EA042007B1 - NEW ANTIBODIES AGAINST THE HUMAN TRANSFERRIN RECEPTOR, ABLE TO OVERCOME THE HEMATOENCEPHALIC BARRIER - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to antibodies against human transferrin receptor for use in conjugation with a compound that should exhibit its function in the central nervous system when administered parenterally (protein or low molecular weight compound, etc.) in order to make this compound able to pass through the blood-brain barrier after parenteral administration, as well as a method for its preparation, as well as a method for its use.

State of the art

Unlike capillaries in other tissues such as muscles, the capillaries that supply blood to most brain tissues with the exception of some areas, including the circumventricular organs (pineal gland, pituitary gland, posteriormost field, etc.), differ in that the endothelial cells that form their endothelium are interconnected by strong intercellular junctions. Passive transfer of substances from the capillaries to the brain is thus limited, and although there are some exceptions, substances are unlikely to enter the brain from the blood except for compounds such as fat-soluble or low molecular weight (less than 200-500 Daltons) and electrically neutral near physiological pH. This system, which limits the exchange of substances between blood and brain tissue fluid through the capillary endothelium in the brain, is called the blood-brain barrier or BBB. The blood-brain barrier not only limits the exchange of substances between the blood and the brain, but also between the tissue fluid of the central nervous system, including the brain and spinal cord, and the blood.

Due to the blood-brain barrier, most cells of the central nervous system avoid exposure to fluctuating concentrations of substances, such as hormones and lymphokines in the blood, and thus maintain their biochemical hemostasis.

However, the blood-brain barrier poses a problem when it comes to pharmaceutical development. For example, for mucopolysaccharidosis type I (Hurler's syndrome), an inherited metabolic disease caused by deficiency of α-L-iduronidase, although enzyme replacement therapy by intravenous administration of recombinant α-L-iduronidase is performed as treatment, the therapy is ineffective due to the marked impairment observed in the central nervous system (CNS) in Hurler syndrome because the enzyme cannot pass through the blood-brain barrier.

Attempts have been made to develop various methods for the macromolecular substances such as proteins and the like to be involved in the central nervous system to pass through the blood-brain barrier. For example, in the case of nerve growth factor, although attempts have been made in a method to cause the factor to cross the blood-brain barrier by allowing factor-encapsulating liposomes to fuse with the cell membrane of endothelial cells in brain capillaries, they have not been realized (Non-Patent Document 1). In the case of α-L-iduronidase, an attempt was made to increase the passive transport of the enzyme across the blood-brain barrier by increasing its concentration in the blood through an increased single dose of the enzyme, and thus, using an animal model of Hurler's syndrome, it was shown that this method improves the disorder in the central nervous system. system (CNS) (non-patent document 2).

In addition, an attempt was also made to bypass the blood-brain barrier by introducing a macromolecular substance directly into the medullary cavity or into the brain. For example, reports have been made on a method in which human α-L-iduronidase (Patent Document 1) was injected into the medullary cavity of a patient with Hurler syndrome (mucopolysaccharidosis type I) (Patent Document 1), a method in which human acidic acid was injected into the ventricles of the brain of a patient with Niemann-Pick disease. sphingomyelinase (Patent Document 2), and a method in which iduronate-2-sulfatase (I2S) was injected into the cerebral ventricles of animals in the Hunter syndrome model (Patent Document 3). Although any of these methods appear to accurately enable the pharmaceutical agent to act in the central nervous system, they have a problem in that they are highly invasive.

Various methods have been reported to ensure that a macromolecular substance enters the brain across the blood-brain barrier, in which the macromolecular substance is modified to give it affinity for membrane proteins located on the endothelial cells of brain capillaries. Examples of these membrane proteins that are found on brain capillary endothelial cells include receptors for compounds such as insulin, transferrin, insulin-like growth factor (IGF-I, IGF-II), LDL, and leptin.

For example, a method has been reported in which nerve growth factor (NGF) was synthesized as an insulin fusion protein, and the fusion protein was able to cross the blood-brain barrier by binding to the insulin receptor (Patent Documents 4-6). In addition, a method was reported in which nerve growth factor (NGF) was synthesized as a fusion protein with an anti-insulin receptor antibody, and this fusion protein was able to pass through the hematoma.

- 1 042007 encephalic barrier due to its binding to the insulin receptor (patent documents 4 and 7). In addition, a method was reported in which nerve growth factor (NGF) was synthesized as a fusion protein with transferrin, and this fusion protein was able to cross the blood-brain barrier by binding to the transferrin receptor (TfR) (Patent Document 8). In addition, a method was reported in which nerve growth factor (NGF) was synthesized as a fusion protein with an anti-transferrin receptor antibody (anti-TfR antibody), and this fusion protein was able to cross the blood-brain barrier by binding to TfR (patent documents 4 and 9).

Considering further methods that use an anti-transferrin receptor antibody, it has been reported that a single-chain antibody can be used in the art for allowing pharmaceuticals to cross the blood-brain barrier by binding to an anti-TfR antibody (Non-Patent Document 3). In addition, it has been reported that anti-hTfR antibodies having relatively high dissociation constants with hTfR (low affinity anti-hTfR antibody) can be successfully used in the method to allow pharmaceuticals to cross the blood-brain barrier (Patent Documents 10 and 11 and Non-Patent Document 4). Moreover, it has also been reported that anti-TfR antibodies whose affinity for hTfR varies with pH can be used as a carrier for passing pharmaceuticals through the blood-brain barrier (Patent Document 12 and Non-Patent Document 5).

Citation list

patent document.

Patent Document 1: JP 2007-504166 A1.

Patent Document 2: JP 2009-525963 A1.

Patent Document 3: JP 2012-62 312 A1.

Patent document 4: US 5154924 B1.

Patent Document 5: JP 2011-144178 A1.

Patent Document 6: US 2004/0101904 A1.

Patent document 7: JP 2006-51151 b A1.

Patent Document 8: JP H06-228199 A1.

Patent document 9: US 5977307 B1.

Patent Document 10: WO 2012/075037.

Patent Document 11: WO 2013/177062.

Patent Document 12: WO 2012/143379.

non-patent document.

Non-Patent Document 1: Xie Y. et al., J. Control Release. 105. 106-19 (2005).

Non-Patent Document 2: Ou L. et al., Mol. Genet. Metab. 111. 116-22 (2014).

Non-Patent Document 3: Li J.Y. protein engineering. 12. 787-96 (1999).

Non-Patent Document 4: Bien-Ly N. et al., J Exp Med. 211. 233-44 (2014).

Non-Patent Document 5: Sada H. PLoS ONE. 9. E96340 (2014).

The essence of the invention

Problems to be solved by the invention.

Against this background, an object of the present invention is to provide an anti-TfR antibody that can be used for conjugation with a compound that should exhibit its function in the central nervous system when administered parenterally (protein or small molecular weight compound and the like) in order to make this compound capable of passing through the blood-brain barrier, as well as the method of its preparation, as well as the method of its application.

Problem solving tools

As a result of intensive studies directed to the above object, the inventors of the present invention have found that antibodies against human transferrin receptor (anti-hTfR antibodies) that recognize the extracellular region of hTfR, which are obtained by the antibody production method detailed in the specification, effectively pass through the blood-brain barrier when introduced into the body, and thereafter completed the present invention. Thus, according to the present invention, the following is provided.

1. An antibody against human transferrin receptor, in which in the variable region of the heavy chain of the antibody:

(a) CDR1 contains the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, (b) CDR2 contains the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and (c) CDR3 contains the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

2. The antibody of claim 1 above, wherein the heavy chain framework region 3 comprises the amino acid sequence of SEQ ID NO: 64.

3. An antibody according to claim 1 or 2 above, wherein in the variable region of the heavy chain:

CDR2 contains an amino acid sequence having at least 80% homology with the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 instead, and

- 2 042007

CDR3 contains an amino acid sequence having at least 80% homology with the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 instead.

4. An antibody according to claim 1 or 2 above, wherein in the variable region of the heavy chain,

CDR2 contains an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 instead, and

CDR3 contains an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 instead.

5. An antibody according to claim 1 or 2 above, wherein the heavy chain variable region contains an amino acid sequence modified from at least one amino acid sequence:

(a) SEQ ID NO: 62 or SEQ ID NO: 63 for CDR1, (b) SEQ ID NO: 13 or SEQ ID NO: 14 for CDR2, (c) SEQ ID NO: 15 or SEQ ID NO: 16 in relation to CDR3 and (d) SEQ ID NO: 64 in relation to framework region 3, by substitution, deletion or addition of 1-5 amino acids instead, while in CDR1 methionine located at position 5 from the N-terminal side of the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63 is also at the same position in the thus modified amino acid sequence, and in framework region 3, the leucine located at position 17 from the N-terminal side of SEQ ID NO: 64 is also at the same position in the thus modified amino acid sequence.

