regarding CDR1, the methionine at position 5 from the N-terminus of amino acid sequence 63 is also located at the same position in the modified amino acid sequence, and regarding frame region 3, the leucine at position 17 from the N-terminus of sequence 64 is also located at the same position in the modified amino acid sequence.

[0066] 16. The antibody according to 12 above, wherein, instead of the amino acid sequence constituting the heavy chain, contains an amino acid sequence modified by substitution, deletion, or addition of 1 to 3 amino acids relative to that sequence.

[0067] Here, regarding CDR1, the methionine at position 5 from the N-terminus of amino acid sequence 63 is also located at the same position in the modified amino acid sequence, and regarding frame region 3, the leucine at position 17 from the N-terminus of sequence 64 is also located at the same position in the modified amino acid sequence.

[0068] 17. An antibody according to any one of 1 to 16 above, wherein, in the variable region of the light chain of the antibody,

[0069] (a) CDR1 contains an amino acid sequence with sequence number 6 or 7.

[0070] (b) CDR2 contains the amino acid sequence of sequence number 8 or 9, or the amino acid sequence Lys-Val-Ser, and

[0071] (c)CDR3 contains the amino acid sequence of sequence number 10.

[0072] 18. The antibody according to 17 above, wherein, in the variable region of the light chain,

[0073] (a) An amino acid sequence that replaces sequence number 6 or 7 of CDR1, containing an amino acid sequence with more than 80% homology to that sequence.

[0074] (b) The amino acid sequence replacing sequence number 8 or 9 of CDR2, or the amino acid sequence Lys-Val-Ser, contains an amino acid sequence with more than 80% homology to that sequence, and

[0075] (c) The amino acid sequence that replaces sequence number 10 of CDR3 contains an amino acid sequence that has more than 80% homology with respect to that sequence.

[0076] 19. The antibody according to 17 above, wherein, in the variable region of the light chain,

[0077] (a) An amino acid sequence that replaces sequence number 6 or 7 of CDR1, containing an amino acid sequence with more than 90% homology to that sequence.

[0078] (b) The amino acid sequence replacing sequence number 8 or 9 of CDR2, or the amino acid sequence Lys-Val-Ser, contains an amino acid sequence with more than 90% homology to that sequence, and

[0079] (c) The amino acid sequence that replaces sequence number 10 of CDR3 contains an amino acid sequence that has more than 90% homology with respect to that sequence.

[0080] 20. According to the antibody in 17 above, wherein, in the variable region of the light chain,

[0081] Replacing at least one amino acid sequence in (a) sequence number 6 or 7 of CDR1, (b) sequence number 8 or 9 of CDR2, or the amino acid sequence Lys-Val-Ser, and (c) sequence number 10 of CDR3, the amino acid sequence contains an amino acid sequence modified by substitution, deletion or addition of 1 to 5 amino acids relative to the sequence.

[0082] 21. According to the antibody in 17 above, wherein, in the variable region of the light chain,

[0083] Replacing at least one amino acid sequence in (a) sequence number 6 or 7 of CDR1, (b) sequence number 8 or 9 of CDR2, or the amino acid sequence Lys-Val-Ser, and (c) sequence number 10 of CDR3, the amino acid sequence contains an amino acid sequence modified by substitution, deletion or addition of 1 to 3 amino acids relative to the sequence.

[0084] 22. An antibody according to any one of 1 to 16 above, wherein the variable region of the light chain of the antibody contains an amino acid sequence of sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21 or sequence number 22.

[0085] 23. The antibody according to 22 above, wherein the amino acid sequence replacing the variable region of the light chain contains an amino acid sequence having more than 80% homology with respect to the sequence.

[0086] 24. The antibody according to 22 above, wherein the amino acid sequence replacing the variable region of the light chain contains an amino acid sequence having more than 90% homology with respect to the sequence.

[0087] 25. The antibody according to 22 above, wherein the amino acid sequence replacing the variable region of the light chain contains an amino acid sequence modified by substitution, deletion or addition of 1 to 5 amino acids relative to the sequence.

[0088] 26. The antibody according to 22 above, wherein the amino acid sequence replacing the variable region of the light chain contains an amino acid sequence modified by substitution, deletion or addition of 1 to 3 amino acids relative to the sequence.

[0089] 27. An antibody according to any one of 1 to 16 above, wherein the light chain of the antibody contains an amino acid sequence of sequence number 23, sequence number 25, sequence number 27 or sequence number 29.

[0090] 28. The antibody according to 27 above, wherein the amino acid sequence replacing the light chain contains an amino acid sequence having more than 80% homology with respect to the sequence.

[0091] 29. The antibody according to 27 above, wherein the amino acid sequence replacing the light chain contains an amino acid sequence having more than 90% homology with respect to the sequence.

[0092] 30. The antibody according to 27 above, wherein the amino acid sequence replacing the light chain contains an amino acid sequence modified by substitution, deletion or addition of 1 to 5 amino acids relative to the sequence.

[0093] 31. The antibody according to 27 above, wherein the amino acid sequence replacing the light chain contains an amino acid sequence modified by substitution, deletion or addition of 1 to 3 amino acids relative to the sequence.

[0094] 32. The antibody according to any one of 1 to 31 above has an affinity for both the extracellular region of human transferrin receptor and the extracellular region of monkey transferrin receptor.

[0095] 33. Based on the antibody in 32 above, its dissociation constant with the extracellular region of the human transferrin receptor is 1 × 10⁻⁶. -10 Below M, the dissociation constant with the extracellular region of the monkey transferrin receptor is 1 × 10⁻⁶. -9 Below M.

[0096] 34. The antibody according to any one of 1 to 33 above is a Fab antibody, an F(ab')2 antibody, or an F(ab') antibody.

[0097] 35. The anti-human transferrin receptor antibody according to any one of 1 to 33 above is a single-chain antibody selected from the group consisting of scFab, scF(ab'), scF(ab')2 and scFv.

[0098] 36. The antibody according to 35 above, wherein its light chain and heavy chain bind through a linker sequence.

[0099] 37. The antibody according to 36 above, wherein the heavy chain is bound to the C-terminal side of the light chain via a linker sequence.

[0100] 38. The antibody according to 36 above, wherein the light chain is bound to the C-terminal side of the heavy chain via a linker sequence.

[0101] 39. An antibody based on any one of 36 to 38 above, wherein the linker sequence consists of 8 to 50 amino acid residues.

[0102] 40. According to the antibody of 39 above, wherein the adapter sequence is selected from the group consisting of amino acid sequences Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly, each amino acid sequence of sequence number 3, sequence number 4, and sequence number 5, an amino acid sequence formed by linking three amino acid sequences of sequence number 3, and an amino acid sequence formed by linking 1 to 10 of these amino acid sequences.

[0103] 41. A fusion protein, which is a fusion protein of an anti-human transferrin receptor antibody and other proteins (A),

[0104] The anti-human transferrin receptor antibody is any one of the antibodies listed in items 1 to 40 above.

[0105] The protein (A) binds to the C-terminal or N-terminal side of the light chain of the antibody.

[0106] 42. The fusion protein according to 41 above, wherein the protein (A) is directly or through a linker bound to the C-terminal or N-terminal side of the light chain.

[0107] 43. The fusion protein according to 41 or 42 above, wherein the protein (A) binds to the C-terminal or N-terminal side of the light chain via a linker.

[0108] 44. The fusion protein according to 43 above, wherein the linker is a peptide composed of 1 to 50 amino acid residues.

[0109] 45. The fusion protein according to 44 above, wherein the linker is a peptide containing an amino acid sequence selected from the group consisting of 1 glycine, 1 serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, and 1 to 10 of these amino acid sequences linked together.

[0110] 46. ​​A fusion protein, which is a fusion protein of an anti-human transferrin receptor antibody and other proteins (A),

[0111] The anti-human transferrin receptor antibody is any one of the antibodies listed in items 1 to 40 above.

[0112] The protein (A) binds to the C-terminal or N-terminal side of the heavy chain of the antibody.

[0113] 47. The fusion protein according to 46 above, wherein the protein (A) binds directly or via a linker to the C-terminal or N-terminal side of the heavy chain.

[0114] 48. The fusion protein according to 46 or 47 above, wherein the protein (A) binds to the C-terminal or N-terminal side of the heavy chain via a linker.

[0115] 49. The fusion protein according to 48 above, wherein the linker sequence is a peptide consisting of 1 to 50 amino acid residues.

[0116] 50. The fusion protein according to 49 above, wherein the linker is a peptide containing an amino acid sequence selected from the group consisting of 1 glycine, 1 serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, and 1 to 10 of these amino acid sequences linked together.

[0117] 51. The fusion protein according to any one of 41 to 50 above, wherein the protein (A) is a human-derived protein.

[0118] 52. A 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-derived neurotrophic factor (GDNF), neurotrophic factor 3, neurotrophic factor 4 / 5, neurotrophic factor 6, neuroregulatory protein 1, erythropoietin, dabepoetin, 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 (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, enzymes with β-amyloid degradation activity, anti-β-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.

[0119] 53. According to the fusion protein in 52 above, wherein protein (A) is a lysosomal enzyme selected from the group consisting of: α-L-iduronidase, iduronate-2-sulfatase, human acidic α-glucosidase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-aminohexosidase A, β-aminohexosidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramide enzyme, sphingolipid activator protein C, arylsulfatase A, α- L-fucosidase, aspartate glucosidase, α-N-acetylgalactosidase, acid sphingomyelinase, α-galactosidase A, β-glucuronidase, heparin N-sulfatase, α-N-acetylglucosidase, acetyl-CoA α-glucosinolate N-acetyltransferase, N-acetylglucosamine-6-sulfatase, acid ceramidinase, amyl-1,6-glucosidase, sialidase, palmitoylprotein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2.

[0120] 54. The fusion protein according to any one of 41 to 51 above, wherein the protein (A) is any one of human iduronate-2-sulfatase, human acid α-glucosidase or human α-L-iduronase.

[0121] 55. Based on the fusion protein of 51 above, wherein protein (A) is human acid α-glucosidase,

[0122] (1) The light chain of this antibody contains the amino acid sequence at sequence number 23, and

[0123] (2) The heavy chain of the antibody binds to human acid α-glucosidase via the amino acid sequence Gly-Ser at its C-terminus, thereby forming an amino acid sequence with sequence number 57 or sequence number 58.

[0124] 56. Based on the fusion protein in 51 above, wherein protein (A) is human acid α-glucosidase,

[0125] (1) The light chain of this antibody contains the amino acid sequence at sequence number 23, and

[0126] (2) The heavy chain of the antibody contains an amino acid sequence of sequence number 66 or 68, which binds to human acid α-glucosidase with an amino acid sequence of sequence number 55 or 56 via the amino acid sequence Gly-Ser at its C-terminus.

[0127] 57. Based on the fusion protein in 51 above, wherein protein (A) is human acid α-glucosidase, and the antibody is a Fab antibody.

[0128] (1) The light chain of this antibody contains the amino acid sequence at sequence number 23, and

[0129] (2) The heavy chain of the antibody binds to human acid α-glucosidase through an amino acid sequence formed by three amino acid sequences with sequence number 3 on its C-terminal side, thereby forming an amino acid sequence with sequence number 89.

[0130] 58. Based on the fusion protein in 51 above, wherein protein (A) is human acid α-glucosidase, and the antibody is a Fab antibody.

[0131] (1) The light chain of this antibody contains the amino acid sequence at sequence number 23, and

[0132] (2) The heavy chain of the antibody contains an amino acid sequence of sequence number 61. The heavy chain binds to human acid α-glucosidase with an amino acid sequence of sequence number 55 or sequence number 56 by an amino acid sequence formed by three amino acid sequences of sequence number 3 linked together on its C-terminal side.

[0133] 59. Based on the fusion protein in 51 above, wherein protein (A) is human α-L-iduronase, and the antibody is a Fab antibody.

[0134] (1) The light chain of this antibody contains the amino acid sequence at sequence number 23, and

[0135] (2) The heavy chain of the antibody binds to human α-L-iduronase with an amino acid sequence formed by three amino acid sequences with sequence number 3 linked together on its C-terminal side, thereby forming an amino acid sequence with sequence number 93.

[0136] 60. Based on the fusion protein of 51 above, wherein protein (A) is human α-L-iduronase, and the antibody is a Fab antibody,

[0137] (1) The light chain of this antibody contains the amino acid sequence at sequence number 23, and

[0138] (2) The heavy chain of the antibody contains the amino acid sequence of sequence number 61. The heavy chain binds to human α-L-iduronase with the amino acid sequence of sequence number 75 or sequence number 76 by the amino acid sequence formed by the C-terminal side of the heavy chain through three amino acid sequences of sequence number 3.

[0139] 61. The fusion protein according to any one of 41 to 60 above, wherein a human IgG Fc region or a portion thereof is introduced between the protein (A) and the antibody.

[0140] 62. The fusion protein according to 61 above, wherein a human IgG Fc region is directly or through a linker sequence bound to the C-terminal side of the protein (A), and the heavy chain or light chain of the antibody is directly or through a linker sequence bound to the C-terminal side of the human IgG Fc region.

[0141] 63. According to the fusion protein of 61 or 62 above, the human IgG Fc region contains the amino acid sequence of sequence number 70.

[0142] 64. The fusion protein according to 63 above, which contains the amino acid sequence of sequence number 71, wherein the amino acid sequence of sequence number 71 is formed by the binding of the human IgG Fc region to the Fab heavy chain composed of the amino acid sequence of sequence number 61 through an adapter sequence.

[0143] 65. A DNA fragment encoding the amino acid sequence of any one of the above 1 to 40 anti-human transferrin receptor antibodies.

[0144] 66. A DNA fragment encoding the amino acid sequence of any one of the fusion proteins described in 41 to 64 above.

[0145] 67. An expression vector that integrates the DNA fragment of the above 65 or 66.

[0146] 68. A mammalian cell transformed with the expression vector described in 67 above.

[0147] 69. An anti-human transferrin receptor antibody-pharmacologically active substance complex, comprising a substance having a low molecular weight pharmacologically active substance, which is required to cross the blood-brain barrier and exert its effect in the brain, bound to either the light chain or the heavy chain of any one of the anti-human transferrin receptor antibodies described in any of the preceding 1 to 40.

[0148] 70. The anti-human transferrin receptor antibody-pharmacologically active substance complex according to 69 above, wherein the pharmacologically active substance is selected from any of the following groups: anticancer agents, Alzheimer's disease treatment agents, Parkinson's disease treatment agents, Huntington's disease treatment agents, schizophrenia treatment agents, depression treatment agents, multiple sclerosis treatment agents, amyotrophic lateral sclerosis treatment agents, treatment agents for central nervous system tumors including brain tumors, treatment agents for lysosomal diseases associated with encephalopathy, glycogen storage disease treatment agents, muscular dystrophy treatment agents, cerebral ischemia treatment agents, prion disease treatment agents, treatment agents for traumatic central nervous system disorders, treatment agents for viral and bacterial central nervous system diseases, agents for postoperative recovery from neurosurgery, agents for postoperative recovery from spinal surgery, siRNA, antisense DNA, and peptides.

[0149] 71. The application of any of the anti-human transferrin receptor antibodies from 1 to 40 above in enabling proteins (A) or low molecular weight pharmacologically active substances to cross the blood-brain barrier and exert their effects in the brain.

[0150] 72. The use of any of the anti-human transferrin receptor antibodies described in 1 to 40 above in the manufacture of a blood-administered pharmaceutical preparation for treating a disease state by binding to a molecule of a physiologically active protein or a pharmacologically active low-molecular-weight compound that targets a disease state of the central nervous system.

[0151] 73. A treatment method for a disease of the central nervous system, comprising the steps of: administering a therapeutically effective amount of a physiologically active protein or a pharmacologically active low-molecular-weight compound targeting the disease in the form of a conjugate of an anti-human transferrin receptor antibody molecule of any one of the above 1 to 40 into the bloodstream to a patient with the disease.

[0152] 74. The application of any of the fusion proteins in items 52 to 58 above in enabling human acidic α-glucosidase to cross the blood-brain barrier and exert its function in the brain.

[0153] 75. The use of any of the fusion proteins in 52 to 58 above in the manufacture of a blood-administered pharmaceutical preparation for treating the disease state of the central nervous system associated with Pompe disease.

[0154] 76. A treatment method for a central nervous system disease associated with Pompe disease, comprising the steps of administering a therapeutically effective amount of any one of the fusion proteins of 52 to 58 above into the bloodstream to a patient with the disease.

[0155] 77. The application of any of the fusion proteins in 52-54, 59 or 60 above in enabling human acidic α-L-iduronase to cross the blood-brain barrier and exert its effects in the brain.

[0156] 78. The use of any of the fusion proteins in 52-54, 59 or 60 above in the manufacture of a blood-administered pharmaceutical preparation for the treatment of disease states of the central nervous system associated with Hurler syndrome or Hurler-Scheie syndrome.

[0157] 79. A treatment method for a central nervous system disorder associated with Hurler syndrome or Hurler-Scheie syndrome, comprising the steps of administering a therapeutically effective amount of any one of the fusion protein of 52-54, 59 or 60 above into the bloodstream to a patient with the disorder.

[0158] Invention Effects

[0159] For many substances that are physiologically or pharmacologically active proteins or low molecular weight substances that can hardly or completely cross the blood-brain barrier and therefore cannot be utilized by blood administration in the past, the present invention enables them to be formed into a form that can cross the blood-brain barrier. Therefore, it is possible to form new blood-administered agents for treating disease states of the central nervous system. Attached Figure Description

[0160] Figure 1 These are alternative photographs illustrating 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 administered (number 3). The lines in the lower right corner of each photograph are scale bars representing 50 μm.

[0161] Figure 2 This image shows the results of immunohistochemical staining of the hippocampus of cynomolgus monkeys after a single intravenous administration of anti-hTfR antibody. (a) No anti-hTfR antibody administered, (b) Anti-hTfR antibody administered (number 3). The line in the lower right corner of each image is a scale bar representing 50 μm.

[0162] Figure 3 These are alternative photographs illustrating the results of immunohistochemical staining of the cerebellum of cynomolgus monkeys after a single intravenous administration of anti-hTfR antibody. (a) No anti-hTfR antibody administered, (b) Anti-hTfR antibody administered (number 3). The lines in the lower right corner of each photograph are scale bars representing 50 μm.

[0163] Figure 4 This graph shows the cumulative amounts of humanized anti-hTfR antibodies in various organs other than the brain of cynomolgus monkeys after a single intravenous administration. The vertical axis represents the amount of humanized anti-hTfR antibody per unit wet weight (μg / g wet weight) in each organ. The white bars, from left to right, represent the cumulative amounts in various organs 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). The black bars represent the cumulative amounts of trastuzumab (Herceptin). TM The cumulative amount in various organs of monkeys. ND indicates not measured.

[0164] Figure 5These are proxy photographs showing the results of immunohistochemical staining of the cerebral cortex of cynomolgus monkeys after a single intravenous administration of humanized anti-hTfR antibody. (a) Herceptin administration, (b) administration of humanized anti-hTfR antibody No. 3, (c) administration of humanized anti-hTfR antibody No. 3-2, (d) administration of humanized anti-hTfR antibody No. 3 (IgG4), (e) administration of humanized anti-hTfR antibody No. 3-2 (IgG4). The line in the lower right corner of each photograph is a scale bar indicating 20 μm.

[0165] Figure 6 This image shows the results of immunohistochemical staining of the hippocampus of cynomolgus monkeys after a single intravenous administration of humanized anti-hTfR antibody. (a) Herceptin administration, (b) administration of humanized anti-hTfR antibody No. 3, (c) administration of humanized anti-hTfR antibody No. 3-2, (d) administration of humanized anti-hTfR antibody No. 3 (IgG4), (e) administration of humanized anti-hTfR antibody No. 3-2 (IgG4). The line in the lower right corner of each image is a scale bar representing 20 μm.

[0166] Figure 7 This image shows the results of immunohistochemical staining of the cerebellum of cynomolgus monkeys after a single intravenous administration of humanized anti-hTfR antibody. (a) Herceptin administration, (b) administration of humanized anti-hTfR antibody No. 3, (c) administration of humanized anti-hTfR antibody No. 3-2, (d) administration of humanized anti-hTfR antibody No. 3 (IgG4), (e) administration of humanized anti-hTfR antibody No. 3-2 (IgG4). The line in the lower right corner of each image is a scale bar representing 20 μm.

[0167] Figure 8 This image shows the results of immunohistochemical staining of the medulla oblongata of cynomolgus monkeys after a single intravenous administration of humanized anti-hTfR antibody. (a) Herceptin administration, (b) administration of humanized anti-hTfR antibody No. 3, (c) administration of humanized anti-hTfR antibody No. 3-2, (d) administration of humanized anti-hTfR antibody No. 3 (IgG4), (e) administration of humanized anti-hTfR antibody No. 3-2 (IgG4). The line in the lower right corner of each image is a scale bar representing 20 μm.

[0168] Figure 9 This image shows the results of immunohistochemical staining of the cerebellum of cynomolgus monkeys after a single intravenous administration of hGAA. (a) hGAA-anti-hTfR antibody 3N (IgG4) administered, (b) hGAA administered. The line in the lower right corner of each image is a scale bar representing 20 μm.

[0169] Figure 10This is a graph showing the efficacy evaluation results of hGAA-anti-hTfR antibody 3N (IgG4) in mice. (a) shows glycogen concentration in the right brain, (b) shows glycogen concentration in the cervical spinal cord, (c) shows glycogen concentration in the heart, (d) shows glycogen concentration in the diaphragm, (e) shows glycogen concentration in the liver, and (f) shows glycogen concentration in the spleen. In each graph, 1 represents the normal control group, 2 represents the disease control group, 3 represents the hGAA 20 mg / kg administration group, and 4-7 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). The vertical lines represent the SD, indicating p < 0.01 compared to the disease control group, p < 0.05 compared to the disease control group, $ indicates p < 0.01 compared to the hGAA-treated group, and $ indicates p < 0.05 compared to the hGAA-treated group (all Tukey HSD tests).

[0170] Figure 11 This is a graph showing the efficacy evaluation results of hGAA-anti-hTfR antibody 3N (IgG4) in mice. (a) represents the glycogen concentration in the quadriceps femoris muscle, (b) represents the glycogen concentration in the gastrocnemius muscle, (c) represents the glycogen concentration in the soleus muscle, (d) represents the glycogen concentration in the tibialis anterior muscle, and (e) represents the glycogen concentration in the extensor digitorum longus muscle and gastrocnemius muscle. In each graph, 1 represents the normal control group, 2 represents the disease control group, 3 represents the hGAA-treated group, and 4-7 represent the hGAA-anti-hTfR antibody 3N (IgG4) treated groups at 2.5 mg / kg, 5.0 mg / kg, 10 mg / kg, and 20 mg / kg, respectively. The vertical axis represents the glycogen concentration (mg / g wet weight). The vertical lines represent the SD, indicating p < 0.01 compared to the disease control group, p < 0.05 compared to the disease control group, $ indicates p < 0.01 compared to the hGAA-treated group, and $ indicates p < 0.05 compared to the hGAA-treated group (all Tukey HSD tests).