6. An antibody according to claim 1 or 2 above, wherein the heavy chain variable region contains an amino acid sequence modified from at least one amino acid sequence:

(a) SEQ ID NO: 62 or SEQ ID NO: 63 for CDR1, (b) SEQ ID NO: 13 or SEQ ID NO: 14 for CDR2, (c) SEQ ID NO: 15 or SEQ ID NO: 16 in relation to CDR3, and (d) SEQ ID NO: 64 in relation to framework region 3, by substitution, deletion or addition of 1-3 amino acids instead, while in CDR1 methionine located at position 5 from the N-terminal side of the amino acid sequence SEQ ID NO: 62 or SEQ ID NO: 63 is also at the same position in the thus modified amino acid sequence, and in framework region 3, the leucine located at position 17 from the N-terminal side of SEQ ID NO: 64 is also at the same position in the thus modified amino acid sequence.

7. The antibody of claim 2 above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 65.

8. The antibody of claim 7 above, wherein the heavy chain variable region comprises, in the region except for the amino acid sequences shown as SEQ ID NO: 62 or SEQ ID NO: 63 CDR1 and SEQ ID NO: 64 of framework region 3, the amino acid sequence having a homology of at least 80% with a site instead of a site.

9. The antibody of claim 7 above, wherein the heavy chain variable region comprises, in the region except for the amino acid sequences shown as SEQ ID NO: 62 or SEQ ID NO: 63 CDR1 and SEQ ID NO: 64 of framework region 3, the amino acid sequence having a homology of at least 90% with a site instead of a site.

10. The antibody according to claim 7 above, wherein the antibody contains an amino acid sequence modified from the amino acid sequence constituting the variable region of the heavy chain, by substitution, deletion or addition of 1-5 amino acids instead, while in CDR1 methionine located at position 5 from the N-terminus of the amino acid sequence of SEQ ID NO: 63 is also at the same position in the thus modified amino acid sequence, and in framework region 3 the leucine located at position 17 from the N-terminal side of SEQ ID NO: 64 is also is in the same position in the thus modified amino acid sequence.

11. The antibody according to claim 7 above, wherein the antibody contains an amino acid sequence modified from the amino acid sequence constituting the variable region of the heavy chain, by substitution, deletion or addition of 1-3 amino acids instead, while in CDR1 methionine located at position 5 from the N-terminus of the amino acid sequence of SEQ ID NO: 63 is also at the same position in the thus modified amino acid sequence, and in framework region 3 the leucine located at position 17 from the N-terminal side of SEQ ID NO: 64 is also is in the same position in the thus modified amino acid sequence.

12. The antibody of claim 7 above, wherein the heavy chain contains the amino acid sequence

- 3 042007

SEQ ID NO: 66 or SEQ ID NO: 68.

13. The antibody of claim 12 above, wherein the heavy chain comprises an amino acid sequence having at least 80% homology with the region except for the amino acid sequences shown as SEQ ID NO: 62 or SEQ ID NO: 63 CDR1 and SEQ ID NO: 64 frame region 3, instead of a plot.

14. The antibody of claim 12 above, wherein the heavy chain comprises an amino acid sequence having at least 90% homology with the region except for the amino acid sequences shown as SEQ ID NO: 62 or SEQ ID NO: 63 CDR1 and SEQ ID NO: 64 frame region 3, instead of a plot.

15. The antibody according to claim 12 above, wherein the antibody contains an amino acid sequence modified from the amino acid sequence constituting the heavy chain, by substitution, deletion or addition of 1-5 amino acids instead, while in CDR1 methionine located at position 5 from the N-terminus of the amino acid sequence of SEQ ID NO: 63 is also at the same position in the thus modified amino acid sequence, and in framework region 3, the leucine located at position 17 from the N-terminal side of SEQ ID NO: 64 is also at the same position in the thus modified amino acid sequence.

16. The antibody according to claim 12 above, wherein the antibody contains an amino acid sequence modified from the amino acid sequence constituting the heavy chain, by substitution, deletion or addition of 1-3 amino acids instead, while in CDR1 methionine located at position 5 from the N-terminus of the amino acid sequence of SEQ ID NO: 63 is also at the same position in the thus modified amino acid sequence, and in framework region 3, the leucine located at position 17 from the N-terminal side of SEQ ID NO: 64 is also at the same position in the thus modified amino acid sequence.

17. An antibody according to any one of claims 1-16 above, wherein in the variable region of the light chain of the antibody:

(a) CDR1 contains the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, (b) CDR2 contains the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence of Lys-Val-Ser, a (c ) CDR3 contains the amino acid sequence of SEQ ID NO: 10.

18. An antibody according to claim 17 above, wherein in the variable region of the light chain:

(a) CDR1 contains an amino acid sequence having at least 80% homology with the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 instead, (b) CDR2 contains an amino acid sequence having at least 80% homology with the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence of Lys-Val-Ser, instead, and (c) CDR3 contains an amino acid sequence having at least 80% homology with the amino acid sequence of SEQ ID NO: 10, instead of her.

19. The antibody of claim 17 above, wherein in the variable region of the light chain:

(a) CDR1 contains an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 instead, (b) CDR2 contains an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence of Lys-Val-Ser, instead, and (c) CDR3 contains an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO: 10, instead of her.

20. The antibody of claim 17 above, wherein the light chain variable region contains an amino acid sequence modified from at least one amino acid sequence:

(a) SEQ ID NO: 6 or SEQ ID NO: 7 for CDR1, (b) SEQ ID NO: 8 or SEQ ID NO: 9 or Lys-Val-Ser amino acid sequence for CDR2, and (c) SEQ ID NO : 10 in relation to CDR3 by substitution, deletion or addition of 1-5 amino acids instead.

21. The antibody of claim 17 above, wherein the light chain variable region contains an amino acid sequence modified from at least one amino acid sequence:

(a) SEQ ID NO: 6 or SEQ ID NO: 7 for CDR1, (b) SEQ ID NO: 8 or SEQ ID NO: 9, or Lys-Val-Ser amino acid sequence for CDR2, and (c) SEQ ID NO: 10 in relation to CDR3 by substitution, deletion or addition of 1-3 amino acids instead.

- 4 042007

22. An antibody according to any one of claims 1-16 above, wherein the variable region of the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO : 21 or SEQ ID NO: 22.

23. The antibody of claim 22 above, wherein the antibody contains an amino acid sequence having at least 80% homology with the light chain variable region amino acid sequence in its place.

24. The antibody of claim 22 above, wherein the antibody contains an amino acid sequence having at least 90% homology with the light chain variable region amino acid sequence in its place.

25. The antibody of claim 22 above, wherein the antibody comprises an amino acid sequence modified from the light chain variable region amino acid sequence by substitution, deletion, or addition of 1-5 amino acids instead.

26. The antibody of claim 22 above, wherein the antibody contains an amino acid sequence modified from the amino acid sequence of the light chain variable region by substitution, deletion, or addition of 1-3 amino acids in its place.

27. An antibody according to any one of claims 1-16 above, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29.

28. The antibody of claim 27 above, wherein the antibody contains an amino acid sequence having at least 80% homology with the light chain amino acid sequence instead.

29. The antibody of claim 27 above, wherein the antibody contains an amino acid sequence having at least 90% homology with the light chain amino acid sequence instead.

30. The antibody of claim 27 above, wherein the antibody contains an amino acid sequence modified from the light chain amino acid sequence by substitution, deletion, or addition of 1-5 amino acids instead.

31. The antibody of claim 27 above, wherein the antibody contains an amino acid sequence modified from the light chain amino acid sequence by substitution, deletion, or addition of 1-3 amino acids instead.

32. An antibody according to any one of claims 1-31 above, wherein the antibody has affinity for both the extracellular region of the human transferrin receptor and the extracellular region of the monkey transferrin receptor.

33. The antibody of claim 32 above, wherein the dissociation constant of its complex with the extracellular region of the human transferrin receptor is no more than ^1x10'10 M, and the dissociation constant of its complex with the extracellular region of the monkey transferrin receptor is no more than 1x10'9 M.

34. An antibody according to any one of claims 1-33 above, wherein the antibody is a Fab antibody, F(ab') ₂ antibody, or F(ab') antibody.

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

36. The antibody of claim 35 above, wherein its light chain and heavy chain are linked via a linker sequence.

37. The antibody of claim 36 above, wherein the heavy chain is linked via a linker sequence to the light chain at its C-terminal side.

38. The antibody of claim 35 above, wherein the light chain is linked via a linker sequence to the heavy chain at its C-terminal side.

39. An antibody according to any one of claims 36-38 above, wherein the linker sequence consists of 850 amino acid residues.

40. The antibody of claim 39 above, wherein the linker sequence is selected from the group consisting of Gly-Ser amino acid sequence, Gly-GlySer amino acid sequence, Gly-Gly-Gly amino acid sequence, amino acid sequences shown as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, an amino acid sequence consisting of three consecutively linked amino acid sequences, each shown as SEQ ID NO: 3, and amino acid sequences consisting of 1-10 of them, which are sequentially linked.

41. A fusion protein of an anti-human transferrin receptor antibody and another protein (A), wherein the anti-human transferrin receptor antibody is an antibody according to any one of claims 1 to 40 above, and the protein (A) is linked to the light chain of the antibody at its C-terminal side or N-terminal side.

42. The fusion protein of claim 41 above, wherein the protein (A) is linked directly or via a linker to the light chain at its C-terminal side or N-terminal side.

43. The fusion protein of claim 41 or 42 above, wherein the protein (A) is linked via a linker to the light chain at its C-terminal side or N-terminal side.

44. The fusion protein according to claim 43 above, in which the linker is a peptide consisting of 1-50 amino acids

- 5 042007 lot balances.

45. The fusion protein of claim 44 above, wherein the linker is a peptide comprising an amino acid sequence selected from the group consisting of one glycine, one serine, Gly-Ser amino acid sequence, Gly-Gly-Ser amino acid sequence, SEQ ID amino acid sequence NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and amino acid sequences of 1-10 of them that are sequentially linked.

46. A fusion protein of an anti-human transferrin receptor antibody and another protein (A), wherein the anti-human transferrin receptor antibody is an antibody according to any one of claims 1 to 40 above, and the protein (A) is linked to the heavy chain of the antibody at its C-terminal side or N-terminal side.

47. The fusion protein of claim 46 above, wherein the protein (A) is linked directly or via a linker to the heavy chain at its C-terminal side or N-terminal side.

48. The fusion protein of claim 46 or 47 above, wherein the protein (A) is linked via a linker to the heavy chain at its C-terminal side or N-terminal side.

49. The fusion protein of claim 48 above, wherein the linker sequence is a peptide consisting of 1-50 amino acid residues.

50. The fusion protein of claim 49 above, wherein the linker is a peptide comprising an amino acid sequence selected from the group consisting of one glycine, one serine, Gly-Ser amino acid sequence, Gly-Gly-Ser amino acid sequence, SEQ ID amino acid sequence NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and amino acid sequences of 1-10 of them that are sequentially linked.

51. A fusion protein according to any one of claims 41-50 above, wherein the protein (A) is a protein derived from a human.

52. The fusion protein of any one of claims 41-51 above, wherein the protein (A) is selected from the group consisting of nerve growth factor (NGF), lysosomal enzymes, ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF ), neurotrophin-3, neurotrophin-4/5, neurotrophin-6, neuregulin-1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (MCSF), cytokines, receptor factor tumor necrosis-α (TNF-α receptor), PD-1, PD-L1, PDL2 ligands, β-amyloid-degrading enzymes, antibodies against β-amyloid, antibodies against BACE, antibodies against EGFR, antibodies against P D-1, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-HER2 antibodies, anti-TNF-α antibodies, anti-CTLA-4 antibodies and other antibody-based drugs.

53. A fusion protein wherein protein (A) is a lysosomal enzyme, wherein the lysosomal enzyme is selected from the group consisting of a-L-iduronidase, iduronate-2-sulfatase, human acid α-glucosidase, glucocerebrosidase, β-galactosidase, activator protein GM2, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, a-L-fucosidase, aspartylglucosaminidase, a-N-acetylgalactosaminidase, acid sphingomyelinase, a-galactosidase A, β-glucuronidase, heparan-N-sulfatase, a-N-acetylglucosaminidase, acetyl-KoA:α-glucosaminide-N-acetyltransferase, N-acetylglucosamine-6-sulfate-sulfatase, acid ceramidase, amyl-1,6-glucosidase, sialidase , palmitoyl protein thioesterase 1, tripeptidyl peptidase 1, hyaluronidase 1, CLN1 and CLN2.

54. A fusion protein according to any of claims 41 to 51 above, wherein protein (A) is human iduronate-2-sulfatase, human acid α-glucosidase, or human α-L-iduronidase.

55. The fusion protein of claim 51 above, wherein protein (A) is human acid α-glucosidase, wherein:

(1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain is linked at its C-terminal side and via the Gly-Ser amino acid sequence to human acid α-glucosidase, thereby forming the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58.

56. The fusion protein of claim 51 above, wherein protein (A) is human acid α-glucosidase, wherein:

(1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68, and the heavy chain is linked at its C-terminal side and through the amino acid Gly-Ser sequences with human acid α-glucosidase having the amino acid sequence

- 6 042007 SEQ ID NO: 55 or 56.

57. The fusion protein of claim 51 above, wherein protein (A) is human acid α-glucosidase, wherein the antibody is a Fab antibody, and wherein:

(1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain is linked at its C-terminal side and through an amino acid sequence consisting of three consecutively linked amino acid sequences, each shown as SEQ ID NO : 3 with human acid α-glucosidase, thereby forming the amino acid sequence of SEQ ID NO: 89.

58. The fusion protein of claim 51 above, wherein protein (A) is human acid α-glucosidase, wherein the antibody is a Fab antibody, and wherein:

(1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 61, and the heavy chain is linked at its C-terminal side and through an amino acid sequence consisting of three consecutive linked amino acid sequences, each shown as SEQ ID NO: 3, with human acid α-glucosidase having the amino acid sequence of SEQ ID NO: 55 or 56.

59. The fusion protein of claim 51 above, wherein protein (A) is human a-L-iduronidase, wherein the antibody is a Fab antibody, and wherein:

(1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain is linked at its C-terminal side and through an amino acid sequence consisting of three consecutively linked amino acid sequences, each shown as SEQ ID NO : 3 with human a-L-iduronidase, thereby forming the amino acid sequence of SEQ ID NO: 93.

60. The fusion protein of claim 51 above, wherein protein (A) is human a-L-iduronidase, wherein the antibody is a Fab antibody, and wherein:

(1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 61, and the heavy chain is linked at its C-terminal side and through an amino acid sequence consisting of three consecutive linked amino acid sequences, each shown as SEQ ID NO: 3, with human a-L-iduronidase having the amino acid sequence of SEQ ID NO: 75 or 76.

61. The fusion protein of claim 41 above, wherein a human IgG Fc region or a portion thereof is inserted between the protein (A) and the antibody.

62. The fusion protein of claim 61 above, wherein the human IgG Fc region is linked directly or via a linker sequence to the protein (A) on its C-terminal side, and the antibody heavy chain or light chain is linked directly or via a linker sequence to the Fc human IgG region on its C-terminal side.

63. The fusion protein of claim 61 or 62 above, wherein the human IgG Fc region contains the amino acid sequence of SEQ ID NO: 70.

64. The fusion protein of claim 63 above, wherein the human IgG Fc region is linked via a linker sequence to a Fab heavy chain consisting of the amino acid sequence of SEQ ID NO: 61 and contains the amino acid sequence of SEQ ID NO: 71 thus formed.

65. A DNA fragment encoding the amino acid sequence of an anti-human transferrin receptor antibody according to any one of claims 1 to 40 above.

66. A DNA fragment encoding the amino acid sequence of a fusion protein according to any one of claims 41-64 above.

67. An expression vector containing a DNA fragment included in it according to paragraph 65 or 66 above.

68. A mammalian cell transformed with the expression vector of claim 67 above.

69. An anti-human transferrin receptor antibody-pharmacologically active compound complex, wherein the light chain and/or heavy chain of an anti-human transferrin receptor antibody according to any one of claims 1 to 40 above is linked to a pharmacologically active small molecular weight compound that needs to be able to pass through the blood-brain barrier and manifestations of its function in the brain.

70. The anti-human transferrin receptor antibody-pharmacologically active compound complex of claim 69 above, wherein the pharmacologically active compound is any compound selected from the group consisting of an anticancer drug, an Alzheimer's disease therapeutic agent, a Parkinson's disease therapeutic agent, a therapeutic agent for Huntington's disease, therapeutic agent for schizophrenia, antidepressant, therapeutic agent for multiple sclerosis, therapeutic agent for amyotrophic lateral sclerosis, therapeutic agent for tumors of the central nervous system, including brain tumor, therapeutic agent for lysosomal storage disease accompanied by encephalitis

- 7 042007 spatula, therapeutic agent for glycogenosis, therapeutic agent for muscular dystrophy, therapeutic agent for cerebral ischemia, therapeutic agent for prion diseases, therapeutic agent for traumatic disorders of the central nervous system, therapeutic agent for viral and bacterial diseases of the central nervous system, pharmaceutical agent used for brain surgery recovery, a pharmaceutical for spinal surgery recovery, siRNA, antisense DNA, and a peptide.

71. The use of an anti-human transferrin receptor antibody according to any one of claims 1 to 40 above, allowing the passage of the protein (A) or pharmacologically active small molecule compound through the blood-brain barrier and its function in the brain.

72. The use of an anti-human transferrin receptor antibody according to any one of claims 1 to 40 above for preparing a parenteral pharmaceutical for treating a central nervous system disease condition by linking a physiologically active protein molecule or a pharmacologically active small molecule compound for the disease state to it.

73. A method of treating a disease state of the central nervous system, comprising parenterally administering to a patient with a disorder a therapeutically effective amount of a physiologically active protein or a pharmacologically active small molecule compound for the disorder in the form of a conjugate with an anti-human transferrin receptor antibody molecule according to any one of claims 1-40 above.

74. The use of a fusion protein according to any one of claims 52-58 above to allow human acid α-glucosidase to cross the blood-brain barrier and function in the brain.

75. The use of a fusion protein according to any one of claims 52-58 above for the preparation of a parenteral pharmaceutical agent for the treatment of a central nervous system disease condition accompanying Pompe's disease.

76. A method of treating a central nervous system disease accompanying Pompe disease, comprising parenterally administering a therapeutically effective amount of a fusion protein according to any one of claims 52-58 above to a patient with the disease.

77. Use of a fusion protein according to any one of claims 52-54, 59 and 60 above to allow human acid α-L-iduronidase to cross the blood-brain barrier and function in the brain.