[0171] Figure 12 This is a graph showing the efficacy evaluation results of Fab GS-GAA in mice. (a) represents glycogen concentration in the right brain, (b) represents glycogen concentration in the heart, (c) represents glycogen concentration in the diaphragm, (d) represents glycogen concentration in the quadriceps femoris muscle, (e) represents glycogen concentration in the soleus muscle, and (f) represents glycogen concentration in the tibialis anterior muscle. In each graph, 1 represents the normal control group, 2 represents the disease control group, 3 represents the hGAA treatment group, and 4 and 5 represent the Fab GS-GAA 5.0 mg / kg and 20 mg / kg treatment groups, respectively. The vertical axis represents glycogen concentration (mg / g wet weight). The vertical lines represent the SD (slow, steady, slow) response. Detailed Implementation

[0172] In this invention, the term "antibody" mainly refers to human antibodies, mouse antibodies, humanized antibodies, chimeric antibodies of human antibodies and antibodies of other mammals, and chimeric antibodies of mouse antibodies and antibodies of other mammals. However, it is not limited to these antibodies as long as they have the property of specifically binding to a specific antigen. In addition, there are no particular restrictions on the animal species of the antibody. Among them, humanized antibodies are preferred.

[0173] In this invention, the term "human antibody" refers to an antibody whose entire protein is encoded by a human gene. Antibodies encoded by genes into which a mutated gene has been introduced into the original human gene without modifying the original amino acid sequence, for purposes such as improving gene expression efficiency, are also included in "human antibodies." Furthermore, antibodies created by combining two or more genes encoding a human antibody, or by replacing a portion of one human antibody with a portion of another, are also considered "human antibodies." Human antibodies possess three complementarity-determining regions (CDRs) of the immunoglobulin light chain and three complementarity-determining regions (CDRs) of the immunoglobulin heavy chain. The three CDRs of the immunoglobulin light chain are named CDR1, CDR2, and CDR3 sequentially, starting from the CDR located at the N-terminus. The three CDRs of the immunoglobulin heavy chain are also named CDR1, CDR2, and CDR3 sequentially, starting from the CDR located at the N-terminus. Antibodies whose antigen specificity, affinity, etc., of a human antibody are altered by replacing the CDR of one human antibody with the CDR of another human antibody are also included in human antibodies.

[0174] In this invention, antibodies that have undergone substitution, deletion, or addition mutations in the amino acid sequence of the original antibody by modifying its gene are also included in the term "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 incorporating these amino acid substitution and deletion mutations 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 of the original antibody, either at the N-terminus or C-terminus. Antibodies incorporating these amino acid addition, substitution, and deletion mutations are also human antibodies. The amino acid sequence of the antibody with the introduced mutation preferably shows more than 80% homology with the amino acid sequence of the original antibody, more preferably more than 90% homology, even more preferably more than 95% homology, and even more preferably more than 98% homology. That is, when referring to "genes derived from humans" in this invention, it includes not only original human genes but also genes obtained by modifying them.

[0175] The homology between the amino acid sequence of the original antibody and the amino acid sequence of the antibody with the introduced mutation can be easily calculated using well-known homology calculation algorithms. For example, such algorithms include BLAST (Altschul SF. JMol. Biol. 215. 403-10, (1990)), Pearson and Lipman's similarity search method (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), Smith and Waterman's local homology algorithm (Adv. Appl. Math. 2. 482-9 (1981)), etc.

[0176] In this invention, when referring to "mouse antibody," it means an antibody whose protein is entirely encoded by genes derived from mice, containing an amino acid sequence identical to that of an antibody. Therefore, antibodies encoded by genes into which a variant gene has been introduced into the original mouse gene without modifying the original amino acid sequence, for purposes such as improving gene expression efficiency, are also included in "mouse antibody." Furthermore, antibodies that combine two or more genes encoding a mouse antibody, or antibodies in which a portion of one mouse antibody is replaced by a portion of another mouse antibody, are also included in mouse antibody. Mouse antibodies possess three complementarity-determining regions (CDRs) of the immunoglobulin light chain and three complementarity-determining regions (CDRs) of the immunoglobulin heavy chain. The three CDRs of the immunoglobulin light chain are named CDR1, CDR2, and CDR3, starting from the CDR located at the N-terminus. The three CDRs of the immunoglobulin heavy chain are named CDR1, CDR2, and CDR3, starting from the CDR located at the N-terminus. For example, antibodies that alter the antigen specificity, affinity, etc. of a mouse antibody by replacing the CDR of one mouse antibody with the CDR of another mouse antibody are also included in mouse antibodies.

[0177] In this invention, antibodies that have undergone substitution, deletion, or addition variations in the amino acid sequence of the original mouse antibody by modifying its gene are also included in the "mouse antibody". When amino acids in the original antibody's amino acid sequence 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 original antibody's amino acid sequence 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 with variations incorporating 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 original antibody's amino acid sequence, either at the N-terminus or C-terminus. Antibodies with variations incorporating these amino acid additions, substitutions, and deletions are also included in the "mouse antibody". The amino acid sequence of the antibody with the introduced mutation preferably shows more than 80% homology with the amino acid sequence of the original antibody, more preferably more than 90% homology, even more preferably more than 95% homology, and even more preferably more than 98% homology. That is, when referring to "genes derived from mice" in this invention, it includes not only the original genes derived from mice, but also genes obtained by modifying them.

[0178] In this invention, the term "humanized antibody" refers to an antibody in which a portion of the variable region (e.g., all or part of the CDR) is derived from a mammal other than a human, while the remaining regions are derived from a human. For example, examples of humanized antibodies include antibodies produced by replacing the three complementarity-determining regions (CDRs) of the immunoglobulin light chain and the three complementarity-determining regions (CDRs) of the immunoglobulin heavy chain that constitute a human antibody with CDRs from other mammals. The biological species of other mammals from which the CDRs are derived for transplantation into the appropriate locations of the human antibody are not particularly limited as long as they are mammals other than humans, but are preferably mice, rats, rabbits, horses, or primates other than humans, more preferably mice and rats, and even more preferably mice.

[0179] In this invention, the term "chimeric antibody" refers to an antibody composed of two or more different antibody fragments from two or more different species linked together.

[0180] Chimeric antibodies, which are a mixture of human antibodies and antibodies from other mammals, are antibodies obtained by replacing a portion of a human antibody with a portion of an antibody from a mammal other than a human. The antibody consists of an Fc region, a Fab region, and a hinge region, as described below. Specific examples of such chimeric antibodies include those where the Fc region is derived from a human antibody and the Fab region is derived from an antibody from another mammal. The hinge region is derived from either the human antibody or an antibody from another mammal. Conversely, examples include chimeric antibodies where the Fc region is derived from another mammal and the Fab region is derived from a human antibody. The hinge region is derived from either the human antibody or an antibody from another mammal.

[0181] Alternatively, it can be said that antibodies consist of variable and constant regions. As another specific example of a chimeric antibody, the constant region (C0) of the heavy chain can be cited. H ) and the constant region of the light chain (C L The variable region (V) of the heavy chain, derived from human antibodies. H ) and the variable region (V) of the light chain L Chimeric antibodies derived from other mammals, conversely, can be listed as constant regions of the heavy chain (C). H ) and the constant region of the light chain (C L The variable region (V) of the heavy chain, derived from antibodies from other mammals. H ) and the variable region (V) of the light chain L Chimeric antibodies derived from human antibodies. Here, the biological species of other mammals are not particularly limited as long as they are not humans, but are preferably mice, rats, rabbits, horses, or primates other than humans, with mice being more preferred.

[0182] Chimeric antibodies, which are mice antibodies combined with antibodies from other mammals, are antibodies obtained by replacing a portion of a mouse antibody with a portion of an antibody from a mammal other than a mouse. Specific examples of such chimeric antibodies include those where the Fc region originates from a mouse antibody and the Fab region originates from an antibody from another mammal; conversely, examples include those where the Fc region originates from another mammal and the Fab region originates from a mouse antibody. Here, the biological species of the other mammal are not particularly limited as long as they are mammals other than mice, but are preferably primates other than rats, rabbits, horses, and humans, and more preferably humans.

[0183] Chimeric antibodies, specifically referring to "human / mouse chimeric antibodies," can be categorized as follows: Human / mouse chimeric antibodies may have an Fc region derived from a human antibody and a Fab region derived from a mouse antibody, or vice versa. The hinge region originates from either a human or mouse antibody. Other specific examples of human / mouse chimeric antibodies include the constant region (Cf) of the heavy chain. H ) and the constant region of the light chain (C L The variable region (V) of the heavy chain, derived from human antibodies. H ) and the variable region (V) of the light chain L Human / mouse chimeric antibodies derived from mouse antibodies, conversely, can be used to enumerate the constant regions of the heavy chain (C). H ) and the constant region of the light chain (C L The variable region (V) of the heavy chain, derived from mouse antibodies. H ) and the variable region (V) of the light chain L Human / mouse chimeric antibodies derived from human antibodies.

[0184] Antibodies originally have a basic structure consisting of four polypeptide chains: two immunoglobulin light chains and two immunoglobulin heavy chains. However, in this invention, the term "antibody" includes not only antibodies with this basic structure,

[0185] (1) An antibody consisting of two polypeptide chains, one immunoglobulin light chain and one immunoglobulin heavy chain, as detailed below.

[0186] (2) A single-chain antibody formed by binding a linker sequence to the C-terminus of the immunoglobulin light chain and further binding the immunoglobulin heavy chain to its C-terminus, and

[0187] (3) A single-chain antibody formed by binding a linker sequence to the C-terminus of the immunoglobulin heavy chain and further binding an immunoglobulin light chain to its C-terminus. Moreover,

[0188] (4) Antibodies consisting of Fab regions that lack the Fc region from the basic structure of antibodies in the original sense, as well as antibodies consisting of all or part of the Fab region and hinge region (including Fab, F(ab') and F(ab')2), are also included in the “antibody” of the present invention.

[0189] Here, Fab refers to: containing variable regions and C L A light chain with a constant region (the region of the light chain) and containing a variable region and C H A molecule formed by the bonding of one heavy chain within region 1 (the constant region 1 of the heavy chain) to each other via disulfide bonds through the cysteine ​​residues present in each chain. In Fab, the heavy chain, in addition to the variable region and C... H Beyond region 1 (part 1 of the constant region of the heavy chain), a portion of a hinge region may be further contained, but this hinge region lacks the cysteine ​​residues present in the hinge region that bind the heavy chains of the antibody together. In Fab, the light and heavy chains are linked by disulfide bonds, which are formed in the constant region (C1) of the light chain. L Cysteine ​​residues in the region and constant regions (C) present in the heavy chain H Between cysteine ​​residues in the hinge region (or region 1) or the linker region. The heavy chain that forms Fab is called the Fab heavy chain. Fab lacks the cysteine ​​residues present in the hinge region that bind the heavy chains of the antibody together, and therefore consists of one light chain and one heavy chain. The light chain that makes up Fab contains a variable region and C. L Regions. The heavy chains that make up a Fab can consist of variable regions and C. H One region can be composed of, but can also exclude variable regions and C. H In addition to region 1, there is also a portion of the hinge region. However, in this case, the hinge region is selected to be free of cysteine ​​residues that bind the heavy chains, thus preventing the formation of disulfide bonds between the two heavy chains via the hinge region. In F(ab'), its heavy chains, in addition to the variable region and C... HOutside of region 1, it contains all or part of a hinge region having cysteine ​​residues that bind the heavy chains together. F(ab')2 refers to a molecule formed by two F(ab') molecules linked together by disulfide bonds through cysteine ​​residues present in their respective hinge regions. The heavy chain forming F(ab') or F(ab')2 is called a Fab' heavy chain. Furthermore, polymers such as dimers and trimers obtained by direct or linker binding of multiple antibodies are also antibodies. Moreover, not limited to these, substances containing a portion of an immunoglobulin molecule and possessing the property of specific binding to an antigen are included in the "antibody" described in this invention. That is, in this invention, when referring to immunoglobulin light chains, it includes substances containing all or part of the amino acid sequence of the variable region derived from immunoglobulin light chains. Similarly, when referring to immunoglobulin heavy chains, it includes substances containing all or part of the amino acid sequence of the variable region derived from immunoglobulin heavy chains. Therefore, as long as it has all or part of the amino acid sequence of the variable region, for example, a substance lacking the Fc region is also an immunoglobulin heavy chain.

[0190] Additionally, here, Fc or Fc region refers to the region in the antibody molecule containing Cc... H Region 2 (part 2 of the constant region of the heavy chain) and C H The region consisting of the segment of region 3 (part 3 of the constant region of the heavy chain).

[0191] Furthermore, in this invention, when referring to "antibody," it also includes:

[0192] (5) scFab, scF(ab'), and scF(ab')2, which are single-chain antibodies formed by binding the light and heavy chains of Fab, F(ab'), or F(ab')2 as shown in (4) above through a linker sequence. Here, for scFab, scF(ab'), and scF(ab')2, the linker sequence can be bound to the C-terminal side of the light chain, and further, the heavy chain can be bound to the C-terminal side. Alternatively, the linker sequence can be bound to the C-terminal side of the heavy chain, and further, the light chain can be bound to the C-terminal side. Furthermore, scFv, a single-chain antibody formed by binding the variable regions of the light chain and the heavy chain through a linker sequence, is also included in the antibody of the present invention. For scFv, the linker sequence can be bound to the C-terminal side of the variable region of the light chain, and further, the variable region of the heavy chain can be bound to the C-terminal side. Alternatively, the linker sequence can be bound to the C-terminal side of the variable region of the heavy chain, and further, the variable region of the light chain can be bound to the C-terminal side.

[0193] Furthermore, the “antibody” described in this specification, in addition to full-length antibodies and the antibodies shown in (1) to (5) above, also includes any form of antigen-binding fragment (antibody fragment) that is missing a portion of a full-length antibody, which is a concept broader than that contained in (4) and (5) above.

[0194] The term "antigen-binding fragment" refers to an antibody fragment that retains at least a portion of its specific binding activity to an antigen. Examples of binding fragments, in addition to those shown in (4) and (5) above, include: Fab, Fab', F(ab')2, a variable region (Fv), and a heavy chain variable region (V) connected to a suitable linker. H ) and light chain variable region (V L Single-chain antibodies (scFv) linked together, containing heavy chain variable regions (V) H ) and light chain variable region (V L A double-chain antibody consisting of a polypeptide dimer, as part of a constant region (C) bound to the heavy chain (H chain) of scFv. H 3) Minibodies, peptide dimers, and other low-molecular-weight antibodies. However, the ability to bind to antigens is not limited to these molecules. In addition, these binding fragments include not only products obtained by treating the full-length antibody protein molecule with appropriate enzymes, but also proteins produced using genetically engineered antibody genes in appropriate host cells.

[0195] In this invention, when referring to "single-chain antibody," it means a protein that specifically binds to a specific antigen by binding a linker sequence to the C-terminus of all or part of the amino acid sequence of the variable region containing the immunoglobulin light chain, and further by binding all or part of the amino acid sequence of the variable region containing the immunoglobulin heavy chain to its C-terminus. For example, the antibodies shown in (2), (3), and (5) above are included in single-chain antibodies. Additionally, a protein that specifically binds to a specific antigen by binding a linker sequence to the C-terminus of all or part of the amino acid sequence of the variable region containing the immunoglobulin heavy chain, and further by binding all or part of the amino acid sequence of the variable region containing the immunoglobulin light chain to its C-terminus is also a "single-chain antibody" in this invention. In the case of single-chain antibodies formed by binding the immunoglobulin light chain to the C-terminus of the immunoglobulin heavy chain via a linker sequence, the Fc region of the immunoglobulin heavy chain is usually missing. The variable region of the immunoglobulin light chain has three complementarity-determining regions (CDRs) related to the antigen specificity of the antibody. Similarly, the variable region of the immunoglobulin heavy chain also has three CDRs. These CDRs are the main regions that determine the antigen specificity of the antibody. Therefore, it is preferable that the single-chain antibody contains all three CDRs of the immunoglobulin heavy chain and all three CDRs of the immunoglobulin light chain. However, as long as the antigen specificity of the antibody can be maintained, it is also possible to use a single-chain antibody that lacks one or more CDRs.

[0196] In single-chain antibodies, the linker sequence configured between the light and heavy chains of immunoglobulins is a peptide chain consisting of preferably 2 to 50, more preferably 8 to 50, further preferably 10 to 30, even more preferably 12 to 18 or 15 to 25, for example 15 or 25 amino acid residues. Regarding such adapter sequences, there are no restrictions on their amino acid sequences as long as the anti-hTfR antibody formed by linking the two chains with them can maintain its affinity for hTfR. Preferred adapter sequences are those consisting only of glycine or of glycine and serine. Examples include adapter sequences containing the amino acid sequences Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly, Gly-Gly-Gly-Gly (Sequence No. 3), Gly-Gly-Gly-Gly-Gly-Ser (Sequence No. 4), Ser-Gly-Gly-Gly-Gly (Sequence No. 5), or sequences in which these amino acid sequences are repeated 2 to 10 times or 2 to 5 times. For example, when the variable region of the immunoglobulin light chain is bound to the C-terminus of the amino acid sequence consisting of the entire variable region of the immunoglobulin heavy chain via a linker sequence, it is preferable to have a linker sequence containing a total of 15 amino acids, which is equivalent to the 3 amino acid sequences Gly-Gly-Gly-Gly-Ser (Sequence No. 3).

[0197] In this invention, the term "human transferrin receptor" or "hTfR" refers to a membrane protein having the amino acid sequence shown in Serial No. 1. In one embodiment, the anti-hTfR antibody of this invention specifically binds to the portion of the amino acid sequence shown in Serial No. 1 from the 89th cysteine ​​residue at the N-terminus to the phenylalanine residue at the C-terminus (the extracellular region of hTfR), but is not limited thereto. Additionally, in this invention, the term "monkey transferrin receptor" or "monkey TfR" specifically refers to a membrane protein derived from the cynomolgus monkey (Macaca fascicularis) having the amino acid sequence shown in Serial No. 2. In one embodiment, the anti-hTfR antibody of this invention also binds to the portion of the amino acid sequence shown in Serial No. 2 from the 89th cysteine ​​residue at the N-terminus to the phenylalanine residue at the C-terminus (the extracellular region of monkey TfR), but is not limited thereto.

[0198] The typical method for producing antibodies against hTfR is as follows: Recombinant human transferrin receptor (rhTfR) is created by introducing cells with an expression vector integrating the hTfR gene. This rhTfR is then used to immunize animals such as mice. Cells producing antibodies against hTfR are then extracted from the immunized animals and fused with myeloma cells to create hybridoma cells capable of producing anti-hTfR antibodies.

[0199] Alternatively, cells producing antibodies against hTfR can be obtained by immunizing immune system cells obtained from animals such as mice with rhTfR using an in vitro immunization method. When using the in vitro immunization method, there are no particular limitations on the species of animal from which the immune system cells are derived; mice, rats, rabbits, guinea pigs, dogs, cats, horses, and primates, including humans, are preferred, mice, rats, and humans are more preferred, and mice and humans are even more preferred. For example, spleen cells prepared from mouse spleens can be used as the immune system cells for mice. For 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.

[0200] After immunizing immune system cells using in vitro immunization, the cells are fused with myeloma cells to create hybridoma cells capable of antibody production. Alternatively, mRNA can be extracted from immunized cells and cDNA synthesized. Using this cDNA as a template, DNA fragments containing genes encoding the light and heavy chains of immunoglobulins can be amplified via PCR, and these fragments can be used to artificially reconstruct antibody genes.

[0201] The hybridoma cells obtained using the above method also contain cells that produce antibodies that recognize proteins other than hTfR as antigens. However, not all hybridoma cells that produce anti-hTfR antibodies will necessarily produce anti-hTfR antibodies that show high affinity for hTfR.

[0202] Similarly, artificially reconstructed antibody genes also contain genes encoding antibodies that recognize proteins other than hTfR as antigens. Furthermore, genes encoding anti-hTfR antibodies may not all possess the desired characteristics, such as those encoding anti-hTfR antibodies that exhibit high affinity for hTfR.

[0203] Therefore, it is necessary to select hybridoma cells from the hybridoma cells obtained above that produce antibodies with the desired characteristics (such as high affinity for hTfR). Furthermore, regarding artificially reconstructed antibody genes, it is necessary to select from these antibody genes the gene encoding an antibody with the desired characteristics (such as high affinity for hTfR). The method detailed below is effective as a method for selecting hybridoma cells that produce antibodies exhibiting high affinity for hTfR (high-affinity antibodies) or genes encoding high-affinity antibodies. It should be noted that antibodies exhibiting high affinity for hTfR refer to those with a dissociation constant (Ki) with hTfR determined using the method described in Example 7. D The preferred value is 1×10 -8 Antibodies with an M value below M are more preferably 1×10 -9 Antibodies with an M value below 1×10 are further preferred. -10Antibodies with an M value or lower are preferred, and 1×10 are further preferred. -11 Antibodies with a dissociation constant of less than M. For example, as a preferred example, antibodies with a dissociation constant of 1 × 10⁻⁶ can be listed. -13 M~1×10 -9 Antibody to M, 1×10 -13 M~1×10 -10 Antibody to M.

[0204] For example, when selecting hybridoma cells that produce antibodies with high affinity for hTfR, the following method can be used: Recombinant hTfR is added to a plate and held therein. Then, culture supernatant of hybridoma cells is added, and antibodies that do not bind to recombinant hTfR are removed from the plate. The amount of antibody retained in the plate is measured. According to this method, the higher the affinity of the antibody in the culture supernatant of the hybridoma cells added to the plate for hTfR, the greater the amount of antibody retained in the plate. Therefore, the amount of antibody retained in the plate can be measured, and hybridoma cells corresponding to plates with more retained antibodies can be selected as cell lines that produce anti-hTfR antibodies with relatively high affinity for hTfR. mRNA is extracted from such selected cell lines and cDNA is synthesized. Using this cDNA as a template, PCR is used to amplify a DNA fragment containing the gene encoding the anti-hTfR antibody, thereby isolating the gene encoding the high-affinity antibody.