78. The use of a fusion protein according to any one of claims 52-54, 59 and 60 above for the preparation of a parenteral pharmaceutical for the treatment of a central nervous system disease condition accompanying Hurler's syndrome or Hurler-Scheie's syndrome.

79. A method of treating a central nervous system disease accompanying Hurler's syndrome or Hurler-Scheie's syndrome, the method comprising parenterally administering a therapeutically effective amount of a fusion protein according to any one of claims 52-54, 59 and 60 above to a patient with the disease.

The results of the invention.

With the present invention, various compounds, such as proteins and small molecule compounds, which, although physiologically or pharmacologically active, were unsuitable for parenteral administration due to their little or no ability to cross the blood-brain barrier, can be provided in a form that allows them to pass. across the blood-brain barrier, thus making them novel parenteral pharmaceuticals for the treatment of disease states of the central nervous system.

Brief description of the drawings

Fig. 1 are substitute photographs for drawings showing the result of immunohistochemical staining of anti-hTfR antibodies in the cerebral cortex of a cynomolgus monkey after a single intravenous administration of anti-hTfR antibody. (a) Anti-hTfR antibody was not injected, (b) Anti-hTfR antibody #3 was injected. The scale in the lower right corner of each photograph is 50 µm.

Fig. 2 is a figure showing the result of immunohistochemical staining of anti-hTfR antibodies in the hippocampus of a cynomolgus monkey after a single intravenous administration of anti-hTfR antibody. (a) Anti-hTfR antibody was not injected, (b) Anti-hTfR antibody #3 was injected. The scale in the lower right corner of each photograph is 50 µm.

Fig. 3 are substitute photographs for drawings showing the result of immunohistochemical staining of anti-hTfR antibodies in the cerebellum of a cynomolgus monkey after a single intravenous injection of anti-hTfR antibody. (a) Anti-hTfR antibody was not injected, (b) Anti-hTfR antibody #3 was injected. The scale in the lower right corner of each photograph has a size of 50 µm.

Fig. 4 is a figure showing the amount of humanized anti-hTfR antibodies accumulated in various organs other than the brain of the cynomolgus monkey after a single intravenous administration. The vertical axis denotes the amount of humanized anti-hTfR antibodies (µg/g crude

- 8 042007 mass) on the wet weight of each organ. The white bars represent, on the left, the amount accumulated in each organ of the monkey after administration of humanized anti-hTfR antibody #3, humanized anti-hTfR antibody #3-2, humanized anti-hTfR antibody #3 (IgG4), and humanized anti-hTfR antibody #3-2 ( IgG4), respectively, and the black bars represent the amount accumulated in the corresponding organs of the monkey after administration of trastuzumab (Herceptin™). ND stands for not detected.

Fig. 5 are replacement photographs for the drawings showing the result of immunohistochemical staining of humanized anti-hTfR antibodies in the cerebral cortex of a cynomolgus monkey after a single intravenous injection, (a) Herceptin was administered, (b) humanized anti-hTfR antibody #3 was administered, (c) humanized antibody was administered against hTfR No. 3-2, (d) a humanized anti-hTfR antibody No. 3 (IgG4) was administered, (e) a humanized anti-hTfR antibody No. 3-2 (IgG4) was administered. The scale in the lower right corner of each photograph is 20 µm.

Fig. 6 are substitute photographs for the drawing showing the result of immunohistochemical staining of humanized anti-hTfR antibodies in the cynomolgus monkey hippocampus after a single intravenous injection. (a) Herceptin was administered, (b) humanized anti-hTfR antibody #3 was administered, (c) humanized anti-hTfR antibody #3-2 was administered, (d) humanized anti-hTfR antibody #3 (IgG4) was administered, (e) humanized antibody was administered against hTfR #3-2 (IgG4). The scale in the lower right corner of each photograph is 20 µm.

Fig. 7 is a figure showing the result of immunohistochemical staining of humanized anti-hTfR antibodies in the cerebellum of a cynomolgus monkey after a single intravenous injection. (a) Herceptin was administered, (b) humanized anti-hTfR antibody #3 was administered, (c) humanized anti-hTfR antibody #3-2 was administered, (d) humanized anti-hTfR antibody #3 (IgG4) was administered, (e) humanized antibody was administered against hTfR #3-2 (IgG4). The scale in the lower right corner of each photograph is 20 μm in size.

Fig. 8 is a figure showing the result of immunohistochemical staining of humanized anti-hTfR antibodies in the medulla oblongata of a cynomolgus monkey after a single intravenous injection. (a) Herceptin was administered, (b) humanized anti-hTfR antibody #3 was administered, (c) humanized anti-hTfR antibody #3-2 was administered, (d) humanized anti-hTfR antibody #3 (IgG4) was administered, (e) humanized antibody was administered against hTfR #3-2 (IgG4). The scale in the lower right corner of each photograph is 20 μm in size.

Fig. 9 is a figure showing the result of immunohistochemical staining of hGAA in the cerebellum of a cynomolgus monkey after a single intravenous injection, (a) hGAA anti-hTfR 3N antibody (IgG4) was administered, (b) hGAA was administered. The scale in the lower right corner of each photograph is 20 µm.

Fig. 10 is a figure showing the result of evaluating the pharmacological action of hGAA antibodies against hTfR 3N (IgG4) using a mouse. Glycogen concentrations are shown in (a) the right hemisphere of the brain, (b) the cervical spinal cord, (c) the heart, (d) the diaphragm, (e) the liver, and (f) the spleen. In each figure, the number 1 denotes the normal control group, the number 2 denotes the disease control group, the number 3 denotes the 20 mg/kg hGAA administration group, and the numbers 4-7 denote the 2.5 mg/kg, 5.0 mg/kg, 10 mg/kg and 20 mg/kg, respectively, hGAA antibodies against hTfR 3N (IgG4). The ordinate indicates the concentration of glycogen (mg/g wet weight). Vertical scale denotes SD, # denotes p<0.01 versus disease control group, ## denotes p<0.05 versus disease control group, $ denotes p<0.01 versus hGAA administration group, a $ $ denotes p<0.05 compared to the hGAA administration group (each based on Tukey's test).

Fig. 11 is a figure showing the result of evaluating the pharmacological action of hGAA antibodies against hTfR 3N (IgG4) using a mouse. Glycogen concentrations in (a) quadriceps femoris, (b) gastrocnemius, (c) soleus, (d) tibialis anterior, and (e) gastrocnemius extensor digitorum longus. In each figure, the number 1 indicates the normal control group, the number 2 indicates the disease control group, the number 3 indicates the hGAA administration group, and the numbers 4-7 indicate the 2.5 mg/kg, 5.0 mg/kg, 10 mg/kg administration groups and 20 mg/kg, respectively, hGAA antibodies against hTfR 3N (IgG4). The ordinate indicates the concentration of glycogen (mg/g wet weight). Vertical scale denotes SD, # denotes p<0.01 versus disease control group, ## denotes p<0.05 versus disease control group, $ denotes p<0.01 versus hGAA administration group, a $ $ denotes p<0.05 compared to the hGAA administration group (each based on Tukey's test).

Fig. 12 is a figure showing the result of the evaluation of the pharmacological action of Fab GS-GAA using a mouse. Glycogen concentrations in (a) right cerebral hemisphere, (b) heart, (c) diaphragm, (d) quadriceps femoris, (e) soleus, and (f) tibialis anterior. In each figure, the number 1 denotes the normal control group, the number 2 denotes the disease control group, the number 3 denotes the hGAA administration group, and the numbers 4 and 5 denote 5.0 mg/kg and 20 mg/kg, respectively, Fab GS-GAA. The ordinate indicates the concentration of glycogen (mg/g wet weight). The vertical scale indicates SD.

- 9 042007

Embodiments of the invention.

According to the present invention, the term antibody mainly refers to a human antibody, a mouse antibody, a humanized antibody, as well as a chimeric antibody between a human antibody and a non-human mammalian antibody, and a chimeric antibody between a mouse antibody and a non-mouse mammalian antibody, but the meaning of the term is not limited to this, since the substance of interest has the property of specifically binding to a particular antigen, and there is no particular limitation on animal species of the antibody. However, a humanized antibody is preferred.

According to the present invention, the term human antibody refers to an antibody whose entire protein is encoded by a gene derived from a human. However, the term human antibody also includes an antibody encoded by a gene obtained by introducing a mutation into the original human gene in order to increase the efficiency of gene expression, for example, without modifying the original amino acid sequence. The term human antibody also includes an antibody that is made by joining two or more genes encoding human antibodies and replacing some part of the human antibody with part of another human antibody. The human antibody contains three complementarity determining regions (CDRs) in the immunoglobulin light chain and three complementarity determining regions (CDRs) in the immunoglobulin heavy chain. The three CDRs in an immunoglobulin light chain are called, on the N-terminal side, CDR1, CDR2, and CDR3, respectively. The three CDRs in the immunoglobulin heavy chain are also called, on the N-terminal side, CDR1, CDR2 and CDR3, respectively. The term human antibody also includes a human antibody obtained by replacing the CDR of a human antibody with the CDR of another human antibody to change properties such as antigen specificity and affinity of the original human antibody, etc.