[0205] When selecting a gene encoding a high-affinity anti-hTfR antibody from the aforementioned artificially reconstructed antibody genes, the artificially reconstructed antibody gene is temporarily integrated into an expression vector, and this expression vector is introduced into a host cell. At this time, any cell used as a host cell that can express the antibody gene through the introduction of an expression vector integrating the artificially reconstructed antibody gene, regardless of whether it is a prokaryotic or eukaryotic cell, is acceptable; there are no particular limitations. Cells derived from mammals such as humans, mice, and Chinese hamsters are preferred, with CHO cells derived from Chinese hamster ovaries or NS / O cells derived from mouse myeloma cells being particularly preferred. Furthermore, the expression vector used to integrate and express the gene encoding the antibody gene can be used without particular limitation, as long as it is a vector that expresses the gene when introduced into mammalian cells. The gene integrated into the expression vector is positioned downstream of a DNA sequence (gene expression control site) that can regulate gene transcription frequency within mammalian cells. Examples of gene expression control sites that can be used in this invention include promoters derived from cytomegalovirus, the SV40 early promoter, the human elongation factor-1α (EF-1α) promoter, and the human ubiquitin C promoter.

[0206] Mammalian cells incorporating such an expression vector can express the artificially reconstructed antibody integrated into the expression vector. When selecting cells from these cells expressing the artificially reconstructed antibody to produce antibodies with high affinity for hTfR, the following method can be used: Recombinant hTfR is added to a plate and held therein. The recombinant hTfR is then brought into contact with the cell culture supernatant. Antibodies not binding to the recombinant hTfR are then removed from the plate, and the amount of antibody remaining in the plate is measured. According to this method, the higher the affinity of the antibody in the cell culture supernatant for hTfR, the greater the amount of antibody remaining in the plate. Therefore, by measuring the amount of antibody remaining in the plate, cells corresponding to plates with more antibodies can be selected as cell lines producing anti-hTfR antibodies with relatively high affinity for hTfR. Furthermore, the gene encoding the anti-hTfR antibody with high affinity for hTfR can be selected. From the selected cell lines, the DNA fragment containing the gene encoding the anti-hTfR antibody is amplified by PCR, thereby isolating the gene encoding the high-affinity antibody.

[0207] The selection of genes encoding high-affinity anti-hTfR antibodies from the artificially reconstructed antibody genes described above can also be performed as follows: The artificially reconstructed antibody gene is integrated into an expression vector, which is then introduced into *E. coli*. Using the culture supernatant obtained from culturing *E. coli* or a solution containing antibodies obtained by lysing *E. coli*, *E. coli* strains with the desired gene are selected using the same method as when selecting hybridoma cells. The selected *E. coli* strain is a cell line expressing a gene encoding an anti-hTfR antibody with relatively high affinity for hTfR. Genes encoding anti-hTfR antibodies with relatively high affinity for hTfR can be selected from this cell line. If the antibody is secreted into the culture supernatant of *E. coli*, the antibody gene is integrated into the expression vector by binding a secretion signal sequence to the N-terminus.

[0208] Another method for selecting genes encoding high-affinity anti-hTfR antibodies is as follows: The antibody encoded by the artificially reconstructed antibody gene is held on a phage particle and expressed. In this case, the antibody gene is reconstructed in the form of a gene encoding a single-chain antibody. Methods for holding antibodies on phage particles are known in international publications (WO1997 / 09436, WO1995 / 11317), etc. When selecting phages that hold antibodies with high affinity for hTfR from phages holding antibodies encoded by artificially reconstructed antibody genes, the following method can be used: Recombinant hTfR is added to a plate and held, then contacted with phages, and phages that do not bind to the recombinant hTfR are removed from the plate, and the amount of phage held in the plate is measured. According to this method, the higher the affinity of the antibody held on the phage particle for hTfR, the greater the amount of phage held in the plate. Therefore, the amount of phage retained in the plate can be determined, and phage particles corresponding to plates containing more phage can be selected as phage particles that produce anti-hTfR antibodies with relatively high affinity for hTfR. Furthermore, the gene encoding anti-hTfR antibodies with high affinity for hTfR can be selected. From the selected phage particles, the DNA fragment containing the gene encoding anti-hTfR antibodies is amplified by PCR, thereby isolating the gene encoding the high-affinity antibody.

[0209] It is possible to: prepare cDNA or phage DNA from cells such as hybridoma cells that produce antibodies with high affinity for hTfR, or from phage particles that have antibodies with high affinity for hTfR, and use these as templates to amplify and separate DNA fragments containing the following genes, such as PCR. These genes are genes encoding the light chain of an anti-hTfR antibody, the heavy chain of an anti-hTfR antibody, or all or part of a single-chain antibody that is an anti-hTfR antibody. Similarly, it is also possible to: amplify and separate DNA fragments containing genes encoding all or part of the variable region of the light chain of an anti-hTfR antibody, or DNA fragments containing genes encoding all or part of the variable region of the heavy chain of an anti-hTfR antibody, using PCR or other methods.

[0210] By integrating all or part of the genes encoding the light and heavy chains of high-affinity anti-hTfR antibodies into an expression vector, and then transforming host cells such as mammalian cells using this expression vector and culturing the resulting transformed cells, high-affinity anti-hTfR antibodies can be produced. Alternatively, the amino acid sequence of the anti-hTfR antibody can be translated from the isolated gene encoding the anti-hTfR antibody, and a DNA fragment encoding this amino acid sequence can be artificially synthesized. During the artificial synthesis of the DNA fragment, the expression level of anti-hTfR antibodies in host cells can be increased by selecting appropriate codons.

[0211] To introduce substitutions, deletions, additions, or other variations into the amino acid sequence of the original anti-hTfR antibody, variations can also be appropriately introduced into the gene encoding the anti-hTfR antibody contained in the isolated DNA fragment. The gene encoding the anti-hTfR antibody after the mutation is introduced preferably has more than 80% homology with the original gene, more preferably more than 90% homology, but there are no particular restrictions on homology. By introducing variations into the amino acid sequence, the number and type of glycans binding to the anti-hTfR antibody can be altered, thereby increasing the stability of the anti-hTfR antibody in vivo.

[0212] When introducing a mutation into all or part of the variable region of the light chain encoding an anti-hTfR antibody, the mutated gene preferably has more than 80% homology with the original gene, more preferably more than 90% homology, but there is no particular limitation on homology. When replacing amino acids in the amino acid sequence of the variable region of the light chain with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When deleting amino acids in the amino acid sequence of the variable region of the light chain, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. In addition, mutations combining these amino acid substitutions and deletions can also be introduced. When adding amino acids to the variable region of the light chain, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of the variable region of the light chain, either to the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the variable region of the mutated light chain preferably has more than 80% homology with the amino acid sequence of the variable region of the original light chain, more preferably more than 90% homology, and even more preferably more than 95% homology. Particularly when replacing amino acids in the CDR amino acid sequence 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 deleting amino acids in the CDR amino acid sequence, the number of deleted amino acids is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. Furthermore, variations involving the substitution and deletion of these amino acids can also be introduced. When adding amino acids, 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, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of each modified CDR preferably has more than 80% homology with the amino acid sequence of the original CDR, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0213] When introducing a mutation into all or part of the variable region of the heavy chain encoding an anti-hTfR antibody, the mutated gene preferably has at least 80% homology with the original gene, more preferably at least 90% homology, but there is no particular limitation on homology. When replacing the amino acid sequence of the variable region of the heavy chain with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When deleting amino acids from the amino acid sequence of the variable region of the heavy chain, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. In addition, mutations combining these amino acid substitutions and deletions can also be introduced. When adding amino acids to the variable region of the heavy chain, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of the variable region of the heavy chain, either to the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the variable region of the mutated heavy chain preferably has more than 80% homology with the amino acid sequence of the variable region of the original heavy chain, more preferably more than 90% homology, and even more preferably more than 95% homology. Specifically, when replacing amino acids in the CDR amino acid sequence 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 deleting amino acids in the CDR amino acid sequence, the number of deleted amino acids is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. Furthermore, variations involving the substitution and deletion of these amino acids can also be introduced. When adding amino acids, 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, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequences of each modified CDR preferably have more than 80% homology with the amino acid sequences of the original CDRs, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0214] The mutations can also be combined with the mutations in the variable regions of the light chain and heavy chain of the anti-hTfR antibody, and introduced into both the light chain and heavy chain variable regions of the anti-hTfR antibody.

[0215] The substitutions made by other amino acids in the amino acid sequences of the light and heavy chains of the aforementioned anti-hTfR antibody include, for example, 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), which are classified into the same group. It is predicted that such substitutions using similar amino acids will not change the phenotype of the protein (i.e., conservative amino acid substitutions). However, the amino acid sequence of the frame region 3 of the hTfR of antibody number 3 (not disclosed at the time of filing of this application), which was first discovered by the inventors and confirmed to have the same effect as the present invention, namely, the 17th position of sequence number 83, is Trp. In contrast, the frame region 3 of the heavy chain of the anti-hTfR antibody number 3N in the present invention has the amino acid sequence of sequence number 68, in which the 17th position from the N-terminus is replaced with Leu. Furthermore, in the amino acid sequence of CDR1, Thr at position 5 of sequence number 12 is replaced with Met, as shown in sequence number 66. Trp and Leu do not have the aforementioned similar relationship, nor do Thr and Met. However, as will be described later, antibody number 3N, containing this substitution, unexpectedly exhibits the same efficacy as antibody number 3, and shows superior efficacy.

[0216] It should be noted that when a mutation is introduced into an anti-hTfR antibody and an amino acid is added to its C-terminus or N-terminus, and this added amino acid is located between the anti-hTfR antibody and protein (A) during the fusion of the anti-hTfR antibody and protein (A), the added amino acid constitutes part of the linker. The linker configured between the anti-hTfR antibody and protein (A) in the fusion protein will be discussed in detail later.

[0217] Anti-hTfR antibodies obtained by culturing cells selected using the methods described above that produce anti-hTfR antibodies with high affinity for hTfR, and anti-hTfR antibodies obtained by expressing genes encoding high-affinity anti-hTfR antibodies, can also be modified to have the desired properties by introducing mutations such as substitutions, deletions, and additions into their amino acid sequences. Introducing mutations into the amino acid sequence of anti-hTfR antibodies is done by introducing mutations into the genes corresponding to their amino acid sequences.

[0218] Anti-hTfR antibodies can have their affinity for hTfR appropriately adjusted by introducing mutations such as substitutions, deletions, and additions into the amino acid sequence of their variable regions. For example, if an antibody has a high affinity for an antigen and a significantly low dissociation constant in aqueous solution, it may not dissociate from the antigen when administered to a living organism, potentially resulting in functional adverse effects. In such cases, the dissociation constant can be adjusted stepwise to 2–5 times, 5–10 times, or 10–100 times that of the original antibody by introducing mutations into the variable region, thus obtaining the optimal antibody that meets the target. Conversely, the dissociation constant can also be adjusted stepwise to 1 / 2–1 / 5 times, 1 / 5–1 / 10 times, or 1 / 10–1 / 100 times that of the original antibody by introducing such mutations.

[0219] The introduction of substitutions, deletions, additions, and other variations into the amino acid sequence of anti-hTfR antibodies can be carried out as follows: using the gene encoding anti-hTfR antibodies as a template, variations can be introduced into specific sites of the gene's base sequence or randomly introduced using methods such as PCR.

[0220] The purpose is to adjust the affinity of the antibody for hTfR by introducing mutations into the amino acid sequence of the anti-hTfR antibody. This can be done, for example, by integrating the gene encoding the anti-hTfR antibody as a single-chain antibody into a phage particle, using the phage particle to create a phage expressing the single-chain antibody on the surface of the capsid protein, introducing mutations into the gene encoding the single-chain antibody by inducing the mutagen, etc., while proliferating the phage, and selecting phages expressing single-chain antibodies with the desired dissociation constant from the proliferated phages using the above method, or by purifying the phages with the desired dissociation constant using an antigen column under certain conditions.

[0221] The antibody with relatively high affinity for hTfR obtained by selecting cells that produce the aforementioned high-affinity antibody is preferably determined using the dissociation constant (K) with hTfR as described in Example 7. D The preferred value is 1×10 -8 For M and below, 1×10 is more preferred. -9 For M and below, 1×10 is further preferred. -10 M or less, and further preferably 1×10 -11 Below M. For example, as a preferred example, a dissociation constant of 1 × 10⁻⁶ can be cited. -13 M~1×10 -9 The antibody for M is 1×10 -13 M~1×10 -10 Antibodies for M. The same applies when the antibody is a single-chain antibody. For temporarily obtained antibodies, appropriate modifications such as introduction of mutations can be made to give them the desired properties.

[0222] By selecting antibodies with affinity for monkey TfR from the anti-hTfR antibodies obtained through the above operations, antibodies with affinity for both human and monkey TfR can be obtained. Antibodies with affinity for monkey TfR can be selected, for example, using an ELISA method employing recombinant monkey TfR, which is produced using gene recombination technology. In this ELISA method, recombinant monkey TfR is added to a plate and held therein, then exposed to anti-hTfR antibodies. Antibodies that do not bind to the recombinant monkey TfR are then removed from the plate, and the amount of antibody remaining in the plate is measured. Higher affinity for recombinant monkey TfR results in a greater amount of antibody remaining in the plate; therefore, the antibody corresponding to the plate with the most antibodies remaining can be selected as the antibody with affinity for monkey TfR. It should be noted that the term "monkey" here preferably refers to an animal classified as an ape other than humans, more preferably an animal classified as a monkey family, and even more preferably an animal classified as a macaque, such as a cynomolgus monkey or a macaque, with cynomolgus monkeys being particularly preferred for evaluation.

[0223] Antibodies with affinity for both human and monkey hTfR have the following advantages: the pharmacokinetic dynamics of the antibody can be observed in vivo using monkeys. For example, in drug development using the anti-hTfR antibody of the present invention, the pharmacokinetic studies of the drug can be conducted using monkeys, thus significantly facilitating drug development.

[0224] In this invention, the antibody exhibits relatively high affinity for hTfR and also for monkey TfR, and particularly when measured using the method described in Example 7, the dissociation constants with human and monkey TfR are as follows:

[0225] (a) Dissociation constant with hTfR: preferably 1×10 -10 M or less, more preferably 2.5 × 10 -11 For values ​​below M, 5×10 is further preferred. -12 For values ​​below M, 1×10 is even more preferred. -12 Below M,

[0226] (b) Dissociation constant with monkey TfR: preferably 1×10 -9 For M and below, 5×10 is more preferred. -10 For M and below, 1×10 is further preferred. -10 Below M, for example, 7.5 × 10 -11 Below M.

[0227] For example, the dissociation constants of hTfR and monkeyTfR are 1×10⁻⁶. -10 Below M and 1×10 -9 Below M, 1×10-11 Below M and 5×10 -10 Below M, 5×10 -12 Below M and 1×10 -10 Below M, 5×10 -12 Below M and 7.5×10 -11 Below M, 1×10 -12 Below M and 1×10 -10 Below M, 1×10 -12 Below M and 7.5×10 -11 Below M. Here, there is no particularly explicit lower limit for the dissociation constant of human TfR; for example, it can be set to 5 × 10⁻⁶. -13 M, 1×10 -13 M, etc. Furthermore, there is no particularly clear lower limit to the dissociation constant of the TfR with monkeys; for example, it can be set to 1 × 10⁻⁶. -11 M, 1×10 -12 M, etc. The same applies when the antibody is a single-chain antibody.

[0228] Humanized antibodies can be prepared by using antibodies from animals other than humans that have relatively high affinity for hTfR obtained through the method described above for selecting cells that produce high-affinity antibodies. A humanized antibody is an antibody obtained by replacing a suitable region of a human antibody (transplanting the sequence into the human antibody) with a portion of the variable region (e.g., all or part of the CDR) of an antibody from an animal other than a human antibody, while maintaining the specificity to the antigen. For example, examples of humanized antibodies include antibodies obtained by replacing the three complementarity-determining regions (CDRs) of the immunoglobulin light chain and the three complementarity-determining regions (CDRs) of the immunoglobulin heavy chain with CDRs from other mammals. The source species of the CDRs integrated into the human antibody are not particularly limited as long as they are mammals other than humans; preferred species include mice, rats, rabbits, horses, and primates other than humans; more preferred are mice and rats; and even more preferred are mice. However, antibodies obtained by replacing a portion of a human antibody with a portion of another human antibody can also be prepared.

[0229] Methods for producing humanized antibodies are well-known in this technical field. The most conventional method is based on the approach devised by Winter et al., which involves replacing the amino acid sequence of the complementarity-determining region (CDR) in the variable region of a human antibody with the CDR of a non-human mammalian antibody (Verhoeyen M. Science. 239. 1534-1536 (1988)). It is known that, in order to reproduce the original activity of the donor antibody, it is sometimes necessary to replace the corresponding position of the human antibody that serves as the receptor with not only the CDR of the non-human mammalian antibody, but also the amino acid sequence of the region outside the CDR involved in CDR structural maintenance or antigen binding (Queen C. Proc. Natl. Acad. Sci. USA. 86. 10029-10033 (1989). Here, the region outside the CDR is also called the frame region (FR).

[0230] Both the heavy and light chains of the antibody contain four frame regions 1 to 4 (FR1 to 4) in their variable regions. FR1 is the region adjacent to CDR1 at its N-terminus and consists of an 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 an amino acid sequence from the amino acid adjacent to the C-terminus of CDR3 to the C-terminus of the variable region. However, this is not a limitation; in this invention, the regions of each of the above-mentioned FR regions other than the 1 to 5 amino acids on its N-terminus side and / or the 1 to 5 amino acids on its C-terminus side may also be used as frame regions.

[0231] The creation of humanized antibodies involves the following steps: transplanting the CDR (and, if necessary, the surrounding FR) of a non-human mammalian antibody to replace the CDR (and, if necessary, the surrounding FR) of the variable region of the human antibody. In this process, the framework region of the variable region that forms the basis of the 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 the 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). Alternatively, DNA 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 Welfare, 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)".

[0232] As described above, in humanized antibodies, the region of the non-human mammalian antibody transplanted into the variable region of the original human antibody typically contains the CDR itself or the CDR and its surrounding FRs. However, FRs transplanted along with the CDR also participate in the structural maintenance of the CDR or binding to the antigen, and are considered to substantially also have a function in determining antibody complementarity. Therefore, in this invention, the term "CDR" refers to the region or transplantable region transplanted from a non-human mammalian antibody into the humanized antibody when manufacturing humanized antibodies. That is, regarding the CDR, even regions that are generally considered FRs are included in this invention as long as they participate in the structural maintenance of the CDR or binding to the antigen and are considered to substantially have a function in determining antibody complementarity.

[0233] Regarding the anti-hTfR antibody of the present invention, when administered to a living organism via intravenous injection or the like, it can efficiently bind to hTfR on the endothelial cells of capillaries present in the brain. The antibody, after binding to hTfR, is taken into the brain via mechanisms such as endocytosis and transcytosis, crossing the blood-brain barrier. Therefore, by binding proteins, low-molecular-weight compounds, etc., that are intended to function in the brain to the anti-hTfR antibody of the present invention, these substances can efficiently cross the blood-brain barrier and reach the brain. Furthermore, the anti-hTfR antibody of the present invention, after crossing the blood-brain barrier, can reach the brain parenchyma, neuron-like cells of the hippocampus, Purkinje cells of the cerebellum, etc., or at least any one of these. It is also expected to reach neuron-like cells of the striatum of the brain and neuron-like cells of the substantia nigra of the midbrain. Therefore, by binding proteins, low-molecular-weight compounds, etc., that act on these tissues or cells to the anti-hTfR antibody of the present invention, these tissues or cells can be reached.

[0234] The anti-hTfR antibody of the present invention may be an effective means of enabling 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 to reach the brain from the blood and exert their effects there. In particular, the anti-hTfR antibody of the present invention, after crossing the blood-brain barrier, reaches the brain parenchyma, neuron-like cells of the hippocampus, Purkinje cells of the cerebellum, or at least any of these. It also holds promise for reaching neuron-like cells of the striatum of the brain and neuron-like cells of the substantia nigra of the midbrain. Therefore, by administering these substances in the blood in a form bound to the anti-hTfR antibody molecule of the present invention via intravenous administration or the like, their functions can be exerted and enhanced in these brain tissues or cells.

[0235] As a method for binding anti-hTfR antibodies to such substances (proteins, low-molecular-weight compounds, etc.), there are methods using either non-peptide linkers or peptide linkers. Non-peptide linkers can be polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitosans, and hyaluronic acid, or derivatives thereof, or combinations thereof. Peptide linkers are peptide chains or derivatives thereof consisting of 1 to 50 amino acids forming peptide bonds, with their N-terminus and C-terminus forming covalent bonds with either the anti-hTfR antibody, protein, or low-molecular-weight compound, thereby binding the anti-hTfR antibody to the protein, low-molecular-weight compound, etc.

[0236] When the anti-hTfR antibody of the present invention is bound to another desired protein (A) using PEG as a non-peptide linker, it is specifically referred to as anti-hTfR antibody-PEG-protein. The anti-hTfR antibody-PEG-protein can be manufactured by binding an anti-hTfR antibody to PEG to form an anti-hTfR antibody-PEG, and then binding the anti-hTfR antibody-PEG to another protein (A). Alternatively, the anti-hTfR antibody-PEG-protein can also be manufactured by binding another protein (A) to PEG to form a protein-PEG, and then binding the protein-PEG to an anti-hTfR antibody. When binding PEG to the anti-hTfR antibody and the other protein (A), PEG modified with functional groups such as carbonates, carbonyl imidazolium, active esters of carboxylic acids, acrylonitriles, cyclic iminothiophenes, isocyanates, isothiocyanates, imine esters, or aldehydes is used. The functional groups introduced into these PEGs primarily react with the amino groups within the molecules of anti-hTfR antibodies and other proteins (A), thereby forming covalent bonds between the PEG and the hTfR antibodies and other proteins (A). There are no particular limitations on the molecular weight and shape of the PEG used, but its average molecular weight (MW) is preferably MW = 500–60,000, more preferably MW = 500–20,000. For example, PEGs with average molecular weights of about 300, about 500, about 1,000, about 2,000, about 4,000, about 10,000, about 20,000, etc., can preferably be used as non-peptide linkers. The same applies when binding anti-hTfR antibodies to desired low-molecular-weight compounds.

[0237] For example, anti-hTfR antibody-PEG can be obtained as follows: An anti-hTfR antibody is mixed with polyethylene glycol (ALD-PEG-ALD) having an aldehyde group as a functional group, with an ALD-PEG-ALD molar ratio to the antibody of 11, 12.5, 15, 110, 120, etc., and a reducing agent such as NaCNBH3 is added to induce a reaction. Then, the anti-hTfR antibody-PEG is reacted with other proteins (A) in the presence of a reducing agent such as NaCNBH3 to obtain anti-hTfR antibody-PEG-protein. Conversely, anti-hTfR antibody-PEG-protein can also be obtained by first binding other proteins (A) to ALD-PEG-ALD to prepare protein-PEG, and then binding protein-PEG to the anti-hTfR antibody.