According to the present invention, the term human antibody also includes an antibody that is obtained by modifying the gene of the original human antibody by introducing a mutation such as substitution, deletion, addition, in the amino acid sequence of the original antibody. When replacing one or more amino acids of the amino acid sequence of the parent antibody with other amino acids, the number of amino acids substituted may preferably be 1-20, more preferably 1-5, and even more preferably 1-3. When deleting one or more amino acids of the amino acid sequence of the parent antibody, the number of amino acids removed may preferably be 1-20, more preferably 1-5, and even more preferably 1-3. An antibody produced by the combined mutation of these amino acid substitutions and deletions is also a human antibody. In some instances, one or more amino acids, preferably 1-20, more preferably 1-5, and even more preferably 1-3 amino acids, may be added within the amino acid sequence of the parent antibody or at its N- or C-terminal side. An antibody produced by a combination mutation of addition, substitution and deletion of amino acids is also a human antibody.

The amino acid sequence of such a mutant antibody has preferably no less than 80%, more preferably no less than 90%, even more preferably no less than 95%, and even more preferably no less than 98% homology with the amino acid sequence of the parent antibody. Thus, according to the present invention, the term human-derived gene includes not only a non-mutated human-derived gene, but also a gene obtained by modifying it.

The homology between the amino acid sequence of a non-mutated antibody and the amino acid sequence of an antibody mutated into it can be easily calculated using well-known homology calculation algorithms. As such algorithms, there are, for example, BLAST (Altschul S.F. J. Mol. Biol. 215. 403-10 (1990)), Pearson and Lipman similarity search (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)) and the Smith-Waterman local homology search algorithm (Adv. Appl. Math. 2. 482-9 (1981)) and the like.

According to the present invention, the term mouse antibody refers to an antibody whose entire protein consists of the same amino acid sequence as that of an antibody encoded by a gene derived from a mouse. Therefore, the term mouse antibody also includes an antibody that is encoded by a gene obtained by introducing a mutation in the original mouse gene that does not cause a change in its amino acid sequence, but, for example, in order to improve the efficiency of gene expression. In addition, the term mouse antibody also includes an antibody obtained by combining two or more genes encoding mouse antibodies by replacing a portion of a mouse antibody with a portion of another mouse antibody. The mouse antibody has three complementarity determining regions (CDRs) in the immunoglobulin light chain and three complementarity determining regions (CDRs) in the immunoglobulin heavy chain. The three CDRs in an immunoglobulin light chain are called, on the N-terminal side, CDR1, CDR2, and CDR3, respectively. The three CDRs in the immunoglobulin heavy chain are also called, on the N-terminal side, CDR1, CDR2 and CDR3, respectively. The term mouse antibody also includes an antibody obtained by replacing the CDR of a mouse antibody with the CDR of another mouse antibody to change the specificity and affinity of the original mice.

- 10 042007 antibodies.

According to the present invention, the term mouse antibody also includes an antibody that is obtained by modifying the gene of the original mouse antibody by introducing a mutation such as substitution, deletion, addition, in the amino acid sequence of the original antibody. When replacing one or more amino acids of the amino acid sequence of the parent antibody with other amino acids, the number of amino acids substituted may preferably be 1-20, more preferably 1-5, and even more preferably 1-3. When deleting one or more amino acids of the amino acid sequence of the parent antibody, the number of amino acids removed may preferably be 1-20, more preferably 1-5, and even more preferably 1-3. An antibody produced by the combined mutation of these amino acid substitutions and deletions is also included in a mouse antibody. When adding one or more amino acids, they can be added inside the amino acid sequence of the original antibody or from its N- or C-terminal side in an amount of preferably 1-20, more preferably 1-5, and even more preferably 1-3. An antibody produced by a combined amino acid addition, substitution, and deletion mutation is also included in a mouse antibody. The amino acid sequence of such a mutant antibody has preferably no less than 80%, more preferably no less than 90%, even more preferably no less than 95%, and even more preferably no less than 98% homology with the amino acid sequence of the parent antibody. Thus, according to the present invention, the term mouse-derived gene includes not only a non-mutated mouse-derived gene, but also a gene obtained by modifying it.

According to the present invention, the term humanized antibody refers to an antibody in which part of the amino acid sequence of its variable region (eg, in particular all or part of its CDRs) is from a non-human mammal, while the remainder is from a human. An example of a humanized antibody is an antibody obtained by replacing the three complementarity determining regions (CDRs) of an immunoglobulin light chain and the three complementarity determining regions (CDRs) of an immunoglobulin heavy chain constituting a human antibody with a CDR from a non-human mammal. Since it is derived from a non-human mammal, there is no particular limitation on the species from which those CDRs are derived that are transplanted into the proper position of a human antibody, although mouse, rat, rabbit, horse, or non-human primate is preferred, mouse and rat are more preferred. and even more preferred is a mouse.

According to the present invention, the term chimeric antibody refers to an antibody obtained by combining fragments of two or more different antibodies derived from two or more different species.

A chimeric antibody between a human antibody and a non-human mammal antibody is an antibody provided by replacing a portion of a human antibody with a portion of a non-human mammal antibody. As explained below, an antibody consists of an Fc region, a Fab region, and a hinge region. A specific example of such chimeric antibodies is a chimeric antibody whose Fc region is derived from a human antibody while its Fab region is derived from a non-human mammalian antibody. The hinge region is either from a human antibody or from a non-human mammalian antibody. In contrast, the term chimeric antibody also includes an antibody whose Fc region is derived from a non-human mammalian antibody while its Fab region is derived from a human antibody. In such a case, too, the hinge region is derived from either a human antibody or a non-human mammalian antibody.

It can be seen that antibodies are composed of a variable region and a constant region. Additional examples of chimeric antibodies include an antibody in which the heavy chain constant region ( _CH ) and light chain constant region (Cl) are both derived from a human antibody, while the heavy chain variable region (VH) and light chain variable region (V _L ) are both are derived from a non-human mammalian antibody, and vice versa, an antibody in which the heavy chain constant region (C _n ) and the light chain constant region (Cl) are both derived from a non-human mammalian antibody, while the heavy chain variable region (VH) and the light chain variable region (V _L ) both are derived from a human antibody. Since it is a non-human mammal, they have no particular restriction on non-human mammal species, although a mouse, rat, rabbit, horse, or non-human primate is preferred, and a mouse is more preferred.

A chimeric antibody between a mouse antibody and a non-mouse mammal antibody is an antibody provided by replacing a portion of a mouse antibody with a portion of a non-mouse mammal antibody. Specific examples of such chimeric antibodies include a chimeric antibody whose Fc region is derived from a mouse antibody while its Fab region is derived from a non-mouse mammalian antibody, and vice versa, a chimeric antibody whose Fc region is derived from a non-mouse mammalian antibody while its fab area

- 11 042007 is derived from a mouse antibody. Since it is a non-mouse mammal, they do not have a particular limitation on non-mouse mammal species, although rat, rabbit, horse, or non-human primate is preferred, and human is more preferred.

A chimeric antibody between a human antibody and a mouse antibody is specifically referred to as a human/mouse chimeric antibody. Examples of human/mouse chimeric antibodies include a chimeric antibody in which the Fc region is derived from a human antibody while the Fab region is derived from a mouse antibody, and vice versa, a chimeric antibody whose Fc region is derived from a mouse antibody while its Fab is region is derived from a human antibody. The hinge region is either from a human antibody or from a mouse antibody. Additional specific examples of human/mouse chimeric antibodies include antibodies whose heavy chain constant region (CH) and light chain constant region ( _CL ) are derived from a human antibody, while their heavy chain variable region (VH) and light chain variable region (V _L ) are derived from a mouse antibody, and vice versa, antibodies whose heavy chain constant region (CH) and light chain constant region ( _CL ) are derived from a mouse antibody, while their heavy chain variable region (V _H ) and light chain variable region ( V _L ) originate from a human antibody.

Initially, an antibody has a basic structure having four polypeptide chains, consisting in total of two immunoglobulin light chains and two immunoglobulin heavy chains. However, according to the present invention, the term antibody, in addition to an antibody having this basic structure, also refers to:

(1) an antibody consisting of two polypeptide chains, one immunoglobulin light chain and one immunoglobulin heavy chain; a sequence which is in turn linked at its C-terminal side to an immunoglobulin heavy chain, (3) single chain antibodies consisting of an immunoglobulin heavy chain which is linked at its C-terminal side to a linker sequence which, in turn, bound at its C-terminal side to an immunoglobulin light chain, and (4) an antibody consisting of a Fab region, i.e. structure remaining after removal of the Fc region from an antibody having a basic structure as a starting point, and an antibody consisting of a Fab region and all or part of the hinge region (including Fab, F(ab') and F(ab') ₂ ), also included in the term antibody according to the present invention.

The term Fab refers to a molecule consisting of one light chain containing a variable region and a CL region (light chain constant region) and one heavy chain containing a variable region and a CH1 region (region 1 of the heavy chain constant region), which are linked by a disulfide bond between their respective cysteine residues. Although the heavy chain in a Fab may contain part of the hinge region in addition to the variable region and the CH1 region (region 1 of the heavy chain constant region), the hinge region would then lack the cysteine residue that would otherwise be present in the hinge region and would serve to link together two heavy chains of an antibody. In Fab, the light chain and heavy chain are connected by a disulfide bond formed between a cysteine residue located in the light chain constant region ( _CL region) and a cysteine residue located in the heavy chain constant region (CH1 region) or hinge region. The heavy chain that forms a Fab is called a Fab heavy chain. Since it does not have a cysteine residue in the hinge region, which serves to link the two heavy chains of the antibody, Fab consists of one light chain and one heavy chain. The light chain constituting the Fab contains a variable region and a CL region. The heavy chain as a Fab component can either consist of a variable region and a CH1 region, or also a part of the hinge region in addition to the variable region and a _CH1 region. However, in the latter case, the hinge region is chosen so that it does not contain a cysteine residue that can link two heavy chains in order to avoid the formation of a disulfide bond between the two heavy chains in their hinge regions. In F(ab'), the heavy chain, in addition to the variable region and the _CH 1 region, contains all or part of a hinge region containing a cysteine residue that can link two heavy chains. F(ab') ₂ is a molecule consisting of two F(ab') linked together via a disulfide bond formed between cysteine residues present in their respective hinge regions. A heavy chain forming F(ab') or F(ab') ₂ is called a Fab' heavy chain. In addition, the term antibody also includes a polymer, such as a dimer and a trimer, which consists of two or more antibodies connected to each other directly or through a linker. In addition, the term antibody of the present invention, in addition to the above, also includes any molecule that contains a portion of an immunoglobulin molecule and has the property of specific antigen binding. Thus, according to the present invention, the term immunoglobulin light chain includes a molecule that is derived from a parent immunoglobulin light chain and has an amino acid

- 12 042007 the sequence of all or part of its variable region. Also, the term immunoglobulin heavy chain includes a molecule that is derived from a parent immunoglobulin heavy chain and has the amino acid sequence of all or part of its variable region. Therefore, because it has all or part of the amino acid sequence of the variable region, the molecule is included in the term immunoglobulin heavy chain even though it does not have its Fc region, for example.

As used above, the term Fc or Fc region refers to a region containing a fragment consisting of a CH2 region (region 2 of the heavy chain constant region) and a CH3 region (region 3 of the heavy chain constant region) in an antibody molecule.

In addition, according to the present invention, the term antibody also includes:

(5) scFab, scF(ab') and scF(ab') ₂ , which are single chain antibodies obtained by binding the light chain to the heavy chain, which form, respectively, Fab, F(ab') and F(ab ') ₂ mentioned in (4) above, via a linker sequence. Such scFab, scF(ab') and scF(ab') ₂ may be a molecule in which either a light chain is linked at its C-terminal side to a linker sequence which is in turn linked at its C-terminal side to a heavy chain, or the heavy chain is linked at its C-terminal side to a linker sequence, which in turn is linked at its C-terminal side to a light chain. In addition, the term antibody of the present invention also includes scFv, which is a single chain antibody provided by linking a light chain variable region to a heavy chain variable region through a linker sequence therebetween. Such an scFv may be a molecule in which either a light chain variable region is linked at its C-terminal side to a linker sequence which is in turn linked at its C-terminal side to a heavy chain variable region, or a heavy chain variable region is linked to its C-terminal side to a linker sequence, which in turn is linked on its C-terminal side to the light chain variable region.

In addition, in addition to the full-length antibody and the antibodies described in (1) to (5) above, the term antibody in the present specification includes any kind of antigen-binding fragment that lacks a portion of the full-length antibody (antibody fragment), a broader concept that includes items (4) and (5) above.

The term antigen-binding fragment refers to an antibody fragment that retains at least some of its specific binding activity for its antigen. In addition to the antibodies described in (4) and (5) above, examples of binding fragments include Fab, Fab', F(ab')2, variable region (Fv); a single chain antibody (scFv) obtained by linking a heavy chain variable region (VH) and a light chain variable region (V _L ) through an appropriate linker between them; a diabody, which is a dimer of a polypeptide that contains a heavy chain variable region (VH) and a light chain variable region (VL); a mini-antibody, which is a dimer of a molecule in which the scFv heavy chain (H chain) is linked to a portion of the constant region ( _CH 3 ), and other low molecular weight antibodies. However, since it has an antigen-binding ability, the term is not limited to these molecules. Such binding fragments include not only fragments obtained by treating the full-length antibody protein molecule with the appropriate enzyme, but also fragments obtained with the appropriate host cells using the genetically engineered antibody gene.

According to the present invention, the term single chain antibody refers to a protein in which an amino acid sequence containing all or part of the variable region of an immunoglobulin light chain is linked at its C-terminal side to a linker sequence which is in turn linked at its C-terminal side to an amino acid the sequence of all or part of the variable region of the heavy chain of an immunoglobulin, and having the ability to specifically bind a specific antigen. For example, the antibodies described in paragraphs (2), (3) and (5) are included in a single chain antibody. In addition, a protein in which an amino acid sequence containing all or part of the variable region of an immunoglobulin heavy chain is linked at its terminal side to a linker sequence which, in turn, is further linked at its C-terminal side to the amino acid sequence of all or part of the variable light chain region of an immunoglobulin, and which has the ability to specifically bind to a certain antigen, is also included in the term single chain antibody of the present invention. In a single chain antibody in which the immunoglobulin heavy chain is linked at the e C-terminal side and via a linker sequence to the immunoglobulin light chain, the immunoglobulin heavy chain typically does not have an Fc region. The immunoglobulin light chain variable region has three complementarity determining regions (CDRs) that are involved in determining the antigen specificity of an antibody. Also, the immunoglobulin heavy chain variable region also has three CDRs. These CDRs are the main regions that determine the specificity of an antibody for an antigen. Therefore, a single chain antibody preferably contains all three immunoglobulin heavy chain CDRs and all three immunoglobulin light chain CDRs. However, it is also possible to provide a single chain antibody in which one or more of these CDRs are deleted in such

- 13 042007 measure to maintain the antigen-specific affinity of the antibody.

In a single chain antibody, the linker sequence placed between the immunoglobulin light chain and heavy chain is preferably a peptide chain, preferably 2-50, more preferably 8-50, even more preferably 10-30, even more preferably 12-18 or 15-25 , for example 15 or 25 amino acid residues. Although there is no particular restriction on the specific amino acid sequence of such a linker sequence, since an anti-hTfR antibody containing both strands linked in this manner retains affinity for hTfR, it is preferred that it be composed of glycine only or of glycine and serine: for example, the amino acid sequence Gly -Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence Gly-Gly-Gly, amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), amino acid sequence Gly-Gly-Gly-Gly-Gly- Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence that contains 2-10 or 2-5 repeats of any of these amino acid sequences. For example, when linking the amino acid sequence of the entire immunoglobulin heavy chain variable region on its Terminal side and via the linker sequence to the immunoglobulin light chain variable region, the linker sequence is preferably a linker sequence containing 15 amino acids corresponding to three of the amino acid sequence Gly-Gly-Gly-Gly- Ser (SEQ ID NO: 3) connected in series.

According to the present invention, the term human transferrin receptor or hTfR refers to a membrane protein having the amino acid sequence of SEQ ID NO: 1. An anti-hTfR antibody of the present invention, in one of its embodiments, is an antibody that specifically binds to the amino acid sequence of SEQ ID NO: 1, in its part, starting from the cysteine residue at position 8 9 from the N-terminal side to the phenylalanine at the C-terminus (ie, the extracellular region of hTfR), although this is not limited to this embodiment. In addition, according to the present invention, the term monkey transferrin receptor or monkey TfR specifically refers to a membrane protein having the amino acid sequence of SEQ ID NO: 2 derived from a crabeater monkey (Macaca fascicularis). An anti-hTfR antibody of the present invention, in one of its embodiments, is an antibody that also binds to the amino acid sequence of SEQ ID NO: 2, in part from the cysteine residue at position 89 from the N-terminal side to the phenylalanine at the C-terminus ( i.e. the extracellular region of the monkey TfR), although this is not limited to this embodiment.

To produce an anti-hTfR antibody, a general method is known in which a recombinant human transferrin receptor (rhTfR) is prepared using cells that have introduced an expression vector having an inserted hTfR gene, and then animals such as mice are immunized with this rhTfR. By harvesting those cells that produce anti-hTfR antibodies from immunized animals and fusing them with myeloma cells, hybridoma cells capable of producing anti-hTfR antibodies can be obtained.

In addition, cells producing anti-hTfR antibodies can also be obtained by harvesting immunocompetent cells from animals such as mice and immunizing them with rhTfR by in vitro immunization. When performing in vitro immunization, there are no particular restrictions on the animal species from which immunocompetent cells are obtained, although mouse, rat, rabbit, guinea pig, dog, cat, horse, and primates, including humans, are preferred, and mouse, rat, and a human, and even more preferred are a mouse and a human. As mouse immunocompetent cells, for example, spleen cells derived from mouse spleen can be used. As human immunocompetent cells, those derived from human peripheral blood, bone marrow, spleen and the like can be used. By immunizing human immunocompetent cells according to in vitro immunization, a human anti-hTfR antibody can be obtained.

After the immunocompetent cells are immunized according to in vitro immunization, the cells can be fused with myeloma cells to obtain hybridoma cells having the ability to produce antibodies. In addition, it is also possible to isolate mRNA from immunized cells, synthesize cDNA, perform a PCR reaction using cDNA as a template for amplifying a DNA fragment containing a gene encoding an immunoglobulin light chain and a heavy chain, and using them to artificially reconstruct the antibody gene.