[0238] Anti-hTfR antibodies and other proteins (A) can also bind to the N-terminus or C-terminus of other proteins (A) via adapter sequences or directly via peptide bonds at the C-terminus or N-terminus of the heavy or light chain of the anti-hTfR antibody. Fusion proteins formed by combining anti-hTfR antibodies and other proteins (A) can be obtained as follows: a DNA fragment encoding an adapter sequence is placed directly or interspersed at the 3' or 5' end of the cDNA encoding the heavy or light chain of the anti-hTfR antibody in a frame-in-frame configuration to obtain a DNA fragment. This DNA fragment is then integrated into a mammalian cell expression vector, and mammalian cells incubated with the expression vector are cultured to obtain the fusion protein. For this mammalian cell, when binding a DNA fragment encoding another protein (A) to the heavy chain, the mammalian cell with a cDNA fragment integrating the light chain encoding the anti-hTfR antibody is also introduced into the same host cell using an expression vector. Similarly, when binding a DNA fragment encoding another protein (A) to the light chain, the mammalian cell with a cDNA fragment integrating the heavy chain encoding the anti-hTfR antibody is also introduced into the same host cell using an expression vector. When the anti-hTfR antibody is a single-chain antibody, the fusion protein formed by the anti-hTfR antibody and other protein (A) can be obtained as follows: A DNA fragment encoding a single-chain anti-hTfR antibody is linked to a DNA fragment encoding a linker sequence directly or interposed at the 5' or 3' end of the cDNA encoding the other protein (A), and integrated into an expression vector (for mammalian cells, eukaryotic cells such as yeast, or prokaryotic cells such as E. coli), allowing it to be expressed in these cells that have been introduced with the expression vector.

[0239] In the case of fusion proteins of the type where other proteins (A) bind to the C-terminus of the light chain of an anti-hTfR antibody, an anti-human transferrin receptor antibody comprises all or part of the amino acid sequence of a variable region containing a light chain and all or part of the amino acid sequence of a variable region containing a heavy chain, with other proteins (A) binding to the C-terminus of the light chain of the anti-human transferrin receptor antibody. Here, the light chain of the anti-hTfR antibody and the other protein (A) can bind either directly or through a linker.

[0240] In the case of fusion proteins of the type where other proteins (A) bind to the C-terminus of the heavy chain of an anti-hTfR antibody, an anti-human transferrin receptor antibody comprises all or part of the amino acid sequence of a variable region containing a light chain and all or part of the amino acid sequence of a variable region containing a heavy chain, with other proteins (A) binding to the C-terminus of the heavy chain of the anti-human transferrin receptor antibody. Here, the heavy chain of the anti-hTfR antibody and the other protein (A) can bind either directly or through a linker.

[0241] In the case of fusion proteins of the type where other proteins (A) bind to the N-terminus of the light chain of an anti-hTfR antibody, an anti-human transferrin receptor antibody comprises all or part of the amino acid sequence of a variable region containing a light chain and all or part of the amino acid sequence of a variable region containing a heavy chain, with other proteins (A) binding to the N-terminus of the light chain of the anti-human transferrin receptor antibody. Here, the light chain of the anti-hTfR antibody and the other protein (A) can bind either directly or through a linker.

[0242] In the case of fusion proteins of the type where other proteins (A) bind to the N-terminus of the heavy chain of an anti-hTfR antibody, an anti-human transferrin receptor antibody comprises all or part of the amino acid sequence of a variable region containing a light chain and all or part of the amino acid sequence of a variable region containing a heavy chain, with other proteins (A) binding to the N-terminus of the heavy chain of the anti-human transferrin receptor antibody. Here, the heavy chain of the anti-hTfR antibody and the other protein (A) can bind either directly or through a linker.

[0243] At this point, the linker sequence configured between the anti-hTfR antibody and other proteins (A) is a peptide chain consisting of preferably 1 to 50, more preferably 1 to 17, further preferably 1 to 10, and even more preferably 1 to 5 amino acids. 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 which it should bind. As long as the anti-hTfR antibody linked by this linker sequence maintains its affinity for hTfR and the other protein (A) linked by this linker sequence can exert its physiological activity under physiological conditions, its amino acid sequence is not limited, but it is preferably composed of glycine and serine. Examples of linker sequences include those containing a sequence consisting of any one amino acid from 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 (sequence number 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (sequence number 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (sequence number 5), or sequences consisting of 1 to 10 or 2 to 5 of these amino acid sequences linked together. Linker sequences can be those 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, a sequence containing the amino acid sequence Gly-Ser is preferred as a linker sequence. Alternatively, a sequence containing a total of 27 amino acids, consisting of five amino acid sequences Gly-Gly-Gly-Gly-Ser (Sequence No. 3) linked after the amino acid sequence Gly-Ser, can be preferred as the adapter sequence. Alternatively, a sequence containing 25 amino acids, consisting of five amino acid sequences Gly-Gly-Gly-Gly-Ser (Sequence No. 3) linked together, can be preferred as the adapter sequence.

[0244] In fusion proteins of anti-hTfR antibodies and other proteins (A), when 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 typically bind through a linker sequence. In this case, provided the anti-hTfR antibody maintains its affinity for hTfR, it can either bind the linker sequence to the C-terminus of the amino acid sequence originating from the light chain, and then bind the amino acid sequence originating from the heavy chain to its C-terminus; or conversely, it can bind the linker sequence to the C-terminus of the amino acid sequence originating from the heavy chain, and then bind the amino acid sequence originating from the light chain to its C-terminus.

[0245] The linker sequence configured between the light and heavy chains of immunoglobulins is a peptide chain consisting of preferably 2 to 50, more preferably 8 to 50, further preferably 10 to 30, even more preferably 12 to 18 or 15 to 25, for example 15 or 25 amino acids. The amino acid sequence of this adapter sequence is not limited as long as the anti-hTfR antibody formed by linking the two chains with it maintains affinity for hTfR and the other protein (A) bound to the antibody can exert its physiological activity under physiological conditions. It is preferably composed of glycine or glycine and serine, for example, containing the amino acid sequences Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly, Gly-Gly-Gly-Gly (Sequence No. 3), Gly-Gly-Gly-Gly-Gly-Ser (Sequence No. 4), Ser-Gly-Gly-Gly-Gly (Sequence No. 5), or sequences composed of 2 to 10 or 2 to 5 of these amino acid sequences. As a preferred form of adapter sequence, an example is an adapter sequence containing 15 amino acids formed by linking three amino acid sequences Gly-Gly-Gly-Gly-Ser (Sequence No. 3).

[0246] As an example of the specific form of the fusion protein of the humanized anti-hTfR antibody of the present invention and other proteins (A) when the anti-hTfR antibody is a single-chain antibody, the following form can be illustrated: a form in which a single-chain antibody is bound to the C-terminus of other proteins (A) via a first adapter sequence, wherein the first adapter sequence consists of a total of 27 amino acids, which are formed by connecting a 5-amino acid sequence Gly-Gly-Gly-Gly-Ser (sequence number 3) after the amino acid sequence Gly-Ser. Here, as a preferred form of the single-chain antibody used, the following forms can be listed: The form in which the variable region of the variable region of the heavy chain of the humanized anti-hTfR antibody No. 3N having the amino acid sequence described in Serial No. 65 is bound to the variable region of the anti-hTfR antibody light chain having the amino acid sequence described in Serial No. 18, i.e., the form having the amino acid sequence described in Serial No. 60, is bound to the first linker sequence consisting of 15 amino acids linked by the three amino acid sequences Gly-Gly-Gly-Gly-Ser (Serial No. 3); The form in which the Fab heavy chain of the humanized anti-hTfR antibody No. 3N having the amino acid sequence described in Serial No. 61 is bound to the C-terminus of the anti-hTfR antibody light chain having the amino acid sequence described in Serial No. 23, i.e., the form having the amino acid sequence described in Serial No. 98, is bound to the first linker sequence consisting of 32 amino acids, consisting of an amino acid sequence linked 6 times with the amino acid sequence shown in Serial No. 3, followed by the amino acid sequence Gly-Gly.

[0247] When the anti-hTfR antibody is a single-chain antibody, such a fusion protein can be manufactured, for example, by transforming a host cell such as a mammalian cell with an expression vector that integrates a DNA fragment having a base sequence encoding the fusion protein and culturing the host cell.

[0248] It should be noted that in this invention, when a peptide chain contains multiple adapter sequences, for convenience, each adapter sequence is named sequentially from the N-terminal side as the first adapter sequence, the second adapter sequence, etc.

[0249] As an example of a specific form of the fusion protein of the humanized anti-hTfR antibody of the present invention and other proteins (A) when the anti-hTfR antibody is Fab, the following form can be listed: A variable region containing the anti-hTfR antibody heavy chain is fused to the C-terminus of the other protein (A) via a linker sequence consisting of a total of 27 amino acids, formed by a 5-amino acid sequence Gly-Gly-Gly-Gly-Ser (serial number 3) linked after Gly-Ser. H The region of region 1. At this point, except for C. H Outside of region 1, there may be a portion of a hinge region, but this hinge region does not contain cysteine ​​residues used to form disulfide bonds between heavy chains.

[0250] As an example of a preferred heavy chain when the anti-hTfR antibody is Fab, an example having the amino acid sequence described in Serial No. 61 can be cited. The amino acid sequence of Serial No. 61 is the Fab heavy chain of the humanized anti-hTfR antibody No. 3N having the amino acid sequence shown in Serial No. 66, and corresponds to the portion from position 1 to position 226 from the N-terminus of the amino acid sequence of Serial No. 66. It should be noted that the portion from position 1 to position 118 from the N-terminus of Serial No. 61 corresponds to the variable region (Serial No. 65), and the portion from position 119 to position 216 corresponds to the C... H The first area corresponds to the second area, and the portion from the 217th to the 226th position corresponds to the hinge area.

[0251] When the anti-hTfR antibody is Fab, the Fc region of other IgGs can be further introduced into the fusion protein. Introducing the Fc region into the fusion protein can improve its stability in organisms such as blood. For example, a Fab heavy chain may have a morphology in which a human IgG Fc region is directly or indirectly bound to the C-terminus of another protein (A) or via a linker sequence, and an anti-human transferrin receptor antibody is directly or indirectly bound to the C-terminus of that human IgG Fc region.

[0252] The linker sequence between other proteins (A) and the human IgG Fc region is preferably composed of 1 to 50 amino acids. Here, the number of amino acids can be 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 a sequence composed of glycine and serine is preferred. Examples of linker sequences include: sequences consisting of any one amino acid from glycine or serine; amino acid sequences Gly-Ser; amino acid sequences Gly-Gly-Ser; amino acid sequences Gly-Gly-Gly-Gly-Ser (sequence number 3); amino acid sequences Gly-Gly-Gly-Gly-Gly-Ser (sequence number 4); amino acid sequences Ser-Gly-Gly-Gly-Gly (sequence number 5); sequences consisting of 50 or fewer amino acids linked together from 1 to 10 or 2 to 5 of these amino acid sequences; and 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 containing 5 amino acid sequences linked together from Gly-Gly-Gly-Gly-Ser (sequence number 3) with a total of 25 amino acids can be preferred as a linker sequence. The linker sequence between the human IgG Fc region and the Fab heavy chain is the same.

[0253] It should be noted that when introducing the human IgG Fc region, the human IgG Fc region can bind to either the heavy chain or the light chain of the anti-human transferrin receptor antibody. Additionally, the antibody can be other antigen-binding fragments containing F(ab')2, F(ab'), or single-chain antibodies.

[0254] The type of IgG introduced into the human IgG Fc region is not particularly limited and can be any of IgG1 to IgG5. Furthermore, the introduced human IgG Fc region can be the entire Fc region or a portion thereof. As a preferred embodiment of this human IgG Fc region, a form having the amino acid sequence shown in sequence number 70, representing the entire Fc region of human IgG1, can be listed. Additionally, as the amino acid sequence of the Fab heavy chain into which the human IgG Fc region is introduced, a form having the amino acid sequence shown in sequence number 71, where a 25-amino acid linker sequence (Gly-Gly-Gly-Gly-Ser, sequence number 3) containing five amino acids linked together at the N-terminus of the Fab heavy chain amino acid sequence (sequence number 61) of the humanized anti-hTfR antibody 3N is attached to the human IgG Fc region having the amino acid sequence shown in sequence number 70.

[0255] Fusion proteins of anti-hTfR antibodies with other proteins (A) can also be modified to have an affinity for albumin. Albumin affinity can be achieved by binding albumin-affinity compounds, peptides, proteins, etc., to the fusion protein. The fusion protein with albumin affinity, at least a portion of which binds to albumin and circulates in the blood. Albumin has the function of stabilizing proteins it binds to. Therefore, by introducing albumin affinity, the half-life of the fusion protein administered to the organism can be prolonged in the blood, thus enhancing its efficacy. The introduction of albumin affinity is more effective when the anti-hTfR antibody is in a morphology lacking the Fc region that contributes to antibody stability, such as Fab.

[0256] Furthermore, in cases where immunogenicity is observed when an anti-hTfR antibody is administered to a host cell in a fusion protein of another protein (A), introducing affinity for albumin is also effective. Because the fusion protein binds to albumin, it prevents the immunogenic site of the fusion protein from being presented to immune cells, thus resulting in weak immunogenicity.

[0257] When introducing affinity for albumin into a fusion protein of an anti-hTfR antibody and other proteins (A), the portion introducing affinity may be any one of the light chain of the anti-hTfR antibody, the heavy chain of the anti-hTfR antibody, other proteins (A), and the linker portion, or more than two of these portions may be introduced.

[0258] As a peptide or protein with affinity for albumin, a peptide can be used, for example, one having the amino acid sequence shown in Serial No. 74, which modifies the albumin-binding domain of a protein derived from Streptococcus strain G418 (Alm T. Biotechnol J. 5. 605-17 (2010)) in a manner that exhibits alkali tolerance, but is not limited thereto. As a method for binding an albumin-affinity peptide or protein (albumin-affinity peptide) to a fusion protein (anti-hTfR antibody-protein (A) fusion protein) of an anti-hTfR antibody and another protein (A), there are methods using either a non-peptide linker or a peptide linker. As a non-peptide linker, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitosans, and hyaluronic acid, or derivatives thereof, or combinations thereof, can be used. A peptide linker is a peptide chain or its derivative consisting of 1 to 50 amino acids that have formed peptide bonds. Its N-terminus and C-terminus form covalent bonds with either an albumin-affinity peptide or a fusion protein, thereby enabling the albumin-affinity peptide to bind to the fusion protein.

[0259] A substance formed by combining albumin affinity peptides and anti-hTfR antibodies with other proteins (A) using PEG as a non-peptide linker is specifically called an albumin affinity peptide-PEG-protein (A) fusion protein. The albumin affinity peptide-anti-hTfR antibody-protein (A) fusion protein can be manufactured by combining an albumin affinity peptide with PEG to form albumin affinity peptide-PEG, and then combining albumin affinity peptide-PEG with anti-hTfR antibody-protein (A) fusion protein. Alternatively, the albumin affinity peptide-anti-hTfR antibody-protein (A) fusion protein can also be manufactured by combining an anti-hTfR antibody-protein (A) fusion protein with PEG to form an anti-hTfR antibody-protein (A) fusion protein, and then combining anti-hTfR antibody-protein (A) fusion protein-PEG with albumin affinity peptide. When binding PEG to albumin-affinity peptides and anti-hTfR antibody-protein (A) fusion proteins, PEG modified with functional groups such as carbonates, carbonyl imidazoles, active esters of carboxylic acids, acrylonitriles, cyclic iminothiophenes, isocyanates, isothiocyanates, imine esters, or aldehydes is used. The functional groups introduced into these PEGs primarily react with the amino groups within the albumin-affinity peptides and anti-hTfR antibody-protein (A) fusion protein molecules, thereby forming covalent bonds between the PEG and the albumin-affinity peptides and anti-hTfR antibody-protein (A) fusion proteins. The molecular weight and shape of the PEG used are not particularly limited, but its average molecular weight (MW) is preferably MW = 500 to 60,000, more preferably MW = 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, about 20,000, etc., can preferably be used as non-peptide linkers.

[0260] For example, albumin affinity peptide-PEG can be obtained as follows: Albumin affinity peptide and aldehyde-modified PEG (ALD-PEG-ALD) are mixed in a molar ratio of PEG to albumin affinity peptide of 11, 12.5, 15, 110, 120, etc., and a reducing agent such as NaCNBH3 is added to induce a reaction. Then, albumin affinity peptide-PEG is reacted with an anti-hTfR antibody-protein (A) fusion protein in the presence of a reducing agent such as NaCNBH3 to obtain an albumin affinity peptide-PEG-anti-hTfR antibody-protein (A) fusion protein. Conversely, albumin affinity peptide-PEG-anti-hTfR antibody-protein (A) fusion protein can also be obtained by first binding the anti-hTfR antibody-protein (A) fusion protein to ALD-PEG-ALD, and then binding the fusion protein-PEG to the albumin affinity peptide.

[0261] It is also possible to fuse an anti-hTfR antibody-protein (A) fusion protein with an albumin affinity peptide. The fusion protein (anti-hTfR antibody-protein (A) fusion protein-albumin affinity peptide) can be obtained as follows: a DNA fragment encoding an albumin affinity peptide is directly or indirectly incorporated into the cDNA encoding the heavy chain (including fusion proteins of the heavy chain and protein (A)) or light chain (including fusion proteins of the light chain and protein (A)) of the anti-hTfR antibody-protein (A) fusion protein at the 3' or 5' end. This DNA fragment is then integrated into a mammalian cell expression vector, and mammalian cells inoculated with the expression vector are cultured. When binding a DNA fragment encoding an albumin affinity peptide to a heavy chain (or a fusion protein of a heavy chain and protein (A), an expression vector for mammalian cells integrating a cDNA fragment encoding a fusion protein (or light chain) of a light chain constituting an anti-hTfR antibody and protein (A) is also introduced into the same host cell. That is, the albumin affinity peptide can bind to either the N-terminal or C-terminal side of the heavy chain (including the fusion protein of the heavy chain and protein (A)) or the light chain (including the fusion protein of the light chain and protein (A)) of the anti-hTfR antibody-protein (A) fusion protein. When the protein (A) is bound to the N-terminal side of the heavy chain of the anti-hTfR antibody, it is preferably bound to the C-terminal side of the anti-hTfR antibody, and particularly preferably bound to the C-terminal side of the heavy chain.

[0262] When fusing the anti-hTfR antibody-protein (A) fusion protein with an albumin affinity peptide, fusion can be performed directly or via a linker sequence. Here, the linker sequence preferably consists of 1 to 50 amino acids. The number of amino acids can be 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 it is preferably composed of glycine and serine. Examples of linker sequences include: sequences containing any one amino acid from glycine or serine; amino acid sequences Gly-Ser; amino acid sequences Gly-Gly-Ser; amino acid sequences Gly-Gly-Gly-Gly-Ser (sequence number 3); amino acid sequences Gly-Gly-Gly-Gly-Gly-Ser (sequence number 4); amino acid sequences Ser-Gly-Gly-Gly-Gly (sequence number 5); sequences consisting of 1 to 10 or 2 to 5 of these amino acid sequences linked together, 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 containing 3 amino acid sequences Gly-Gly-Gly-Gly-Ser (sequence number 3) linked together, totaling 15 amino acids, can be preferred as a linker sequence.

[0263] The binding affinity of the anti-hTfR antibody-protein (A) fusion protein, incorporating an albumin-affinity peptide, to albumin is preferably 1 × 10⁻⁶ when measured using the biomembrane interferometry method described in Example 7. -7 For M and below, 5×10 is more preferred. -7 For M and below, 1×10 is further preferred. -8 For values ​​below M, 1×10 is even more preferred. -9 Below M.

[0264] Fab antibodies can also be stabilized in the blood by methods other than introducing the Fc region or albumin-affinity peptides. For example, Fab antibodies can be stabilized by PEGylation of the Fab antibody itself or a fusion of the Fab antibody with other proteins. This method is frequently used in the field of protein pharmaceuticals, and PEGylated erythropoietin, interferon, etc., are already in practical use as drugs. Additionally, Fab antibodies can be stabilized by introducing mutations. For example, stabilizing a Fab antibody can be achieved by replacing the methionine at position 4 from the N-terminus of the light chain with leucine. However, the method of introducing mutations is not limited to this; mutations can also be introduced into the heavy chain. Furthermore, the stabilization methods for Fab antibodies are not limited to these; all known methods can be used.

[0265] There are no particular limitations on other proteins (A) that should bind to anti-hTfR antibodies; they are substances that can exert physiological activity in vivo, especially proteins that, although they should reach the brain to exert their effects, cannot cross the blood-brain barrier in their original state and therefore cannot be expected to exert their effects in the brain through intravenous administration. Examples of such proteins include: nerve growth factor (NGF), α-L-iduronidase (IDUA), iduronate-2-sulfatase (IDS), glucocerebrosidase (GBA), β-galactosidase, GM2 activator protein, β-aminohexosidase A, β-aminohexosidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase (LAMAN), β-mannosidase, galactosylceramide enzyme (GALC), sphingolipid activator protein C, arylsulfatase A (ARSA), α-L-fucosidase (FUCA1), aspartate glucosidase, and α-N-acetylgalactosidase. Enzymes, including acid sphingomyelinase (ASM), α-galactosidase A, β-glucuronidase (GUSB), heparin N-sulfatase (SGSH), α-N-acetylglucosidase (NAGLU), acetyl-CoA α-glucosinolate N-acetyltransferase, N-acetylglucosamine-6-sulfatase, acid ceramide (AC), amyl-1,6-glucosidase, sialidase, aspartate glucosidase, palmitoyl protein thioesterase-1 (PPT-1), tripeptidyl peptidase-1 (TPP-1), hyaluronidase-1, acid α-glucosidase (GAA), CLN1, CLN2, and other lysosomal enzymes.

[0266] Nerve growth factor (NGF) bound to anti-hTfR antibodies can be used as a treatment for dementia in Alzheimer's disease; α-L-iduronase (IDUA) fused to anti-hTfR antibodies can be used as a treatment for central nervous system disorders in Hurler syndrome or Hurler-Scheie syndrome; iduronate-2-sulfatase (IDS) fused to anti-hTfR antibodies can be used as a treatment for central nervous system disorders in Hunter syndrome; glucocerebrosidase (GBA) can be used as a treatment for central nervous system disorders in Gaucher disease; and β-galactosidase can be used as a treatment for central nervous system disorders in GM1-ganglioside deposition syndrome types 1-3. GM2 activator protein can be used as a treatment for central nervous system disorders in GM2-gangliosidosis AB variant; β-aminohexosidase A can be used as a treatment for central nervous system disorders in Sandhoff's disease and Ty-Sachs disease; β-aminohexosidase B can be used as a treatment for central nervous system disorders in Sandhoff's disease; N-acetylglucosamine-1-phosphotransferase can be used as a treatment for central nervous system disorders in I-cell disease; α-mannosidase (LAMAN) can be used as a treatment for central nervous system disorders in α-mannoside hyperactivity; β-mannosidase can be used as a treatment for central nervous system disorders in β-mannoside hyperactivity; galactosylceramide enzyme (GALC)... It can be used as a central nervous system disorder treatment for Clapey's disease; sphingolipid-activated protein C can be used as a central nervous system disorder treatment for Gaucher's disease-like accumulation disease; arylsulfatase A (ARSA) can be used as a central nervous system disorder treatment for metachromatic leukodystrophy; α-L-fucosidase (FUCA1) can be used as a central nervous system disorder treatment for fucosylation disorders; aspartate glucosidase can be used as a central nervous system disorder treatment for aspartate glucosamineuria; α-N-acetylgalactosidase can be used as a central nervous system disorder treatment for Schindler's disease and Kawasaki disease; acid sphingomyelinase (ASM) can... It can be used as a central nervous system disorder treatment for Niemann-Pick disease; α-galactosidase A can be used as a central nervous system disorder treatment for Fabry disease; β-glucuronidase (GUSB) can be used as a central nervous system disorder treatment for Sly syndrome; heparin N-sulfatase (SGSH), α-N-acetylglucosidase (NAGLU), acetyl-CoA α-glucosinolate N-acetyltransferase, and N-acetylglucosamine-6-sulfatase can be used as central nervous system disorder treatments for Sanfilippo syndrome; acid ceramidinase (AC) can be used as a central nervous system disorder treatment for Farber disease; starch-1,6-Glucosidase can be used as a treatment for central nervous system disorders in Cori disease (Forbes-Cori disease); sialidase can be used as a treatment for central nervous system disorders in sialidase deficiency; palmitoylprotein thioesterase-1 (PPT-1) can be used as a treatment for central nervous system disorders in neuronal ceroid lipofuscin deposition disease or Santavuori-Haltia disease; tripeptidyl peptidase-1 (TPP-1) can be used as a treatment for central nervous system disorders in neuronal ceroid lipofuscin deposition disease or Jansky-Bielschowsky disease; hyaluronidase-1 can be used as a treatment for central nervous system disorders in hyaluronidase deficiency; acid α-glucosidase (GAA) can be used as a treatment for central nervous system disorders in Pompe disease; and CLN1 and CLN2 can be used as treatments for central nervous system disorders in Batten disease. In particular, the anti-hTfR antibody of the present invention is expected to reach the brain parenchyma, hippocampal neuron-like cells, Purkinje cells of the cerebellum, etc., after crossing the blood-brain barrier, or even neuron-like cells of the striatum and substantia nigra of the midbrain. Therefore, by fusing with proteins that are intended to exert their pharmacological effects in these tissues or cells, the efficacy of those proteins can be enhanced. However, the drug's use is not limited to these diseases.

[0267] It should be noted that, in this invention, the substances used as therapeutic agents can also be used to prevent the onset of diseases.

[0268] Other proteins that can exert their therapeutic effect by binding to anti-hTfR antibodies include: lysosomal enzymes, ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurotrophic factor 3, neurotrophic factor 4 / 5, neurotrophic factor 6, neuroregulatory protein 1, erythropoietin, dabepoetin, 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 interleukins. 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 α receptor (TNF-α receptor), PD-1 ligand, PD-L1, PD-L2, enzymes with β-amyloid degradative activity, anti-β-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, etc.

[0269] Lysosomal enzymes that bind to anti-hTfR antibodies can be used as therapeutic agents for central nervous system disorders of lysosomal diseases; CNTF can be used as a therapeutic agent for amyotrophic lateral sclerosis; GDNF, neurotrophic factor 3, and neurotrophic factor 4 / 5 can be used as therapeutic agents for cerebral ischemia; GDNF can be used as a therapeutic agent for Parkinson's disease; neuroregulatory protein 1 can be used as a therapeutic agent for schizophrenia; erythropoietin and dabepone can be used as therapeutic agents for cerebral ischemia; bFGF and FGF2 can be used for traumatic central nervous system disorders. Treatment agents for systemic disorders, recovery after neurosurgery and spinal surgery; enzymes with β-amyloid degradative activity, anti-β-amyloid antibodies, and anti-BACE antibodies can be used as treatment 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 treatment agents for central nervous system tumors, including brain tumors; and TNFαR-anti-hTfR antibodies can be used as treatment agents for cerebral ischemia and inflammatory brain diseases.

[0270] Treatment agents for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease; mental 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 dystrophy; cerebral ischemia; inflammatory brain diseases; prion diseases; and traumatic central nervous system disorders can often be used as other proteins (A) to fuse with anti-hTfR antibodies. Furthermore, treatment agents for viral and bacterial central nervous system diseases can also often be used as other proteins (A) to fuse with anti-hTfR antibodies. Moreover, medications used for recovery after neurosurgery and spinal surgery can also often be used as other proteins (A) to fuse with anti-hTfR antibodies.

[0271] As other proteins (A) that should bind to anti-hTfR antibodies, analogues obtained by replacing one or more amino acids in these proteins with other amino acids or by deletion, in addition to the natural (wild-type) proteins mentioned above, are also included in these proteins as long as they completely or partially possess the function of these proteins. When amino acids are replaced with other amino acids, the number of amino acids replaced 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 deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. In addition, combinations of these amino acid replacements and deletions can also form desired analogues. Furthermore, substances in which one or more amino acids are added to the amino acid sequence of natural (wild-type) proteins or their analogues, either at the N-terminus or C-terminus, are also included in these proteins as long as they completely or partially possess the function of 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. Combining the addition, substitution, and deletion of these amino acids can also form the desired analogues of these proteins.

[0272] It should be noted that when mutations are introduced into these other proteins (A) and amino acids are added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is located between the protein and the anti-hTfR antibody when the protein is fused with the anti-hTfR antibody.

[0273] The native human acid α-glucosidase (hGAA) is a lysosomal enzyme consisting of 883 amino acids, as shown in SEQ ID NO: 55. However, the substance consisting of 896 amino acids, as shown in SEQ ID NO: 56, formed by adding 13 amino acids to the N-terminus of the amino acid sequence shown in SEQ ID NO: 55, is also included in the native hGAA.

[0274] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native hGAA is fused to the C-terminus of the anti-hTfR antibody heavy chain via the Gly-Ser amino acid sequence as a linker sequence. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hGAA is bound to the C-terminus via a peptide bond through the Gly-Ser linker sequence. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody. Examples of base sequences encoding the substance having the amino acid sequence of Serial No. 66 include the base sequence shown in Serial No. 67, and examples of base sequences encoding the substance having the amino acid sequence of Serial No. 68 include the base sequence shown in Serial No. 69.

[0275] Human acidic alpha-glucosidase (hGAA), also known as alpha-1,4-glucosidase or acidic maltase, has the activity of breaking down glycogen by hydrolyzing the alpha-1,4- and alpha-1,6-glycosidic bonds of glycogen in lysosomes. Pompe disease, also known as glycogen storage disease type II (GSD II), is a disease caused by intracellular glycogen accumulation accompanied by a deficiency in the activity of acidic alpha-glucosidase (acidic maltase) in lysosomes. Pompe disease patients sometimes experience central nervous system disorders. hGAA bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with Pompe disease.

[0276] In this invention, the term "human GAA" or "hGAA" specifically refers to an hGAA having an amino acid sequence identical to that of the natural hGAA, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of the natural hGAA, as long as they possess hGAA activity, are also included in the hGAA. When replacing amino acids in the amino acid sequence of the hGAA with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When amino acids are deleted from the amino acid sequence of the hGAA, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can also be introduced. When adding amino acids to the hGAA, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of the hGAA, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the introduced hGAA preferably has more than 80% homology with the original hGAA amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0277] It should be noted that when hGAA is described as having hGAA activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the natural hGAA. However, this activity relative to the natural hGAA is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hGAA fused with the anti-hTfR antibody.

[0278] A fusion protein of anti-hTfR antibody and hGAA can be manufactured, for example, as follows: An expression vector integrating a DNA fragment having a base sequence encoding the amino acid sequence shown in SEQ ID NO. 59, and an expression vector integrating a DNA fragment having a base sequence encoding the light chain of the anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO. 23, is transformed into a host cell such as a mammalian cell and the host cell is cultured. The resulting fusion protein can be used as a therapeutic agent for Pompe disease, particularly for the central nervous system disorders associated with Pompe disease. The fusion protein obtained in this way is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hGAA.

[0279] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hGAA and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and the hGAA.

[0280] Natural human I2S (hI2S) is a lysosomal enzyme consisting of a 525-amino acid sequence as shown in Serial No. 50. As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, a fusion protein in which natural human I2S is fused to the C-terminus of the anti-hTfR antibody heavy chain via an amino acid sequence Gly-Ser as a linker sequence can be cited. An example of such a fusion protein is a fusion protein in which the light chain consists of the amino acid sequence described in Serial No. 23 and the heavy chain binds to human I2S via a peptide bond formed by a linker consisting of the amino acid sequence Gly-Ser at its C-terminus to form the amino acid sequence of Serial No. 53. The fusion protein having the amino acid sequence of Serial No. 53 is formed by fusing the heavy chain of an IgG1 type anti-hTfR antibody with hI2S, the heavy chain portion having the amino acid sequence of Serial No. 66. By replacing this heavy chain portion with the amino acid sequence of Serial No. 68, a fusion protein formed by fusing an IgG4 type anti-hTfR antibody with hI2S can also be produced.

[0281] Human I2S (hI2S) possesses the activity to hydrolyze the sulfate ester bonds of heparan sulfate and dermatan sulfate, both glycosaminoglycans. Patients with Hunter syndrome, who have a genetic abnormality in this enzyme, accumulate some of the breakdown products of heparan sulfate and dermatan sulfate in tissues such as the liver and spleen due to abnormal metabolism, leading to symptoms such as skeletal abnormalities. Furthermore, Hunter syndrome patients sometimes experience central nervous system disorders. hI2S bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with Hunter syndrome.

[0282] In this invention, the term "human I2S" or "hI2S" specifically refers to hI2S having an amino acid sequence identical to that of the natural type hI2S, but is not limited thereto. Substances with I2S activity, such as substitutions, deletions, or additions, introduced into the amino acid sequence of the natural type hI2S are also included in hI2S. When amino acids in the amino acid sequence of hI2S are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further 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 deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Furthermore, variations combining these amino acid substitutions and deletions can also be introduced. When adding amino acids to hI2S, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of hI2S, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the modified hI2S preferably has more than 80% homology with the original hI2S amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0283] It should be noted that when hI2S is described as having I2S activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the native hI2S. However, this activity is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more, relative to the native hI2S. The same applies when a variant is introduced into the hI2S fused with the anti-hTfR antibody.

[0284] A fusion protein of anti-hTfR antibody and hGAA can be manufactured, for example, as follows: An expression vector integrating a DNA fragment containing a base sequence of sequence number 54 encoding amino acid sequence number 53, and an expression vector integrating a DNA fragment containing a base sequence of sequence number 24 encoding amino acid sequence number 23 (anti-hTfR antibody light chain), is transformed into host cells such as mammalian cells and the host cells are cultured. The resulting fusion protein can be used as a therapeutic agent for Pompe disease, particularly for the central nervous system disorders associated with Pompe disease. The fusion protein obtained in this way is a fusion protein of humanized anti-hTfR antibody and hGAA of type IgG1.

[0285] It should be noted that when a mutation is introduced into an anti-hTfR antibody or human I2S and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and human I2S.

[0286] Natural human α-L-iduronidase (hIDUA) is a lysosomal enzyme composed of the amino acid sequence shown in sequence number 75 or 76. The hIDUA shown in sequence number 76 is a type of substance with Ala-Pro added to the N-terminus of the sequence shown in sequence number 75.

[0287] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native hIDUA is fused to the C-terminus of the anti-hTfR antibody heavy chain via an amino acid sequence Gly-Ser as a linker sequence. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hIDUA is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0288] Human α-L-iduronidase (hIDUA) is a lysosomal enzyme that hydrolyzes the iduronic acid bonds in dermatin sulfate and heparan sulfate molecules. Hurler syndrome, also known as mucopolysaccharidosis type I, is a disease caused by the accumulation of intracellular dermatin sulfate and other substances, accompanied by a deficiency in lysosomal α-L-iduronidase activity. Patients with Hurler syndrome sometimes experience central nervous system disorders. hIDUA bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with Hurler syndrome.

[0289] In this invention, the term "human IDUA" or "hIDUA" specifically refers to hIDUA having an amino acid sequence identical to that of natural hIDUA, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of natural hIDUA, as long as they possess hIDUA activity, are also included in hIDUA. When an amino acid in the amino acid sequence of hIDUA is substituted with another amino acid, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of hIDUA is deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can also be introduced. When an amino acid is added to hIDUA, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of hIDUA, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the introduced hIDUA preferably has more than 80% homology with the original hIDUA amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0290] It should be noted that when hIDUA is described as having hIDUA activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the natural type of hIDUA. However, this activity relative to the natural type of hIDUA is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hIDUA fused with the anti-hTfR antibody.

[0291] A fusion protein of anti-hTfR antibody and hIDUA can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 90 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for Hurler syndrome, particularly for the treatment of central nervous system disorders associated with Hurler syndrome. The fusion protein thus obtained is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hIDUA.

[0292] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hIDUA and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hIDUA.

[0293] Natural human palmitoyl protein thioesterase-1 (hPPT-1) is a lysosomal enzyme composed of the amino acid sequence shown in Serial No. 77.

[0294] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein 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. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hPPT-1 is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0295] Neuronal ceroid lipofuscinosis (including Santavuori-Haltia disease) is a disease caused by the accumulation of intracellular ceroid lipofuscin associated with a deficiency in lysosomal palmitoylprotein thioesterase-1 activity. Patients with neuronal ceroid lipofuscinosis sometimes also experience central nervous system disorders. hPPT-1 bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with neuronal ceroid lipofuscinosis.

[0296] In this invention, the term "human PPT-1" or "hPPT-1" specifically refers to hPPT-1 having an amino acid sequence identical to that of the natural type hPPT-1, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of the natural type hPPT-1, as long as they possess hPPT-1 activity, are also included in hPPT-1. When amino acids in the amino acid sequence of hPPT-1 are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further 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 deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Furthermore, variations combining these amino acid substitutions and deletions can also be introduced. When adding amino acids to hPPT-1, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of hPPT-1, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of hPPT-1 with the introduced variation preferably has more than 80% homology with the original hPPT-1 amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0297] It should be noted that when hPPT-1 is described as having hPPT-1 activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the native hPPT-1. However, this activity is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more, relative to the native hPPT-1. The same applies when a variant is introduced into the hPPT-1 fused with the anti-hTfR antibody.

[0298] A fusion protein of anti-hTfR antibody and hPPT-1 can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 100 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for neuronal ceroid lipofuscinosis, particularly as a therapeutic agent for central nervous system disorders caused by neuronal ceroid lipofuscinosis. The fusion protein thus obtained is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hPPT-1.

[0299] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hPPT-1 and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hPPT-1.

[0300] Natural human acid sphingomyelinase (hASM) is a lysosomal enzyme composed of the amino acid sequence shown in Serial No. 78.

[0301] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native hASM is fused to the C-terminus of the anti-hTfR antibody heavy chain via an amino acid sequence Gly-Ser as a linker sequence. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hASM is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0302] Human acid sphingomyelinase (hASM) is a lysosomal enzyme that hydrolyzes sphingomyelin into phosphocholine and ceramide. Niemann-Pick disease is classified into types A through F based on its etiology and symptoms. Types A and B are caused by a hereditary deficiency of acid sphingomyelinase (ASM). Niemann-Pick disease is caused by the accumulation of intracellular sphingomyelin accompanying a deficiency in lysosomal acid sphingomyelinase activity. Patients with Niemann-Pick disease sometimes experience central nervous system disorders. hASM bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with Niemann-Pick disease.

[0303] In this invention, the term "human ASM" or "hASM" specifically refers to hASM having an amino acid sequence identical to that of natural hASM, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of natural hASM, as long as they possess hASM activity, are also included in hASM. When amino acids in the amino acid sequence of hASM are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further 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 deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can also be introduced. When amino acids are added to hASM, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of hASM, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the modified hASM preferably has more than 80% homology with the original hASM amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0304] It should be noted that when hASM is described as having hASM activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the natural hASM. However, this activity relative to the natural hASM is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hASM fused with the anti-hTfR antibody.

[0305] A fusion protein of anti-hTfR antibody and hASM can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 106 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for Niemann-Pick disease, particularly for the treatment of central nervous system disorders in Niemann-Pick disease. The fusion protein thus obtained is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hASM.

[0306] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hASM and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hASM.

[0307] Natural human arylsulfatase A (hARSA) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 79.

[0308] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native 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. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hARSA is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0309] Metachromatic leukodystrophy (MRLE) is a disease caused by the accumulation of intracellular thiocerebrosides and other substances, accompanied by a deficiency in lysosomal arylsulfatase A activity. Patients with MRLE sometimes experience central nervous system disorders. hARSA bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with MRLE.

[0310] In this invention, the term "human ARSA" or "hARSA" specifically refers to hARSA having an amino acid sequence identical to that of natural hARSA, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of natural hARSA, as long as they possess hARSA activity, are also included in hARSA. When amino acids in the amino acid sequence of hARSA are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further 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 deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can also be introduced. When amino acids are added to hARSA, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of hARSA, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the modified hARSA preferably has more than 80% homology with the original hARSA amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0311] It should be noted that when hARSA is described as having hARSA activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in native hARSA. However, this activity relative to the activity inherent in native hARSA is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hARSA fused with the anti-hTfR antibody.

[0312] A fusion protein of anti-hTfR antibody and hARSA can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 115 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for metachromatic leukoencephalopathy, particularly as a therapeutic agent for central nervous system disorders related to metachromatic leukoencephalopathy. The fusion protein thus obtained is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hARSA.

[0313] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hARSA and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hARSA.

[0314] Natural human heparin N-sulfatase (hSGSH) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 80.

[0315] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native 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. Examples of such fusion proteins include: a fusion protein in which the light chain consists of the amino acid sequence described in Serial No. 23 and the heavy chain consists of the amino acid sequence described in Serial No. 66 or 68, and hSGSH is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0316] Sanfilippo syndrome, also known as mucopolysaccharidosis type III (MPS III), is a disease caused by the accumulation of intracellular heparan sulfate, accompanied by a deficiency in lysosomal heparan N-sulfatase activity. However, deficiencies in other enzymes, such as α-N-acetylglucosidase, can sometimes also be a contributing factor. Central nervous system disorders are sometimes present in patients with Sanfilippo syndrome. hSGSH bound to anti-hTfR antibodies can be used as a treatment for the central nervous system disorders associated with Sanfilippo syndrome.

[0317] In this invention, the term "human SGSH" or "hSGSH" specifically refers to hSGSH having an amino acid sequence identical to that of natural hSGSH, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of natural hSGSH, as long as they possess hSGSH activity, are also included in hSGSH. When amino acids in the amino acid sequence of hSGSH are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further 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 deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can also be introduced. When amino acids are added to hSGSH, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of hSGSH, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the introduced hSGSH preferably has more than 80% homology with the original hSGSH amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0318] It should be noted that when hSGSH is described as having hSGSH activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in native hSGSH. However, this activity relative to the activity inherent in native hSGSH is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hSGSH fused with the anti-hTfR antibody.

[0319] A fusion protein of anti-hTfR antibody and hSGSH can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 124 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for Sanfilippo syndrome, particularly for central nervous system disorders caused by Sanfilippo syndrome. The fusion protein obtained in this way is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hSGSH.

[0320] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hSGSH and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hSGSH.

[0321] Natural human glucocerebroside lipase (hGBA) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 81.

[0322] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native 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. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hGBA is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0323] Human glucocerebroside lipase (hGBA) is a lysosomal enzyme that hydrolyzes the glycolipid glucocerebroside (or glucosylceramide). Gaucher's disease is caused by the accumulation of intracellular glucocerebrosides associated with a deficiency in lysosomal glucocerebroside lipase activity. Patients with Gaucher's disease sometimes experience central nervous system disorders. hGBA bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with Gaucher's disease.

[0324] In this invention, the term "human GBA" or "hGBA" specifically refers to hGBA having an amino acid sequence identical to that of natural hGBA, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of natural hGBA, as long as they possess hGBA activity, are also included in hGBA. When amino acids in the amino acid sequence of hGBA are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further 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 deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can also be introduced. When amino acids are added to hGBA, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of hGBA, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the introduced hGBA preferably has more than 80% homology with the original hGBA amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0325] It should be noted that when hGBA is described as having hGBA activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the native hGBA. However, this activity relative to the native hGBA is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hGBA fused with the anti-hTfR antibody.

[0326] A fusion protein of anti-hTfR antibody and hGBA can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 130 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for Gaucher's disease, particularly for the treatment of central nervous system disorders caused by Gaucher's disease. The fusion protein thus obtained is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hGBA.

[0327] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hGBA and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hGBA.

[0328] Natural human tripeptidyl peptidase-1 (hTPP-1) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 82.

[0329] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native 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. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hTPP-1 is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0330] Neuronal ceroid lipofuscinosis (including Santavuori-Haltia disease) is a disease caused by the accumulation of intracellular lipofuscin associated with a deficiency in tripeptidyl peptidase-1 activity within lysosomes. Central nervous system disorders are sometimes present in patients with neuronal ceroid lipofuscinosis. hTPP-1 bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with neuronal ceroid lipofuscinosis.

[0331] In this invention, the term "human TPP-1" or "hTPP-1" specifically refers to hTPP-1 having an amino acid sequence identical to that of the natural type hTPP-1, but is not limited thereto. Any substance exhibiting hTPP-1 activity, including substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of the natural type hTPP-1, is also included in hTPP-1. When amino acids in the amino acid sequence of hTPP-1 are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further 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 deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Furthermore, variations combining these amino acid substitutions and deletions can also be introduced. When adding amino acids to hTPP-1, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of hTPP-1, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the modified hTPP-1 preferably has more than 80% homology with the original hTPP-1 amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0332] It should be noted that when referring to hTPP-1 having hTPP-1 activity, this means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the native hTPP-1. However, this activity relative to the native hTPP-1 is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hTPP-1 fused with the anti-hTfR antibody.