The hybridoma cells just obtained above also include those cells that produce antibodies that recognize proteins other than hTfR. In addition, not all hybridoma cells that produce anti-hTfR antibodies necessarily produce anti-hTfR antibodies that show high affinity for hTfR.

Also, engineered antibody genes include those genes that encode antibodies that recognize proteins other than hTfR as antigens. In addition, not all genes encoding anti-hTfR antibodies necessarily have the required properties, such as encoding an anti-hTfR antibody with high affinity for hTfR.

- 14 042007

Therefore, a selection step is needed to select hybridoma cells producing antibodies having desired properties (such as high affinity for hTfR) from the hybridoma cells just obtained above. In addition, in the case where antibody genes are artificially rearranged, a selection step is necessary to select from the antibody genes a gene encoding an antibody having desired properties (such as high affinity for hTfR). For selection of hybridoma cells that produce antibodies having high affinity for hTfR (high affinity antibodies), or for selection of genes encoding high affinity antibodies, the following methods are effective, explained in detail below. In addition, antibodies having high affinity for hTfR are those whose dissociation constant (K _D ) with hTfR, when measured by the method described in Example 7, is preferably no more than 1x10' ⁸ M, more preferably no more than 1x10' ⁹ M, even more preferably not more than 1x10'1° M, and even more preferably not more than 1x10'11 M. For example, antibodies having a dissociation constant from 1x10' ¹³ M to 1x10' ⁹ M or from 1x10' ¹³ M to 1x1°' ¹⁰ M.

For example, to select hybridoma cells that produce high-affinity anti-hTfR antibodies, a method is used in which recombinant hTfR is added to the plate and maintained on it, then hybridoma cell culture supernatant is added, and after removing antibodies not bound to recombinant hTfR from the plate, , measure the amount of antibodies contained on the tablet. According to this method, the higher the affinity for hTfR of the antibodies contained in the hybridoma cell culture supernatant added to the plate, the greater the amount of antibodies contained in the plate becomes. Therefore, by measuring the amount of antibodies contained in the plate, those hybridoma cells corresponding to the plates containing the most antibodies can be selected as cell lines producing anti-hTfR antibodies having a relatively high affinity for hTfR. It is also possible to isolate the gene encoding the high affinity antibody by isolating the mRNA from each cell line thus selected, synthesizing cDNA, and amplifying the DNA fragment containing the gene encoding the anti-hTfR antibody by PCR using the cDNA as a template.

In order to select a gene encoding a high affinity anti-hTfR antibody from the above engineered antibody genes, the engineered antibody genes are inserted into an expression vector once, and then the expression vector is introduced into host cells. Although there are no particular restrictions on cells to be used as host cells, whether they are prokaryotic, eukaryotic, as long as they can express the antibody gene after introduction of an expression vector having an engineered antibody gene inserted, cells derived from mammals such as such as human, mouse, Chinese hamster and the like, and especially preferred are CHO cells derived from Chinese hamster ovary cells or NS/0 cells derived from mouse myeloma. In addition, there are no particular restrictions on the expression vector to be used for inserting the antibody-encoding gene and its expression, and any expression vector can be used as long as it can express the gene when inserted into mammalian cells. The gene inserted into the expression vector is located after the DNA sequence that can regulate the transcription frequency of the gene in mammalian cells (gene expression regulation site). Examples of gene expression regulation sites that can be used according to the present invention include a cytomegalovirus-derived promoter, SV40 early promoter, human elongation factor-1a (EF-1a) promoter, human ubiquitin C promoter.

Mammalian cells having such an introduced expression vector will express artificially reshaped antibodies inserted into the expression vector. In order to select those cells that produce high-affinity anti-hTfR antibodies from the above cells expressing engineered antibodies, a method is used in which recombinant hTfR is added to and maintained on a plate, then the recombinant hTfR is contacted with the culture supernatant cells, and after removing antibodies not associated with recombinant hTfR from the plate, the amount of antibodies contained on the plate is measured. According to this method, the higher the affinity of anti-hTfR antibodies contained in the cell culture supernatant, the greater the amount of antibodies contained on the plate becomes. Therefore, by measuring the amount of antibodies contained on the plate, those cells corresponding to the plate containing the most antibodies can be selected as an anti-hTfR antibody-producing cell line having relatively high affinity anti-hTfR antibodies, and ultimately a gene encoding an anti-hTfR antibody having a high affinity of the anti-hTfR antibody for hTfR. Using the cell line thus selected, PCR can be performed to amplify a DNA fragment containing a gene encoding an anti-hTfR antibody to isolate a gene encoding high affinity antibodies.

Selection of a gene encoding a high affinity anti-hTfR antibody from the above engineered antibody genes can also be performed by incorporating artificial

- 15 042007 vein-engineered antibody genes into an expression vector, introducing the expression vector into E. coli cells, culturing E. coli cells, and selecting E. coli cells having the desired gene in the same manner as in the above selection of hybridoma cells using the supernatant cultures of E. coli cells or an antibody-containing solution obtained by lysis of E. coli cells. The E. coli cells thus selected express a gene encoding an anti-hTfR antibody with relatively high affinity for hTfR. From this cell line, a gene encoding an anti-hTfR antibody having a relatively high affinity of the anti-hTfR antibody for hTfR can be selected. In order to allow antibody secretion into the E. coli culture supernatant, the antibody gene can be inserted into an expression vector such that the secretion signal sequence is attached to the N-terminal side of the gene.

Yet another method for selecting a gene encoding a high affinity anti-hTfR antibody is a method in which an antibody encoded by the above engineered antibody gene is expressed and retained on phage particles. To do this, the antibody gene is reconstructed as a gene encoding a single chain antibody. A method for holding phage particles to hold an antibody on their surface is disclosed in international publications WO 1997/09436 and WO 1995/11317 and the like, and is thus well known. In order to select phages retaining high-affinity anti-hTfR antibodies from phages retaining antibodies encoded by artificially reshaped antibody genes, a method is used in which recombinant hTfR from a plate is added to the plate and maintained therein in contact with phages, and after removal phages not associated with recombinant hTfR, measure the number of phages contained on the plate. According to this method, the higher the affinity for hTfR of the antibodies contained on the phage particles, the greater the number of phages contained on the plate becomes. Therefore, by measuring the number of phages contained in the plate, phage particles corresponding to the plate where the phages were present in a larger quantity can be selected as phage particles producing an anti-hTfR antibody having a relatively high affinity of the anti-hTfR antibody for hTfR, and ultimately a gene encoding an anti-hTfR antibody with high affinity for hTfR can be selected. Using the thus selected phage particles, PCR can be performed to amplify a DNA fragment containing a gene encoding an anti-hTfR antibody and isolate the gene encoding a high affinity antibody.

It is possible to obtain cDNA or phage DNA from the above cells such as hybridoma cells producing high-affinity anti-hTfR antibodies or from the above phage particles holding high-affinity anti-hTfR antibodies, and perform PCR and the like using it as a template for amplification and isolating a DNA fragment containing a gene encoding all or part of an anti-hTfR antibody light chain, an anti-hTfR antibody heavy chain, or a single chain antibody as an anti-hTfR antibody. In the same manner, it is also possible to carry out PCR and the like to amplify and isolate a DNA fragment containing a gene encoding all or part of the light chain variable region of an anti-hTfR antibody, or a DNA fragment containing a gene encoding all or part of the heavy chain variable region of an anti-hTfR antibody. hTfR.

A high affinity anti-hTfR antibody can be obtained by inserting all or part of the gene encoding the light chain and heavy chain of this high affinity anti-hTfR antibody into an expression vector, transforming host cells such as mammalian cells with the expression vector, and culturing the resulting transformed cells. Using the nucleotide sequence of an isolated gene encoding an anti-hTfR antibody, it is also possible to translate the amino acid sequence of an anti-hTfR antibody and artificially synthesize a DNA fragment encoding the same amino acid sequence. By artificially synthesizing a DNA fragment, appropriate codon selection can increase the level of expression of the anti-hTfR antibody in host cells.

In order to introduce a mutation such as substitution, deletion, addition, and the like into the amino acid sequence of the original anti-hTfR antibody, the mutation can be introduced into the gene encoding the anti-hTfR antibody contained in the isolated DNA fragment, if necessary. Although the gene encoding the mutant anti-hTfR antibody has a homology with the parent gene preferably not less than 80%, more preferably not less than 90%, there is no particular limitation on the level of homology. By introducing mutations in the amino acid sequence to modify the number or type of sugar chains associated with the hTfR antibody, the stability of the hTfR antibody in the body can also be improved.