[0333] A fusion protein of anti-hTfR antibody and hTPP-1 can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 136 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for neuronal ceroid lipofuscinosis, particularly as a therapeutic agent for central nervous system disorders caused by neuronal ceroid lipofuscinosis. The fusion protein thus obtained is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hTPP-1.

[0334] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hTPP-1 and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hTPP-1.

[0335] Natural human α-N-acetylglucosidase (hNAGLU) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 83.

[0336] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native 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. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hNAGLU is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0337] Sanfilippo syndrome is a disease caused by the accumulation of intracellular heparan sulfate, which is associated with a deficiency in lysosomal α-N-acetylglucosidase activity. Patients with Sanfilippo syndrome sometimes experience central nervous system disorders. hNAGLU bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with Sanfilippo syndrome.

[0338] In this invention, the term "human NAGLU" or "hNAGLU" specifically refers to hNAGLU having an amino acid sequence identical to that of natural hNAGLU, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of natural hNAGLU, as long as they possess hNAGLU activity, are also included in hNAGLU. When amino acids in the amino acid sequence of hNAGLU are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further 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 deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Furthermore, variations combining these amino acid substitutions and deletions can also be introduced. When adding amino acids to hNAGLU, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of hNAGLU, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the modified hNAGLU preferably has more than 80% homology with the original hNAGLU amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0339] It should be noted that when hNAGLU is described as having hNAGLU activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the native hNAGLU. However, this activity relative to the native hNAGLU is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hNAGLU fused with the anti-hTfR antibody.

[0340] A fusion protein of anti-hTfR antibody and hNAGLU can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 142 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for Sanfilippo syndrome, particularly for central nervous system disorders associated with Sanfilippo syndrome. The fusion protein thus obtained is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hNAGLU.

[0341] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hNAGLU and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hNAGLU.

[0342] Natural human β-glucuronidase (hGUSB) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 84.

[0343] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which 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. Examples of such fusion proteins include: a fusion protein in which the light chain consists of the amino acid sequence described in Serial No. 23 and the heavy chain consists of the amino acid sequence described in Serial No. 66 or 68, and hGUSB is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0344] Sly syndrome, also known as mucopolysaccharidosis type VII (MPS VII), is a disorder caused by the accumulation of intracellular mucopolysaccharides associated with a deficiency in lysosomal β-glucuronidase activity. Sly syndrome patients sometimes experience central nervous system disorders. hGUSB bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with Sly syndrome.

[0345] In this invention, the term "human GUSB" or "hGUSB" specifically refers to hGUSB having an amino acid sequence identical to that of natural hGUSB, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of natural hGUSB, as long as they possess hGUSB activity, are also included in hGUSB. When an amino acid in the amino acid sequence of hGUSB is substituted with another amino acid, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of hGUSB is deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can also be introduced. When an amino acid is added to hGUSB, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of hGUSB, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the introduced hGUSB preferably has more than 80% homology with the original hGUSB amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0346] It should be noted that when hGUSB is described as having hGUSB activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the natural hGUSB. However, this activity is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more, relative to the activity inherent in the natural hGUSB. The same applies when a variant is introduced into the hGUSB fused with the anti-hTfR antibody.

[0347] A fusion protein of anti-hTfR antibody and hGUSB can be manufactured, for example, as follows: An expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 150 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23 are transformed into host cells such as mammalian cells, and the host cells are cultured. The resulting fusion protein is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hGUSB. The manufactured fusion protein can be used as a therapeutic agent for Sly syndrome, particularly for the treatment of central nervous system disorders caused by Sly syndrome.

[0348] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hGUSB and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hGUSB.

[0349] Natural human galactosylceramide enzyme (hGALC) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 85.

[0350] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein 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. Examples of such fusion proteins include: a fusion protein in which the light chain consists of the amino acid sequence described in Serial No. 23 and the heavy chain consists of the amino acid sequence described in Serial No. 66 or 68, and which binds hGALC via a peptide bond through the linker sequence Gly-Ser at its C-terminus. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0351] Clapey disease is a disorder caused by a deficiency of galactosylceramidinase activity. Patients with Clapey disease sometimes experience central nervous system disorders. hGALC bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with Clapey disease.

[0352] In this invention, the term "human GALC" or "hGALC" specifically refers to hGALC having an amino acid sequence identical to that of natural hGALC, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of natural hGALC, as long as they possess hGALC activity, are also included in hGALC. When amino acids in the amino acid sequence of hGALC are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When amino acids in the amino acid sequence of hGALC are deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can also be introduced. When amino acids are added to hGALC, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of hGALC, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the modified hGALC preferably has more than 80% homology with the original hGALC amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0353] It should be noted that when hGALC is described as having hGALC activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in native hGALC. However, this activity is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more, relative to the activity inherent in native hGALC. The same applies when a variant is introduced into the hGALC fused with the anti-hTfR antibody.

[0354] A fusion protein of anti-hTfR antibody and hGALC can be manufactured, for example, as follows: An expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 158, and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, are transformed into host cells such as mammalian cells and the host cells are cultured. The resulting fusion protein can be used as a therapeutic agent for Clabbe's disease, particularly for the treatment of central nervous system disorders caused by Clabbe's disease. The fusion protein obtained in this way is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hGALC.

[0355] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hGALC and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and the hGALC.

[0356] Natural human acid ceramide (hAC) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 86.

[0357] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native 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. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hAC is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0358] Farber's disease is caused by the accumulation of intracellular ceramides, which is associated with a deficiency in the activity of acid ceramidinase in lysosomes. Patients with Farber's disease sometimes experience central nervous system disorders. hAC bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with Farber's disease.

[0359] In this invention, the term "human AC" or "hAC" specifically refers to an hAC having an amino acid sequence identical to that of natural hAC, but is not limited thereto. Any substance possessing hAC activity, including substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of natural hAC, is also included in the hAC. When amino acids in the hAC amino acid sequence are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When amino acids are deleted from the hAC amino acid sequence, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Furthermore, variations combining these amino acid substitutions and deletions can also be introduced. When amino acids are added to hAC, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the hAC amino acid sequence, either at the N-terminus or C-terminus. Variations combining these amino acid additions, substitutions, and deletions can also be introduced. The amino acid sequence of the modified hAC preferably has more than 80% homology with the original hAC amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0360] It should be noted that when hAC is described as having hAC activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the native hAC. However, this activity relative to the native hAC is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hAC fused with the anti-hTfR antibody.

[0361] A fusion protein of anti-hTfR antibody and hAC can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 166 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for Farber's disease, particularly for the treatment of central nervous system disorders caused by Farber's disease. The fusion protein thus obtained is a fusion protein of humanized anti-hTfR antibody and hAC of type IgG4.

[0362] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hAC and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and the hAC.

[0363] Natural human α-L-fucosidase (hFUCA1) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 87.

[0364] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein 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. Examples of such fusion proteins include: a fusion protein in which the light chain is composed of the amino acid sequence described in Serial No. 23 and the heavy chain is composed of the amino acid sequence described in Serial No. 66 or 68, and hFUCA1 is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0365] Fucoidopathies are diseases caused by the accumulation of intracellular fucoid oligosaccharides associated with a deficiency in lysosomal α-L-fucosidase activity. Patients with fucoidopathies sometimes experience central nervous system disorders. hFUCA1, bound to anti-hTfR antibodies, can be used as a therapeutic agent for the central nervous system disorders associated with fucoidopathies.

[0366] In this invention, the term "human FUCA1" or "hFUCA1" specifically refers to hFUCA1 having an amino acid sequence identical to that of the natural type hFUCA1, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of the natural type hFUCA1, as long as they possess hFUCA1 activity, are also included in hFUCA1. When amino acids in the amino acid sequence of hFUCA1 are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further 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 deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Furthermore, variations combining these amino acid substitutions and deletions can also be introduced. When adding amino acids to hFUCA1, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of hFUCA1, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the modified hFUCA1 preferably has more than 80% homology with the original hFUCA1 amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0367] It should be noted that when hFUCA1 is described as having hFUCA1 activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the native hFUCA1. However, this activity relative to the native hFUCA1 is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hFUCA1 fused with the anti-hTfR antibody.

[0368] A fusion protein of anti-hTfR antibody and hFUCA1 can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 174 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for fucosylation disorders, particularly for central nervous system disorders caused by fucosylation. The fusion protein obtained in this way is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hFUCA1.

[0369] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hFUCA1 and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hFUCA1.

[0370] Natural human α-mannosidase (hLAMAN) is a lysosomal enzyme consisting of the amino acid sequence shown in Serial No. 88.

[0371] As an example of the specific form of the fusion protein of the anti-hTfR antibody and other proteins (A) of the present invention, the following type can be listed: a fusion protein in which native hLAMAN is fused to the C-terminus of the anti-hTfR antibody heavy chain via an amino acid sequence Gly-Ser as a linker sequence. Examples of such fusion proteins include: a fusion protein in which the light chain consists of the amino acid sequence described in Serial No. 23 and the heavy chain consists of the amino acid sequence described in Serial No. 66 or 68, and hLAMAN is bound to the C-terminus via a peptide bond through the linker sequence Gly-Ser. The substance having the amino acid sequence shown in Serial No. 66 is an IgG1 type anti-hTfR antibody, and the substance having the amino acid sequence shown in Serial No. 68 is an IgG4 type anti-hTfR antibody.

[0372] Human α-mannosidase (hLAMAN) is a lysosomal enzyme that hydrolyzes α-mannose. Hypermannosidemia is a disease caused by the accumulation of intracellular mannose-containing oligosaccharides, accompanied by a deficiency in lysosomal α-mannosidase activity. Patients with hypermannosidemia sometimes experience central nervous system disorders. hLAMAN bound to anti-hTfR antibodies can be used as a therapeutic agent for the central nervous system disorders associated with hypermannosidemia.

[0373] In this invention, the term "human LAMAN" or "hLAMAN" specifically refers to an hLAMAN having an amino acid sequence identical to that of a natural hLAMAN, but is not limited thereto. Substances with substitutions, deletions, additions, or other variations introduced into the amino acid sequence of a natural hLAMAN, as long as they possess hLAMAN activity, are also included in the hLAMAN. When an amino acid in the amino acid sequence of an hLAMAN is substituted with another amino acid, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of an hLAMAN is deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can also be introduced. When an amino acid is added to an hLAMAN, it is preferable to add 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids to the amino acid sequence of the hLAMAN, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequence of the introduced hLAMAN preferably has more than 80% homology with the original hLAMAN amino acid sequence, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0374] It should be noted that when hLAMAN is described as having hLAMAN activity, it means that when fused with an anti-hTfR antibody, it has 3% or more of the activity inherent in the natural hLAMAN. However, this activity relative to the natural hLAMAN is preferably 10% or more, more preferably 20% or more, further preferably 50% or more, and even more preferably 80% or more. The same applies when a variant is introduced into the hLAMAN fused with the anti-hTfR antibody.

[0375] A fusion protein of anti-hTfR antibody and hLAMAN can be manufactured, for example, by transforming host cells such as mammalian cells with an expression vector integrating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 182 and an expression vector integrating a DNA fragment encoding the light chain of an anti-hTfR antibody with the amino acid sequence shown in SEQ ID NO: 23, and culturing the host cells. The resulting fusion protein can be used as a therapeutic agent for alpha-mannosinolate hyperasthenia, particularly as a therapeutic agent for central nervous system disorders caused by alpha-mannosinolate hyperasthenia. The fusion protein thus obtained is a fusion protein of humanized anti-hTfR antibody of type IgG4 and hLAMAN.

[0376] It should be noted that when a mutation is introduced into an anti-hTfR antibody or hLAMAN and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is configured between the anti-hTfR antibody and hLAMAN.

[0377] As described above, examples of fusion proteins of anti-hTfR antibodies and other proteins (A) include hGAA, hI2S, hIDUA, hPPT-1, hASM, hARSA, hSGSH, hGBA, hTTP-1, hNAGLU, hGUSB, hAC, hFUCA1, and hLAMAN. However, in a preferred embodiment of the fusion protein of anti-hTfR antibody and other proteins (A) containing these, 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 a specific affinity for hTfR.

[0378] However, the anti-TfR antibody used here, especially when measured using the method described in Example 7, preferably has a dissociation constant of 1 × 10⁻⁶ with human TfR. -10 For M and below, 1×10 is more preferred. -11 For values ​​below M, 5×10 is further preferred. -12 For values ​​below M, 1×10 is even more preferred. -12 Below M, the dissociation constant of the monkey's TfR is preferably 1×10⁻⁶. -9 For M and below, 5×10 is more preferred. -10 For M and below, 1×10 is further preferred. -10 Below M, for example, 7.5 × 10 -11 Below M.

[0379] For example, the dissociation constants of human TfR and monkey TfR are 1×10⁻⁶. -10 Below M and 1×10 -9 Below M, 1×10 -11 Below M and 5×10 -10 Below M, 5×10 -12 Below M and 1×10 -10 Below M, 5×10 -12 Below M and 7.5×10 -11 Below M, 1×10 -12 Below M and 1×10 -10 Below M, 1×10 -12 Below M and 7.5×10 -11 Below M. Here, there is no particularly explicit lower limit to the dissociation constant of human TfR; it can be set, for example, 5 × 10⁻⁶. -13 M, 1×10-13 M, etc. Furthermore, the dissociation constant of the TfR in monkeys does not have a particularly clear lower limit; it can be set, for example, 1 × 10⁻⁶. -11 M, 1×10 -12 M, etc. The same applies when the antibody is a single-chain antibody.

[0380] Shorter peptide chains can also be bound to anti-hTfR antibodies using the same method as for binding other proteins (A). The peptide chain to be bound to the anti-hTfR antibody is not limited as long as it possesses the desired physiological activity; examples include peptide chains containing the amino acid sequences of various physiologically active regions of different proteins. The chain length is not particularly limited, but preferably consists of 2 to 200 amino acids, for example, 5 to 50 amino acids.

[0381] When binding low molecular weight substances to anti-hTfR antibodies, there are no particular restrictions on the low molecular weight substances that can be candidates. They are low molecular weight substances that, although they should be allowed to reach the brain and exert their effects, cannot cross the blood-brain barrier in their original state and cannot be expected to exert their effects in the brain by intravenous administration. For example, examples of low-molecular-weight substances include: cyclophosphamide, ifosfamide, melphalan, busulfan, thiotepa, nimustine, ramustine, dacarbazine, methylbenzylhydrazine, temozolomide, carmustine, streptozotocin, bendamustine, cisplatin, carboplatin, oxaliplatin, nedaplatin, 5-fluorouracil, sulfadiazine, sulfamethoxazole, methotrexate, trimethoprim, pyrimethamine, fluorouracil, flucytosine, azathioprine, pentostatin, hydroxyurea, fludarabine, cytarabine, gemcitabine, irinotecan, doxorubicin, etoposide, levofloxacin, ciprofloxacin, vincristine, paclitaxel, docetaxel, mitomycin C, doxorubicin, epirubicin, and other anticancer agents. Other low-molecular-weight substances that can bind to anti-hTfR antibodies include siRNA, antisense DNA, and short peptides.

[0382] When anti-hTfR antibodies are bound to low molecular weight substances, the low molecular weight substances may bind to either the light chain or the heavy chain, or they may bind to both the light chain and the heavy chain separately. Furthermore, anti-hTfR antibodies, provided they possess affinity for hTfR, may contain all or part of the amino acid sequence of the variable region containing the light chain and / or all or part of the amino acid sequence of the variable region containing the heavy chain.

[0383] Treatment agents for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease; mental disorders such as schizophrenia and depression; multiple sclerosis; amyotrophic lateral sclerosis (ALS); central nervous system tumors, including brain tumors; lysosomal diseases associated with encephalopathy; glycogen storage diseases; muscular dystrophy; cerebral ischemia; inflammatory brain diseases; prion diseases; and traumatic central nervous system disorders are often candidates for low-molecular-weight substances that should fuse with anti-hTfR antibodies. Additionally, treatment agents for viral and bacterial central nervous system diseases are also often candidates for low-molecular-weight substances that should fuse with anti-hTfR antibodies. Furthermore, medications used for recovery after neurosurgery and spinal surgery are also often candidates for low-molecular-weight substances that should fuse with anti-hTfR antibodies.

[0384] When anti-hTfR antibodies are derived from animals other than humans, there is a significant concern that administering them to humans could trigger an antigen-antibody reaction and cause undesirable side effects. Humanizing these non-human animal antibodies can reduce this antigenicity, thus suppressing the occurrence of antigen-antibody reaction-related side effects when administered to humans. Furthermore, according to reports from monkey studies, humanized antibodies are more stable in the blood than mouse antibodies, suggesting a potentially longer duration of therapeutic effect. Therefore, using human antibodies as anti-hTfR antibodies can also help suppress antigen-antibody reaction-related side effects.

[0385] The following describes the case of anti-hTfR antibodies as either humanized antibodies or human antibodies in more detail. Human antibodies have light chains of λ and κ. The light chain constituting an anti-hTfR antibody can be either a λ or κ chain. Additionally, human antibodies have heavy chains of γ, μ, α, σ, and ε, corresponding to IgG, IgM, IgA, IgD, and IgE, respectively. The heavy chain constituting an anti-hTfR antibody can be any one of γ, μ, α, σ, and ε chains, preferably a γ chain. Furthermore, the γ chain of the heavy chain of a human antibody can be γ1, γ2, γ3, and γ4, corresponding to IgG1, IgG2, IgG3, and IgG4, respectively. When the heavy chain constituting an anti-hTfR antibody is a γ chain, this γ chain can be any one of γ1, γ2, γ3, and γ4 chains, preferably γ1 or γ4. When the anti-hTfR antibody is a humanized antibody or a human antibody that is IgG, the light chain of the human antibody can be either a λ chain or a κ chain, and the heavy chain of the human antibody can be either a γ1 chain, a γ2 chain, a γ3 chain, or a γ4 chain, preferably a γ1 chain or a γ4 chain. For example, as a preferred embodiment of the anti-hTfR antibody, an anti-hTfR antibody with a λ chain as the light chain and a γ1 chain as the heavy chain can be cited.

[0386] When anti-hTfR antibodies are humanized or human antibodies, they can bind to the C-terminus (or N-terminus) of other proteins (A) via a linker sequence or directly via peptide bonds at the N-terminus (or C-terminus) of the heavy or light chain of the anti-hTfR antibody. When binding other proteins (A) to the N-terminal side (or C-terminus) of the heavy chain of the anti-hTfR antibody, they can bind to the C-terminus (or N-terminus) of the γ, μ, α, σ, or ε chain of the anti-hTfR antibody via a linker sequence or directly via peptide bonds. When binding other proteins (A) to the N-terminal side (or C-terminus) of the light chain of the anti-hTfR antibody, they can bind to the C-terminus (or N-terminus) of the λ or κ chain of the anti-hTfR antibody via a linker sequence or directly via peptide bonds. However, when the anti-hTfR antibody is an antibody composed of the Fab region or an antibody composed of all or part of the Fab region and the hinge region (Fab, F(ab')2 and F(ab')), the C-terminus (or N-terminus) of other proteins (A) can be bound by a linker sequence or directly by peptide bonds to the N-terminus (or C-terminus) of the heavy or light chain constituting Fab, F(ab')2 and F(ab').

[0387] Regarding fusion proteins (A) in which the light chain of an anti-hTfR antibody, which is a humanized antibody or a human antibody, is bound to the C-terminal or N-terminal side of another protein (A), an anti-human transferrin receptor antibody comprises all or part of the amino acid sequence of the variable region containing the light chain and all or part of the amino acid sequence of the variable region containing the heavy chain. Here, the light chain of the anti-hTfR antibody can bind to the other protein (A) either directly or through a linker.

[0388] Regarding fusion proteins (A) in which the heavy chain of an anti-hTfR antibody, which is a humanized antibody or a human antibody, is bound to the C-terminal or N-terminal side of another protein (A), an anti-human transferrin receptor antibody comprises all or part of the amino acid sequence of a variable region containing a light chain and all or part of the amino acid sequence of a variable region containing a heavy chain. Here, the heavy chain of the anti-hTfR antibody can bind to the other protein (A) either directly or through a linker.

[0389] When configuring a linker sequence between an anti-hTfR antibody or a human antibody and another protein (A), the linker sequence configured between the anti-hTfR antibody and the other protein (A) is preferably a peptide chain consisting of 1 to 50 amino acids. The number of amino acids constituting the linker sequence can be appropriately adjusted to 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, etc., depending on the other protein (A) to which the anti-hTfR antibody is to bind. The amino acid sequence of this adapter sequence is not limited as long as the anti-hTfR antibody and other proteins (A) linked by the adapter sequence maintain their respective functions (affinity for hTfR and activity or function under physiological conditions). A sequence composed of glycine and serine is preferred. For example, an amino acid sequence containing any one amino acid from 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 (Sequence No. 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (Sequence No. 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (Sequence No. 5), or sequences consisting of 1 to 10 or 2 to 5 of these amino acid sequences linked together. For example, a sequence containing a total of 27 amino acids, consisting of 5 amino acid sequences Gly-Gly-Gly-Gly-Ser (Sequence No. 3) linked after the amino acid sequence Gly-Ser, is preferred as an adapter sequence. In addition, a sequence containing 25 amino acids linked together by the five amino acid sequences Gly-Gly-Gly-Gly-Ser (sequence number 3) can also be preferred as an adapter sequence.

[0390] It should be noted that when referring to the other protein (A) fused with the anti-hTfR antibody or human antibody maintaining the activity or function of that other protein (A) under physiological conditions, or simply referring to having activity, it means maintaining at least 3% of the activity or function inherent in the native type of the other protein (A). However, its activity or function relative to the activity inherent in the native type of the other protein (A) is preferably at least 10%, more preferably at least 20%, further preferably at least 50%, and even more preferably at least 80%. The same applies when there are variations in the introduction of the other protein (A) fused with the anti-hTfR antibody.

[0391] As a more specific example of the fusion protein of the humanized anti-hTfR antibody or human antibody with other protein (A) of the present invention, the following form can be listed: other protein (A) is fused to the C-terminus of the anti-hTfR antibody heavy chain via a linker sequence consisting of a total of 27 amino acids, which is formed by linking a 5-amino acid sequence Gly-Gly-Gly-Gly-Ser (Sequence No. 3) after the amino acid sequence Gly-Ser.

[0392] As an example of the specific form of the humanized anti-hTfR antibody or human antibody fusion protein (A) of the present invention when the anti-hTfR antibody is Fab, the following form can be listed: A variable region containing the anti-hTfR antibody heavy chain is fused to the C-terminus of the other protein (A) via a linker sequence consisting of 25 amino acids linked by a 5-amino acid sequence Gly-Gly-Gly-Gly-Ser (Sequence No. 3). H The shape of region 1. At this point, besides C... H Outside of region 1, there may be a portion of a hinge region, but this hinge region does not contain cysteine ​​residues used to form disulfide bonds between heavy chains.

[0393] The specific affinity of anti-hTfR antibodies for hTfR mainly depends on the amino acid sequence of the CDRs of the heavy and light chains of the anti-hTfR antibody. There are no particular restrictions on the amino acid sequence of these CDRs as long as they enable the anti-hTfR antibody to have specific affinity for hTfR and monkey hTfR.

[0394] However, in this invention, the humanized anti-hTfR antibody or human antibody, which has relatively high affinity for hTfR and affinity for both human and monkey TfR, especially when measured using the method described in Example 7, preferably has a dissociation constant of 1 × 10⁻⁶ with human TfR. -10 M or less, more preferably 2.5 × 10 -11 For values ​​below M, 5×10 is further preferred. -12 For values ​​below M, 1×10 is even more preferred. -12 Below M, the dissociation constant of the monkey's TfR is preferably 1×10⁻⁶. -9 For M and below, 5×10 is more preferred. -10 For M and below, 1×10 is further preferred. -10 Below M, for example, 7.5 × 10 -11 Below M.

[0395] For example, the dissociation constants of human TfR and monkey TfR are 1×10⁻⁶. -10 Below M and 1×10 -9 Below M, 1×10 -11 Below M and 5×10-10 Below M, 5×10 -12 Below M and 1×10 -10 Below M, 5×10 -12 Below M and 7.5×10 -11 Below M, 1×10 -12 Below M and 1×10 -10 Below M, 1×10 -12 Below M and 7.5×10 -11 Below M. Here, there is no particularly explicit lower limit to the dissociation constant of human TfR; it can be set, for example, 5 × 10⁻⁶. -13 M, 1×10 -13 M, etc. Furthermore, the dissociation constant of the TfR in monkeys does not have a particularly clear lower limit; it can be set, for example, 1 × 10⁻⁶. -11 M, 1×10 -12 M, etc. The same applies when the antibody is a single-chain antibody.

[0396] As a preferred embodiment of an antibody with affinity for hTfR, an example can be described as an antibody in the variable region of the heavy chain,

[0397] (a) CDR1 contains the amino acid sequence of sequence number 62 or 63.

[0398] (b) CDR2 contains the amino acid sequence of sequence number 13 or 14, and

[0399] (c) CDR3 contains an amino acid sequence of sequence number 15 or sequence number 16.

[0400] A more specific embodiment of an antibody with affinity for hTfR can be exemplified by an antibody in the variable region of the heavy chain,

[0401] (a) CDR1 contains the amino acid sequence with sequence number 62.

[0402] (b) CDR2 contains the amino acid sequence of sequence number 13, and

[0403] (c) CDR3 contains the amino acid sequence described in sequence number 15.

[0404] In the preferred embodiment of the antibody with affinity for hTfR described above, and in a more specific embodiment of the antibody with affinity for hTfR, the preferred amino acid sequence for the frame region 3 of the antibody heavy chain may include the amino acid sequence containing sequence number 64.

[0405] Preferred combinations of light and heavy chains for antibodies with affinity for hTfR include combinations having the following amino acid sequences in the variable region:

[0406] (a) CDR1 contains the amino acid sequence of sequence number 6 or 7.

[0407] (b) CDR2 contains the amino acid sequence of sequence number 8 or 9, or the amino acid sequence Lys-Val-Ser, and

[0408] (c) CDR3 contains a light chain with the amino acid sequence number 10, and...

[0409] (d) CDR1 contains the amino acid sequence of sequence number 62 or 63.

[0410] (e) CDR2 contains the amino acid sequence of sequence number 13 or 14, and

[0411] (f)CDR3 is a combination of heavy chains containing the amino acid sequence of sequence number 15 or sequence number 16.

[0412] A specific form of the combination of light and heavy chains of an antibody with affinity for hTfR can be exemplified by a form having the following amino acid sequence in the variable region. That is:

[0413] A combination of light chains containing amino acid sequence number 6 in CDR1, amino acid sequence number 8 in CDR2, and amino acid sequence number 10 in CDR3, and heavy chains containing amino acid sequence number 62 in CDR1, amino acid sequence number 13 in CDR2, and amino acid sequence number 15 in CDR3.

[0414] In the above-mentioned preferred combination of light and heavy chains of antibodies with affinity for hTfR, and in the specific form of the combination of light and heavy chains of antibodies with affinity for hTfR, the preferred amino acid sequence for the frame region 3 of the antibody heavy chain can be the amino acid sequence having sequence number 64.

[0415] As a preferred embodiment of a humanized antibody with affinity for hTfR, an example can be a humanized antibody having the amino acid sequence shown below. That is:

[0416] An anti-hTfR antibody, wherein the variable region of the light chain contains an amino acid sequence of sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21 or sequence number 22 and the variable region of the heavy chain contains an amino acid sequence of sequence number 65.

[0417] In the amino acid sequences of the variable regions of the light chains shown in Serial Numbers 17, 18, 19, 20, 21, and 22, CDR1 contains the amino acid sequence of Serial Number 6 or 7, CDR2 contains the amino acid sequence of Serial Number 8 or 9, and CDR3 contains the amino acid sequence of Serial Number 10. However, regarding the amino acid sequences of the variable regions of the light chains described in Serial Numbers 17–22, when referring to CDRs, the sequence of CDRs is not limited to these; regions containing the amino acid sequences of these CDRs, or any consecutive sequence of three or more amino acids containing the amino acid sequences of these CDRs, can also be considered as CDRs.

[0418] The amino acid sequences of the variable region of the heavy chain shown in Serial No. 65 are (a) CDR1 containing the amino acid sequence of Serial No. 62 or 63, (b) CDR2 containing the amino acid sequence of Serial No. 13 or 14, and (c) CDR3 containing the amino acid sequence of Serial No. 15 or 16, and the amino acid sequence containing Serial No. 64 is the amino acid sequence of frame region 3. However, regarding the amino acid sequences of the variable region of the heavy chain shown in Serial No. 65, when referring to CDRs, the CDR sequence is not limited to these; regions containing the amino acid sequences of these CDRs, and any consecutive amino acid sequence of three or more amino acids containing the amino acid sequences of these CDRs, can also be considered CDRs. The same applies to frame regions.

[0419] More specific embodiments of humanized antibodies with affinity for hTfR can be listed as follows:

[0420] Humanized antibodies containing amino acid sequence number 18 in the variable region of the light chain and amino acid sequence number 65 in the variable region of the heavy chain.

[0421] Humanized antibodies containing amino acid sequence number 20 in the variable region of the light chain and amino acid sequence number 65 in the variable region of the heavy chain.

[0422] Humanized antibodies containing the amino acid sequence at sequence number 21 in the variable region of the light chain and the amino acid sequence at sequence number 65 in the variable region of the heavy chain, and

[0423] The humanized antibody has a variable region of the light chain containing the amino acid sequence of sequence number 22 and a variable region of the heavy chain containing the amino acid sequence of sequence number 65.

[0424] More specific embodiments of humanized antibodies with affinity for hTfR can be listed as follows:

[0425] Humanized antibodies containing amino acid sequence number 23 in the light chain and amino acid sequence number 66 in the heavy chain.

[0426] Humanized antibodies containing amino acid sequence number 25 in the light chain and amino acid sequence number 66 in the heavy chain.

[0427] Humanized antibodies containing amino acid sequence number 27 in the light chain and amino acid sequence number 66 in the heavy chain.

[0428] Humanized antibodies containing amino acid sequence number 29 in the light chain and amino acid sequence number 66 in the heavy chain.

[0429] Humanized antibodies containing amino acid sequence number 23 in the light chain and amino acid sequence number 68 in the heavy chain.

[0430] Humanized antibodies containing amino acid sequence number 25 in the light chain and amino acid sequence number 68 in the heavy chain.

[0431] Humanized antibodies containing amino acid sequence number 27 in the light chain and amino acid sequence number 68 in the heavy chain, and

[0432] Humanized antibodies containing amino acid sequence number 29 in the light chain and amino acid sequence number 68 in the heavy chain.

[0433] It should be noted that, in the specific embodiments described above, the humanized anti-hTfR antibody heavy chain containing the amino acid sequence of sequence number 66 is an IgG1 type antibody, and the antibody containing the amino acid sequence of sequence number 68 is an IgG4 type antibody. The variable region contains the amino acid sequence of sequence number 65.

[0434] Furthermore, more specific embodiments of Fab-based humanized antibodies with affinity for hTfR can be listed as follows:

[0435] Humanized antibodies containing amino acid sequence number 23 in the light chain and amino acid sequence number 61 in the Fab heavy chain.

[0436] Humanized antibodies containing amino acid sequence number 25 in the light chain and amino acid sequence number 61 in the Fab heavy chain.

[0437] Humanized antibodies containing amino acid sequence number 27 in the light chain and amino acid sequence number 61 in the Fab heavy chain.

[0438] The humanized antibody has an amino acid sequence containing sequence number 29 in the light chain and an amino acid sequence containing sequence number 61 in the Fab heavy chain.

[0439] When the anti-hTfR antibody is Fab, specific examples of introducing other Fc regions into the Fab heavy chain can be listed as follows:

[0440] An antibody containing the amino acid sequence number 23 in the light chain and the Fab region of the Fc region, and the amino acid sequence number 71 in the heavy chain.

[0441] An antibody containing the amino acid sequence at sequence number 25 in the light chain and the Fab region inserted into the Fc region, and the amino acid sequence at sequence number 71 in the heavy chain.

[0442] An antibody containing the amino acid sequence number 27 in the light chain and the Fab region with an Fc region, and the amino acid sequence number 71 in the heavy chain.

[0443] An antibody containing the amino acid sequence number 29 in the light chain and the Fab heavy chain containing the amino acid sequence number 71 in the Fc region.

[0444] When other Fc regions are introduced into the aforementioned Fab heavy chain, for example, the Fab heavy chain with the Fc region can be introduced directly to the C-terminus of another protein (A) or through an adapter sequence. It should be noted that the heavy chain with the amino acid sequence shown in sequence number 71 is a heavy chain with the human IgG Fc region shown in sequence number 70, which is formed by linking a 5-amino acid sequence (Gly-Gly-Gly-Gly-Ser, sequence number 3) to the N-terminus of the Fab heavy chain of humanized anti-hTfR antibody 3N through an adapter sequence of 25 amino acids.

[0445] Preferred embodiments of antibodies with affinity for hTfR are illustrated as described above. Regarding the light and heavy chains of these anti-hTfR antibodies, variations such as substitutions, deletions, and additions can be appropriately introduced into the amino acid sequences of their variable regions for purposes such as adjusting the affinity of the anti-hTfR antibody for hTfR to a desired level.

[0446] When replacing amino acids in the variable region of the light chain with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When deleting amino acids in the variable region of the light chain, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. Additionally, variations combining these amino acid substitutions and deletions can be introduced.

[0447] When adding amino acids to the variable region of the light chain, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of the variable region of the light chain, either on the N-terminal side or the C-terminal side. Variations combining these amino acid additions, substitutions, and deletions can also be introduced. The amino acid sequence of the variable region of the light chain with the introduced variation preferably has more than 80% homology with the amino acid sequence of the original variable region of the light chain, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0448] Specifically, when replacing amino acids in the amino acid sequences of each CDR or framework region of the light chain with other amino acids, the number of substituted amino acids is preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2, and even more preferably 1. When deleting amino acids in the amino acid sequences of each CDR or framework region, the number of deleted amino acids is preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2, and even more preferably 1. Furthermore, variations combining these amino acid substitutions and deletions can also be introduced.

[0449] When adding amino acids to the amino acid sequences of each CDR or framework region of the light chain, preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2, and even more preferably 1 amino acid is added to the amino acid sequence, either at the N-terminus or C-terminus. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequences of each CDR or framework region with introduced variations preferably have more than 80% homology with the original amino acid sequences of each CDR, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0450] When the amino acid sequence shown as sequence number 65, which is the variable region of the heavy chain, is replaced with other amino acids, the number of amino acids replaced is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. When the amino acid sequence in the variable region of the heavy chain is deleted, the number of amino acids deleted is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. In addition, variations combining these amino acid substitutions and deletions can also be introduced.

[0451] When adding amino acids to the amino acid sequence shown as sequence number 65, which is the variable region of the heavy chain, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of the variable region of the heavy chain, either on the N-terminal side or the C-terminal side. Variations combining these amino acid additions, substitutions, and deletions can also be introduced. The amino acid sequence of the variable region of the heavy chain with the introduced variation preferably has more than 80% homology with the amino acid sequence of the original variable region of the heavy chain, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0452] In particular, when the amino acids in the amino acid sequences of each CDR or each frame region in the amino acid sequence shown in sequence number 65 are replaced with other amino acids, the number of replaced amino acids is preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2, and even more preferably 1. When the amino acids in the amino acid sequences of each CDR are deleted, the number of deleted amino acids is preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2, and even more preferably 1. In addition, variations combining these amino acid substitutions and deletions can also be introduced.

[0453] When adding amino acids to the amino acid sequences of each CDR or each frame region in the amino acid sequence shown in sequence number 65, preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2, and even more preferably 1 amino acid is added to the amino acid sequence, either on the N-terminal side or the C-terminal side. Variations involving the addition, substitution, and deletion of these amino acids can also be introduced. The amino acid sequences of each CDR with introduced variations preferably have more than 80% homology with the original amino acid sequences of each CDR, more preferably more than 90% homology, and even more preferably more than 95% homology.

[0454] It should be noted that when substitutions, deletions, additions, or other variations are introduced into the amino acid sequence of sequence number 65, which is the variable region of the heavy chain used as an anti-hTfR antibody, as described above, it is preferable that the amino acid at position 5 from the N-terminus of CDR1 (as shown in sequence numbers 62 or 63), i.e., methionine, and the amino acid at position 17 from the N-terminus of framework region 3 (as shown in sequence number 64), i.e., leucine, are conserved at the same positions as before. Furthermore, it is preferable that the amino acid sequences of CDR1 and framework region 3 of the heavy chain are also conserved at the same positions as before.

[0455] Furthermore, the mutations in the variable region of the light chain of the aforementioned anti-hTfR antibody can be combined with the mutations in the variable region of the heavy chain of the aforementioned anti-hTfR antibody to introduce mutations into both the variable regions of the light chain and the heavy chain of the anti-hTfR antibody.

[0456] The substitutions of amino acids in the variable regions of the heavy and light chains of the aforementioned anti-hTfR antibodies for other amino acids can be exemplified by, for example, the substitutions of 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), which are classified into the same group.

[0457] It should be noted that when a mutation is introduced into an anti-hTfR antibody and an amino acid is added to the C-terminus or N-terminus, the added amino acid constitutes part of the linker if it is located between the anti-hTfR antibody and the other protein (A) when the anti-hTfR antibody fuses with the other protein (A).

[0458] In the preferred embodiments of antibodies, including humanized antibodies, that have affinity for hTfR as illustrated above, there are no particular limitations on the amino acid sequences of the CDRs of the heavy and light chains of the anti-hTfR antibody, as long as the antibody has specific affinity for hTfR and monkey TfR.

[0459] However, in this invention, the humanized antibody, which has relatively high affinity for hTfR and affinity for both human and monkey TfR, preferably has a dissociation constant of 1 × 10⁻⁶ when measured using the method described in Example 7. -10 M or less, more preferably 2.5 × 10 -11 For values ​​below M, 5×10 is further preferred. -12 For values ​​below M, 1×10 is even more preferred. -12 Below M, the dissociation constant of the monkey's TfR is preferably 1×10⁻⁶. -9 For M and below, 5×10 is more preferred. -10 For M and below, 1×10 is further preferred. -10 Below M, for example, 7.5 × 10 -11 Below M.

[0460] For example, the dissociation constants of human TfR and monkey TfR are 1×10⁻⁶. -10 Below M and 1×10 -9 Below M, 1×10 -11 Below M and 5×10 -10 Below M, 5×10 -12 Below M and 1×10 -10 Below M, 5×10 -12 Below M and 7.5×10 -11 Below M, 1×10 -12 Below M and 1×10-10 Below M, 1×10 -12 Below M and 7.5×10 -11 Below M. Here, there is no particularly explicit lower limit to the dissociation constant of human TfR; it can be set, for example, 5 × 10⁻⁶. -13 M, 1×10 -13 M, etc. Furthermore, there is no particularly clear lower limit to the dissociation constant of the TfR in monkeys; it can be set, for example, 5 × 10⁻⁶. -11 M, 1×10 -11 M, 1×10 -12 M, etc. The same applies when the antibody is a single-chain antibody.

[0461] As a specific embodiment of the fusion protein of the humanized antibody with affinity for hTfR and other proteins (A) shown in the above specific embodiments, examples include: other proteins (A) being fusion proteins of human acid α-glucosidase (hGAA), human iduronate-2-sulfatase (hI2S), human α-L-iduronase (hIDUA), human palmitoyl protein thioesterase-1 (hPPT-1), human acid sphingomyelinase (hASM), human aryl sulfatase A (hARSA), human heparin N-sulfatase (hSGSH), human glucocerebrosidase (hGBA), human tripeptidyl peptidase-1 (hTPP-1), human α-N-acetylglucosidase (hNAGLU), human β-glucuronidase (hGUSB), human acid ceramide enzyme (hAC), human α-L-fucosidase (hFUCA1), and α-mannosidase (hLAMAN).

[0462] Specific examples of fusion proteins of other proteins (A) that are human acid α-glucosidase (hGAA) can be listed as follows:

[0463] (1) A fusion protein consisting of a structure formed by hGAA binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0464] (2) A fusion protein consisting of a structure formed by hGAA binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0465] (3) A fusion protein consisting of a structure formed by hGAA binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0466] (4) A fusion protein consisting of a structure formed by hGAA binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0467] Here, the linker sequence is preferably composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence formed by linking 1 to 10 of these amino acid sequences.

[0468] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody. Additionally, hGAA is a protein or a variant thereof with amino acid sequence number 55 or 56.

[0469] A more specific example of a fusion protein of other protein (A) as a human acid α-glucosidase (hGAA) can be illustrated as follows:

[0470] (1) A fusion protein consisting of a structure formed by hGAA binding 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 is shown in Serial No. 57, and the amino acid sequence of the latter is shown in Serial No. 23.

[0471] (2) A fusion protein consisting of a structure formed by hGAA binding 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 is shown in Serial No. 58, and the amino acid sequence of the latter is shown in Serial No. 23.

[0472] (1): The antibody is of type IgG1, (2): The antibody is of type IgG4. In addition, hGAA is a protein with an amino acid sequence number 55.

[0473] Here, in (1) above, the amino acid sequence of the heavy chain of hTfR contained in sequence number 57 is as shown in sequence number 66. That is, in the fusion protein of (1) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain contains the amino acid sequence of sequence number 66. In addition, in (2) above, the amino acid sequence of the heavy chain of hTfR contained in sequence number 58 is as shown in sequence number 68. That is, in the fusion protein of (2) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain contains the amino acid sequence of sequence number 68.

[0474] Specific examples of fusion proteins in which other protein (A) is human acid α-glucosidase (hGAA) and the humanized antibody is a Fab antibody can be cited as follows:

[0475] (1) A fusion protein consisting of a structure formed by hGAA binding to the C-terminus or N-terminus of the Fab heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the Fab heavy chain is shown in Serial No. 61, and the amino acid sequence of the light chain is shown in Serial No. 23.

[0476] (2) A fusion protein consisting of a structure formed by hGAA binding to the C-terminus or N-terminus of the Fab heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the Fab heavy chain is shown in Serial No. 61, and the amino acid sequence of the light chain is shown in Serial No. 25.

[0477] (3) A fusion protein consisting of a structure formed by hGAA binding to the C-terminus or N-terminus of the Fab heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the Fab heavy chain is shown in Serial No. 61, and the amino acid sequence of the light chain is shown in Serial No. 27.

[0478] (4) A fusion protein consisting of a structure formed by hGAA binding to the C-terminal or N-terminal side of the Fab heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the Fab heavy chain is shown in Serial No. 61, and the amino acid sequence of the light chain is shown in Serial No. 29.

[0479] Here, the linker sequence is preferably composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence formed by linking 1 to 10 of these amino acid sequences.

[0480] A more specific example of a fusion protein in which other protein (A) is human acidic α-glucosidase (hGAA) and the humanized antibody is a Fab antibody can be described as follows: a fusion protein consisting of a linker formed by hGAA linked by three amino acid sequences shown in Serial No. 3 and attached to the C-terminal side of the heavy chain (Fab) of hTfR, and a fusion protein consisting of the light chain of hTfR, the former having the amino acid sequence shown in Serial No. 89 and the latter having the amino acid sequence shown in Serial No. 23.

[0481] Other protein (A) is a fusion protein of human acid α-glucosidase (hGAA) that has affinity for both human and monkey TfR. When measured using the method described in Example 7, the dissociation constant with monkey TfR is preferably 1 × 10⁻⁶. -10 For M and below, 5×10 is more preferred.-11 Below M, the dissociation constant with human TfR is preferably 1×10⁻⁶. -10 For M and below, 5×10 is more preferred. -11 For M and below, 1×10 is further preferred. -11 For values ​​below M, 1×10 is even more preferred. -12 Below M.

[0482] For example, the dissociation constants of TfR in monkeys and TfR in humans are 1×10⁻⁶. -10 Below M and 1×10 -10 Below M, 1×10 -10 Below M and 1×10 -11 Below M, 1×10 -10 Below M and 1×10 -12 Below M, 5×10 -11 Below M and 1×10 -11 Below M, 5×10 -11 Below M and 1×10 -11 Below M, 5×10 -11 Below M and 1×10 -12 Below M. Here, there is no particularly explicit lower limit to the dissociation constant of the monkey's TfR; it can be set, for example, 1 × 10⁻⁶. -11 M, 1×10 -12 M, 1×10 -13 M et al. The dissociation constant of TfR in humans does not have a particularly clear lower limit; it can be set, for example, 1 × 10⁻⁶. -12 M, 5×10 -13 M, 1×10 -13 M, etc. The same applies when the antibody is a single-chain antibody.

[0483] Specific examples of other proteins (A) that are fusion proteins of human iduronate-2-sulfatase (hI2S) can be cited as follows:

[0484] (1) A fusion protein consisting of a structure formed by hI2S binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0485] (2) A fusion protein consisting of a structure formed by hI2S binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0486] (3) A fusion protein consisting of a structure formed by hI2S binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0487] (4) A fusion protein consisting of a structure formed by hI2S binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0488] Here, the linker sequence is preferably composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence formed by linking 1 to 10 of these amino acid sequences.

[0489] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody.

[0490] A more specific example of a fusion protein of other proteins (A) being human iduronate-2-sulfatase (hI2S) can be illustrated by a fusion protein consisting of a structure formed by hI2S bound to the C-terminus of the heavy chain of hTfR via the amino acid sequence Gly-Ser, and a light chain of hTfR, the amino acid sequence of which is shown in Serial No. 53 and the amino acid sequence of which is shown in Serial No. 23. In this case, the antibody is of type IgG1. The amino acid sequence of the heavy chain of hTfR contained in Serial No. 53 is shown in Serial No. 66. That is, in this fusion protein, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of Serial No. 23 and whose heavy chain contains the amino acid sequence of Serial No. 66. Alternatively, the antibody can be of type IgG4, in which case the heavy chain is replaced by a heavy chain containing the amino acid sequence of Serial No. 68.

[0491] Specific examples of fusion proteins in which other protein (A) is human iduronate-2-sulfatase (hI2S) and the humanized antibody is a Fab antibody can be cited:

[0492] (1) A fusion protein consisting of a structure formed by hI2S binding to the C-terminus or N-terminus of the Fab heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the Fab heavy chain is shown in Serial No. 61, and the amino acid sequence of the light chain is shown in Serial No. 23.

[0493] (2) A fusion protein consisting of a structure formed by hI2S binding to the C-terminus or N-terminus of the Fab heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the Fab heavy chain is shown in Serial No. 61, and the amino acid sequence of the light chain is shown in Serial No. 25.

[0494] (3) A fusion protein consisting of a structure formed by hI2S binding to the C-terminus or N-terminus of the Fab heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the Fab heavy chain is shown in Serial No. 61, and the amino acid sequence of the light chain is shown in Serial No. 27.

[0495] (4) A fusion protein consisting of a structure formed by hI2S binding to the C-terminal or N-terminal side of the Fab heavy chain of hTfR through a linker sequence and the light chain of hTfR. The amino acid sequence of the Fab heavy chain is shown in Serial No. 61, and the amino acid sequence of the light chain is shown in Serial No. 29.

[0496] Here, the linker sequence is preferably composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence formed by linking 1 to 10 of these amino acid sequences.

[0497] Specific examples of fusion proteins where other proteins (A) are (hIDUA) can be listed as follows:

[0498] (1) A fusion protein consisting of a structure formed by hIDUA bound to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0499] (2) A fusion protein consisting of a structure formed by hIDUA bound to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0500] (3) A fusion protein consisting of a structure formed by hIDUA bound to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0501] (4) A fusion protein consisting of a structure formed by hIDUA binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0502] Here, the linker sequence is preferably a linker sequence composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence composed of 1 to 10 or 1 to 20 of these amino acid sequences linked together.

[0503] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody. Additionally, hIDUA is a protein or a variant thereof with amino acid sequence number 75 or 76.

[0504] As a more specific example of a fusion protein where other proteins (A) are (hIDUA), the following can be illustrated:

[0505] (1) A fusion protein consisting of a structure formed by hIDUA 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 is shown in Serial No. 90, and the amino acid sequence of the latter is shown in Serial No. 23.

[0506] (2) A fusion protein consisting of hIDUA and a light chain of hTfR, formed by a linker consisting of 42 amino acids (8 amino acids as shown in Serial No. 3) attached to the C-terminus of the heavy chain of hTfR. The amino acid sequence of the former is shown in Serial No. 91, and the amino acid sequence of the latter is shown in Serial No. 23.

[0507] (3) A fusion protein consisting of a linker formed by hIDUA linked by 20 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 92, and the amino acid sequence of the latter is shown in sequence number 23.

[0508] (4) A fusion protein consisting of a linker formed by hIDUA linked by three amino acid sequences shown in Serial No. 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 93, and the amino acid sequence of the latter is shown in Serial No. 23.

[0509] (5) A fusion protein consisting of a linker formed by hIDUA linked by five amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 94, and the amino acid sequence of the latter is shown in sequence number 23.

[0510] (6) A fusion protein consisting of a linker formed by linking hIDUA through 10 amino acid sequences shown in Serial No. 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 95, and the amino acid sequence of the latter is shown in Serial No. 23.

[0511] (7) A fusion protein consisting of a linker formed by linking hIDUA through 20 amino acid sequences shown in Serial No. 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 96, and the amino acid sequence of the latter is shown in Serial No. 23.

[0512] (8) A fusion protein consisting of a structure formed by hIDUA linked to the C-terminus of the heavy chain (Fab) of hTfR, which has a linker with six amino acid sequences as shown in Serial No. 3 at the C-terminus, and a light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 97, and the amino acid sequence of the latter is shown in Serial No. 23.

[0513] (9) hIDUA binds to the fusion protein shown in sequence number 99 on the C-terminus side of the single-chain humanized anti-hTfR antibody No. 3N(2) shown in sequence number 98 via a linker consisting of a 7-amino acid sequence Gly-Gly linked to the amino acid sequence shown in sequence number 3.

[0514] (1)~(3): Antibody is IgG1 type, (4)~(9): Antibody is Fab type.

[0515] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (3) above is shown in sequence number 68. That is, in the fusion proteins (1) to (3) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain contains the amino acid sequence of sequence number 68. In addition, the amino acid sequence of the heavy chain of hTfR in (4) to (9) above is shown in sequence number 61. That is, in the fusion proteins (4) to (9) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain (Fab) contains the amino acid sequence of sequence number 61.

[0516] Specific examples of fusion proteins where other proteins (A) are (hPPT-1) can be listed as follows:

[0517] (1) A fusion protein consisting of a structure formed by hPPT-1 binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0518] (2) A fusion protein consisting of a structure formed by hPPT-1 binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0519] (3) A fusion protein consisting of a structure formed by hPPT-1 binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0520] (4) A fusion protein consisting of a structure formed by hPPT-1 binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0521] Here, the linker sequence is preferably a linker sequence composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence composed of 1 to 10 or 1 to 20 of these amino acid sequences linked together.

[0522] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody. Additionally, hPPT-1 is a protein or a variant thereof with the amino acid sequence having sequence number 77.

[0523] A more specific example of a fusion protein where other proteins (A) are (hPPT-1) is as follows:

[0524] (1) A fusion protein consisting of a structure formed by hPPT-1 binding 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 is shown in sequence number 100, and the amino acid sequence of the latter is shown in sequence number 23.

[0525] (2) A fusion protein consisting of a linker formed by hPPT-1 linked by five amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 101, and the amino acid sequence of the latter is shown in sequence number 23.

[0526] (3) A fusion protein consisting of a linker formed by hPPT-1 linked by 10 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 102, and the amino acid sequence of the latter is shown in sequence number 23.

[0527] (4) A fusion protein consisting of a linker formed by hPPT-1 linked by 20 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 103, and the amino acid sequence of the latter is shown in sequence number 23.

[0528] (5) A fusion protein consisting of a linker formed by hPPT-1 linked by three amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 104, and the amino acid sequence of the latter is shown in sequence number 23.

[0529] (6) A fusion protein consisting of a structure formed by hPPT-1 linked to the C-terminus of the heavy chain (Fab) of hTfR, which has a linker with six amino acid sequences as shown in Serial No. 3 at the C-terminus, and a light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 105, and the amino acid sequence of the latter is shown in Serial No. 23.

[0530] (1)~(4): The antibody is of type IgG1, (5)~(6): The antibody is of type Fab.

[0531] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is shown in sequence number 68. That is, in the fusion proteins of (1) to (4) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain contains the amino acid sequence of sequence number 68. In addition, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is shown in sequence number 61. That is, in the fusion proteins of (5) and (6) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain (Fab) contains the amino acid sequence of sequence number 61.

[0532] Specific examples of fusion proteins where other proteins (A) are (hASM) can be listed as follows:

[0533] (1) A fusion protein consisting of a structure formed by hASM binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0534] (2) A fusion protein consisting of a structure formed by hASM binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0535] (3) A fusion protein consisting of a structure formed by hASM binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0536] (4) A fusion protein consisting of a structure formed by hASM binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0537] Here, the linker sequence is preferably a linker sequence composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence composed of 1 to 10 or 1 to 20 of these amino acid sequences linked together.

[0538] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody. Additionally, hASM is a protein or a variant thereof with the amino acid sequence having sequence number 78.

[0539] A more specific example of a fusion protein where other proteins (A) are (hASM) is:

[0540] (1) A fusion protein consisting of a structure formed by hASM binding 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 is shown in sequence number 106, and the amino acid sequence of the latter is shown in sequence number 23.

[0541] (2) A fusion protein consisting of a linker formed by hASM linked by five amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 107, and the amino acid sequence of the latter is shown in sequence number 23.

[0542] (3) A fusion protein consisting of a linker formed by hASM linked by 10 amino acid sequences as shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 108, and the amino acid sequence of the latter is shown in sequence number 23.

[0543] (4) A fusion protein consisting of a linker formed by hASM linked by 20 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 109, and the amino acid sequence of the latter is shown in sequence number 23.

[0544] (5) A fusion protein consisting of a linker formed by hASM linked by three amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 110, and the amino acid sequence of the latter is shown in sequence number 23.

[0545] (6) A fusion protein consisting of a linker formed by hASM linked by five amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 111, and the amino acid sequence of the latter is shown in sequence number 23.

[0546] (7) A fusion protein consisting of a linker formed by linking hASM through 10 amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 112, and the amino acid sequence of the latter is shown in sequence number 23.

[0547] (8) A fusion protein consisting of a linker formed by linking hASM through 20 amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 113, and the amino acid sequence of the latter is shown in sequence number 23.

[0548] (9) A fusion protein consisting of a structure formed by hASM binding to the C-terminus of the heavy chain (Fab) of hTfR, which is linked to two linkers with six amino acid sequences shown in Serial No. 3 at the C-terminus, and a light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 114, and the amino acid sequence of the latter is shown in Serial No. 23.

[0549] (1)~(4): Antibody is IgG1 type, (5)~(9): Antibody is Fab type.

[0550] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is shown in sequence number 68. That is, in the fusion proteins of (1) to (4) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain contains the amino acid sequence of sequence number 68. In addition, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is shown in sequence number 61. That is, in the fusion proteins of (5) and (6) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain (Fab) contains the amino acid sequence of sequence number 61.

[0551] Specific examples of fusion proteins for which other proteins (A) are (hARSA) can be listed as follows:

[0552] (1) A fusion protein consisting of a structure formed by hARSA binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0553] (2) A fusion protein consisting of a structure formed by hARSA binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0554] (3) A fusion protein consisting of a structure formed by hARSA binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0555] (4) A fusion protein consisting of a structure formed by hARSA binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0556] Here, the linker sequence is preferably a linker sequence composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence composed of 1 to 10 or 1 to 20 of these amino acid sequences linked together.

[0557] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody. Additionally, hARSA is a protein or a variant thereof with the amino acid sequence number 79.

[0558] A more specific example of a fusion protein of other proteins (A) being (hARSA) is as follows:

[0559] (1) A fusion protein consisting of a structure formed by hARSA binding 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 is shown in sequence number 115, and the amino acid sequence of the latter is shown in sequence number 23.

[0560] (2) A fusion protein consisting of a linker formed by hARSA linked by five amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 116, and the amino acid sequence of the latter is shown in sequence number 23.

[0561] (3) A fusion protein consisting of a linker formed by linking hARSA through 10 amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 117, and the amino acid sequence of the latter is shown in sequence number 23.

[0562] (4) A fusion protein consisting of a linker formed by linking hARSA through 20 amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 118, and the amino acid sequence of the latter is shown in sequence number 23.

[0563] (5) A fusion protein consisting of a linker formed by hARSA linked by three amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 119, and the amino acid sequence of the latter is shown in sequence number 23.

[0564] (6) A fusion protein consisting of a linker formed by linking hARSA through five amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 120, and the amino acid sequence of the latter is shown in sequence number 23.

[0565] (7) A fusion protein consisting of a linker formed by linking hARSA through 10 amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 121, and the amino acid sequence of the latter is shown in sequence number 23.

[0566] (8) A fusion protein consisting of a linker formed by linking hARSA through 20 amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 122, and the amino acid sequence of the latter is shown in sequence number 23.

[0567] (9) A fusion protein consisting of a structure formed by hARSA linked to the C-terminus of the heavy chain (Fab) of hTfR, which has a linker with six amino acid sequences as shown in Serial No. 3 at the C-terminus, and a light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 123, and the amino acid sequence of the latter is shown in Serial No. 23.

[0568] (1)~(4): Antibody is IgG1 type, (5)~(9): Antibody is Fab type.

[0569] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is shown in sequence number 68. That is, in the fusion proteins (1) to (4) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain contains the amino acid sequence of sequence number 68. In addition, the amino acid sequence of the heavy chain of hTfR in (5) to (9) above is shown in sequence number 61. That is, in the fusion proteins (5) to (9) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain (Fab) contains the amino acid sequence of sequence number 61.

[0570] Specific examples of fusion proteins where other proteins (A) are (hSGSH) can be listed as follows:

[0571] (1) A fusion protein consisting of a structure formed by hSGSH binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0572] (2) A fusion protein consisting of a structure formed by hSGSH binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0573] (3) A fusion protein consisting of a structure formed by hSGSH binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0574] (4) A fusion protein consisting of a structure formed by hSGSH binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0575] Here, the linker sequence is preferably a linker sequence composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence composed of 1 to 10 or 1 to 20 of these amino acid sequences linked together.

[0576] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody. Additionally, hSGSH is a protein or a variant thereof with the amino acid sequence having sequence number 80.

[0577] A more specific example of a fusion protein where other proteins (A) are (hSGSH) is:

[0578] (1) A fusion protein consisting of a structure formed by hSGSH binding 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 is shown in sequence number 124, and the amino acid sequence of the latter is shown in sequence number 23.

[0579] (2) A fusion protein consisting of a linker formed by hSGSH linked by five amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 125, and the amino acid sequence of the latter is shown in sequence number 23.

[0580] (3) A fusion protein consisting of a linker formed by hSGSH linked by 10 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 126, and the amino acid sequence of the latter is shown in sequence number 23.

[0581] (4) A fusion protein consisting of a linker formed by hSGSH linked by 20 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 127, and the amino acid sequence of the latter is shown in sequence number 23.

[0582] (5) A fusion protein consisting of a linker formed by hSGSH linked by three amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 128, and the amino acid sequence of the latter is shown in sequence number 23.

[0583] (6) A fusion protein consisting of a linker consisting of two heavy chains (Fab) of hTfR linked together by six amino acid sequences (as shown in Serial No. 3) linked together at the C-terminus, with hSGSH attached to the C-terminus, and a light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 129, and the amino acid sequence of the latter is shown in Serial No. 23.

[0584] (1)~(4): Antibody is IgG1 type, (5) and (6): Antibody is Fab type.

[0585] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is shown in sequence number 68. That is, in the fusion proteins of (1) to (4) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain contains the amino acid sequence of sequence number 68. In addition, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is shown in sequence number 61. That is, in the fusion proteins of (5) and (6) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain (Fab) contains the amino acid sequence of sequence number 61.

[0586] Specific examples of fusion proteins where other proteins (A) are (hGBA) can be listed as follows:

[0587] (1) A fusion protein consisting of a structure formed by hGBA binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0588] (2) A fusion protein consisting of a structure formed by hGBA binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0589] (3) A fusion protein consisting of a structure formed by hGBA binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0590] (4) A fusion protein consisting of a structure formed by hGBA binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0591] Here, the linker sequence is preferably a linker sequence composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence composed of 1 to 10 or 1 to 20 of these amino acid sequences linked together.

[0592] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody. Additionally, hGBA is a protein or a variant thereof with the amino acid sequence number 81.

[0593] A more specific example of a fusion protein where other proteins (A) are (hGBA) is:

[0594] (1) A fusion protein consisting of a structure formed by hGBA binding 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 is shown in Serial No. 130, and the amino acid sequence of the latter is shown in Serial No. 23.

[0595] (2) A fusion protein consisting of a linker formed by hGBA linked by five amino acid sequences shown in Serial No. 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 131, and the amino acid sequence of the latter is shown in Serial No. 23.

[0596] (3) A fusion protein consisting of a linker formed by hGBA linked by 10 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 132, and the amino acid sequence of the latter is shown in sequence number 23.

[0597] (4) A fusion protein consisting of a linker formed by hGBA linked by 20 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 133, and the amino acid sequence of the latter is shown in sequence number 23.

[0598] (5) A fusion protein consisting of a linker formed by hGBA linked by three amino acid sequences shown in SEQ ID NO: 3, which is attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in SEQ ID NO: 134, and the amino acid sequence of the latter is shown in SEQ ID NO: 23.

[0599] (6) A fusion protein consisting of a linker consisting of two heavy chains (Fab) of hTfR linked together by six amino acid sequences (as shown in Serial No. 3) linked together at the C-terminus, with hGBA attached to the C-terminus, and a light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 135, and the amino acid sequence of the latter is shown in Serial No. 23.

[0600] (1)~(4): Antibody is IgG1 type, (5) and (6): Antibody is Fab type.

[0601] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is shown in sequence number 68. That is, in the fusion proteins of (1) to (4) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain contains the amino acid sequence of sequence number 68. In addition, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is shown in sequence number 61. That is, in the fusion proteins of (5) and (6) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain (Fab) contains the amino acid sequence of sequence number 61.

[0602] Specific examples of fusion proteins where other proteins (A) are (hTPP-1) can be listed as follows:

[0603] (1) A fusion protein consisting of a structure formed by hTPP-1 binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0604] (2) A fusion protein consisting of a structure formed by hTPP-1 binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0605] (3) A fusion protein consisting of a structure formed by hTPP-1 binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0606] (4) A fusion protein consisting of a structure formed by hTPP-1 binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0607] Here, the linker sequence is preferably a linker sequence composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence composed of 1 to 10 or 1 to 20 of these amino acid sequences linked together.

[0608] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody. Additionally, hTPP-1 is a protein or a variant thereof with the amino acid sequence having sequence number 82.

[0609] A more specific example of a fusion protein where other proteins (A) are (hTPP-1) is as follows:

[0610] (1) A fusion protein consisting of a structure formed by hTPP-1 binding 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 is shown in sequence number 136, and the amino acid sequence of the latter is shown in sequence number 23.

[0611] (2) A fusion protein consisting of a linker formed by hTPP-1 linked by five amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 137, and the amino acid sequence of the latter is shown in sequence number 23.

[0612] (3) A fusion protein consisting of a linker formed by hTPP-1 linked by 10 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 138, and the amino acid sequence of the latter is shown in sequence number 23.

[0613] (4) A fusion protein consisting of a linker formed by hTPP-1 linked by 20 amino acid sequences as shown in sequence number 3, which is attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 139, and the amino acid sequence of the latter is shown in sequence number 23.

[0614] (5) A fusion protein consisting of a linker formed by hTPP-1 linked by three amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain (Fab) of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 140, and the amino acid sequence of the latter is shown in sequence number 23.

[0615] (6) A fusion protein consisting of a linker consisting of two heavy chains (Fab) of hTfR linked together by six amino acid sequences (as shown in Serial No. 3) linked together at the C-terminus, with hTPP-1 attached to the C-terminus, and a light chain of hTfR. The amino acid sequence of the former is shown in Serial No. 141, and the amino acid sequence of the latter is shown in Serial No. 23.

[0616] (1)~(4): Antibody is IgG1 type, (5) and (6): Antibody is Fab type.

[0617] Here, the amino acid sequence of the heavy chain of hTfR in (1) to (4) above is shown in sequence number 68. That is, in the fusion proteins of (1) to (4) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain contains the amino acid sequence of sequence number 68. In addition, the amino acid sequence of the heavy chain of hTfR in (5) and (6) above is shown in sequence number 61. That is, in the fusion proteins of (5) and (6) above, the humanized antibody is a humanized antibody whose light chain contains the amino acid sequence of sequence number 23 and whose heavy chain (Fab) contains the amino acid sequence of sequence number 61.

[0618] Specific examples of fusion proteins for which other proteins (A) are (hNAGLU) can be listed as follows:

[0619] (1) A fusion protein consisting of a structure formed by hNAGLU binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 23.

[0620] (2) A fusion protein consisting of a structure formed by hNAGLU binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 25.

[0621] (3) A fusion protein consisting of a structure formed by hNAGLU binding to the C-terminus or N-terminus of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 27.

[0622] (4) A fusion protein consisting of a structure formed by hNAGLU binding to the C-terminal or N-terminal side of the heavy chain of hTfR via a linker sequence and the light chain of hTfR. The amino acid sequence of the heavy chain is shown in sequence number 66 or 68, and the amino acid sequence of the light chain is shown in sequence number 29.

[0623] Here, the linker sequence is preferably a linker sequence composed of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of sequence number 3, the amino acid sequence of sequence number 4, the amino acid sequence of sequence number 5, or an amino acid sequence composed of 1 to 10 or 1 to 20 of these amino acid sequences linked together.

[0624] Here, the antibody with sequence number 66 on the heavy chain is an IgG1 type antibody, and the antibody with sequence number 68 is an IgG4 type antibody. Additionally, hNAGLU is a protein or a variant thereof with the amino acid sequence number 83.

[0625] A more specific example of a fusion protein for which other proteins (A) are (hNAGLU) is:

[0626] (1) A fusion protein consisting of a structure formed by hNAGLU binding 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 is shown in sequence number 142, and the amino acid sequence of the latter is shown in sequence number 23.

[0627] (2) A fusion protein consisting of a linker formed by hNAGLU linked by five amino acid sequences shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the former is shown in sequence number 143, and the amino acid sequence of the latter is shown in sequence number 23.

[0628] (3) A fusion protein consisting of a linker formed by hNAGLU linked by 10 amino acid sequences as shown in sequence number 3 and attached to the C-terminus of the heavy chain of hTfR, and the light chain of hTfR. The amino acid sequence of the form...

Claims

1. A fusion protein, which is a fusion protein of an anti-human transferrin receptor antibody and human heparin N-sulfatase, wherein, This antibody is a Fab antibody. (1) The light chain of this antibody is composed of the amino acid sequence of sequence number 23, and (2) The heavy chain of the antibody binds to the human heparin N-sulfatase through an amino acid sequence formed by three amino acid sequences numbered 3 at its C-terminus, thereby forming the amino acid sequence numbered 128.

2. A DNA fragment encoding the amino acid sequence of the fusion protein of claim 1.

3. An expression vector integrating the DNA fragment of claim 2.

4. A mammalian cell transformed with the expression vector of claim 3.

Images