When a mutation is introduced into a gene encoding all or part of the light chain variable region of an anti-hTfR antibody, the gene thus mutated has a homology with the original gene that is preferably not less than 80%, more preferably not less than 90%, although in terms of the level of homology there are no special restrictions. When replacing one or more amino acids of the amino acid sequence of the light chain variable region with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, and even more preferably 1 or 2. When deleting one or more amino acids amino acid sequence of the light chain variable region number to be deleted

- 16 042007 amino acids is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to perform a combined mutation of these amino acid substitutions and deletions. When one or more light chain variable region amino acids are added, they can be added inside or at the N-terminal side or C-terminal side of the light chain variable region amino acid sequence, and the number of added amino acids is preferably 1-10, more preferably 1-5, even more preferably 1-3 and even more preferably 1 or 2. It is also possible to carry out a combined mutation of these additions, substitutions and deletions of amino acids. Thus, the mutated light chain variable region amino acid sequence has homology to the amino acid sequence of the original light chain variable region, which is preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%. In particular, when replacing one or more amino acids of the CDR amino acid sequence with other amino acids, the number of amino acids substituted is preferably 1-5, more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids of the CDR amino acid sequence, the number of amino acids to be deleted is preferably is 1-5, more preferably 1-3, even more preferably 1 or 2. Combined mutation of these amino acid substitutions and deletions can also be performed. When adding one or more amino acids, they can be added inside or on the N-terminal side or C-terminal side of the amino acid sequence, and the number of amino acids added is preferably 1-5, more preferably 1-3, even more preferably 1 or 2. a combined mutation of these additions, substitutions, and deletions of amino acids. The amino acid sequence of the corresponding mutant CDR has homology to the amino acid sequence of the original CDR, which is preferably not less than 80%, more preferably not less than 90%, and even more preferably not less than 95%.

When a mutation is introduced into a gene encoding all or part of the heavy chain variable region of an anti-hTfR antibody, the gene thus mutated has a homology with the original gene that is preferably not less than 80%, more preferably not less than 90%, although there is no particular limitation in regarding the level of homology. When replacing one or more amino acids of the heavy chain variable region amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, and even more preferably 1 or 2. When deleting one or more amino acids amino acid sequence of the heavy chain variable region, the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. Combined mutation of these amino acid substitutions and deletions can also be carried out. When one or more amino acids are added to the heavy chain variable region, they can be added inside or at the N-terminal side or C-terminal side of the amino acid sequence of the heavy chain variable region, and the number of added amino acids is preferably 1-10, more preferably 1-5, even more preferably 1-3 and even more preferably 1 or 2. It is also possible to carry out a combined mutation of these additions, substitutions and deletions of amino acids. The amino acid sequence of the heavy chain variable region thus mutated has homology to the amino acid sequence of the original heavy chain variable region, which is preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%. In particular, when replacing one or more amino acids of the CDR amino acid sequence with other amino acids, the number of amino acids substituted is preferably 1-5, more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids of the CDR amino acid sequence, the number of amino acids to be deleted is preferably is 1-5, more preferably 1-3, even more preferably 1 or 2. Combined mutation of these amino acid substitutions and deletions can also be performed. When adding one or more amino acids, they can be added inside or on the N-terminal side or C-terminal side of the amino acid sequence, and the number of amino acids added is preferably 1-5, more preferably 1-3, even more preferably 1 or 2. a combined mutation of these additions, substitutions, and deletions of amino acids. The amino acid sequence of the corresponding mutant CDR has homology to the amino acid sequence of the original CDR, which is preferably not less than 80%, more preferably not less than 90%, and even more preferably not less than 95%.

A mutation can be introduced into both the light chain and heavy chain variable regions of an anti-hTfR antibody by combining the aforementioned mutation in the light chain variable region of the anti-hTfR antibody and the aforementioned mutation in the heavy chain variable region of the anti-hTfR antibody.

Examples of the above substitution of one or more amino acids in the amino acid sequence of the light chain and the heavy chain of an anti-hTfR antibody include amino acids classified into the same groups, such as aromatic amino acids (Phe, Tip, Tyr), aliphatic

- 17 042007 amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having a hydroxy group (Ser, Thr) . Substitution between such similar amino acids is not expected to result in any change in the phenotype of the protein (i.e., it is a conservative amino acid substitution). However, the amino acid sequence of framework region 3 (SEQ ID NO: 83) of hTfR antibody No. 3 (not published at the time of filing of the present application), previously discovered by the present inventor and confirmed to have the same type of effect as in the present application, has Trp at its position 17. In contrast, the heavy chain framework region 3 of the anti-hTfR antibody No. 3N of the present invention has the amino acid sequence of SEQ ID NO: 68 and is substituted at position 17 on its N-terminal side with Leu. In addition, in the amino acid sequence of CDR1, Thr at position 5 of SEQ ID NO: 12 is replaced by Met as shown in SEQ ID NO: 66. Trp and Leu do not have the analogous relationship mentioned above, and Thr and Met do not have the analogous relationship mentioned above. However, as mentioned below, antibody No. 3N containing a substitution unexpectedly has the same type of action as the action of antibody No. 3 and shows excellent results.

In addition, in the case where a mutation is introduced into the anti-hTfR antibody by adding one or more amino acids to the C-terminus or N-terminus, if the anti-hTfR antibody and the other protein (A) are fused by placing the added amino acids between them, the added amino acids form part linker. A detailed explanation of the linker that is between the anti-hTfR antibody and the protein (A) in the fusion protein of the anti-hTfR antibody and protein (A) will be given below.

Anti-hTfR antibodies obtained by culturing cells selected by the above methods and the like as producing anti-hTfR antibodies which have a relatively high affinity of anti-hTfR antibodies for hTfR, and anti-hTfR antibodies obtained by expressing a gene encoding a high-affinity anti-hTfR antibody , can be modified by introducing mutations such as substitution, deletion, addition into their amino acid sequences to give them the desired properties. Introduction of a mutation into the amino acid sequence of an anti-hTfR antibody can be accomplished by introducing a mutation into a gene corresponding to the amino acid sequence.

The affinity of an anti-hTfR antibody for hTfR can be adjusted, if desired, by introducing mutations such as substitution, deletion, and addition into the amino acid sequence of the antibody's variable region. For example, if an antibody has such a high affinity for its antigen that it results in a dissociation constant in water that is too low, there is a possibility that, once introduced into the body, the antibody will not be able to dissociate from the antigen, thus leading to dysfunction. In such a case, most preferred antibodies suitable for this purpose can be obtained by introducing a mutation into the variable region of the antibody in order to incrementally adjust its dissociation constant, so that 2-5 times, 5-10 times, 10-100 times, and so on increase the dissociation constant of the original antibody. Conversely, the dissociation constant can be adjusted step by step to reduce the dissociation constant of the original antibody by 1/2-1/5-fold, 1/5-1/10-fold, 1/10-1/100-fold, and so on by introducing a mutation.

The introduction of a mutation such as substitution, deletion and addition to the amino acid sequence of an anti-hTfR antibody can be performed by introducing a mutation at certain positions of the nucleotide sequence of the gene, either, for example, by PCR and the like, using the gene encoding the anti-hTfR antibody as a template, or by accidental introduction of a mutation.

The introduction of a mutation in the amino acid sequence of an anti-hTfR antibody to control the affinity of an anti-hTfR antibody can be accomplished, for example, by inserting a gene encoding an anti-hTfR antibody as a single-chain antibody into a phagemid, using this phagemid to obtain a phage with an expressed single-chain antibody on the surface of its capsid, allowing the phage to be multiplied while at the same time introducing a mutation into a gene encoding a single chain antibody by using a mutagen and the like, and selecting from the multiplied phages a phage expressing a single chain antibody having a desired dissociation constant, either by the method described above or by purification using column with antigen under certain conditions.

The antibodies having relatively high affinity for hTfR obtained by the above-mentioned method of selecting cells producing high affinity antibodies are antibodies whose dissociation constant (KD) with hTfR, when measured by the method described in Example 7, is preferably not more than 1 x 10' ⁸ M, more preferably not more than 1 x 10' ⁹ M, even more preferably not more than 1 x 10' ¹⁰ M, and even more preferably not more than 1 x 10 ^-11 M. For example, antibodies having a dissociation constant from 1 x 10' ¹³ M to 1 x 10' ⁹ M or from 1 x 10' ¹³ M to 1 x 10' ¹⁰ M. The same also applies if the antibodies are single chain antibodies. Once antibodies have been obtained, they can be modified as necessary, for example by introducing a mutation to give them the desired property.

Antibodies with affinity for both human and monkey TfRs can be obtained by selecting antibodies with affinity for monkey TfRs from anti-hTfR antibodies that have been

- 18 042007 cheny, as described above. Selection of antibodies with affinity for monkey TfR can be performed, for example, by ELISA using recombinant monkey TfR, which is obtained using recombinant DNA techniques. In such an ELISA, recombinant monkey TfR is added to and contained on a plate and contacted with anti-hTfR antibodies, and after the antibodies not bound to recombinant monkey TfR are removed from the plate, the amount of antibodies contained on the plate is measured. The higher the affinity of the antibodies for the recombinant monkey hTfR, the greater the amount of antibodies contained in the plate. Therefore, antibodies corresponding to the plate containing more antibodies can be selected as monkey TfR-affinity antibodies. Here, the term monkey is preferably classified as non-human monkeys, more preferably marmosets, even more preferably macaques, and, for example, crabeater monkey or rhesus monkey, among which the crabeater monkey is convenient for use in the study.

Antibodies having affinity for both human and monkey hTfRs offer the advantage that they provide pharmacokinetic observations for antibodies administered using the monkey. For example, when developing a drug using such anti-hTfR antibodies according to the present invention, the development progress can be markedly accelerated, since its pharmacokinetic study can be performed using a monkey.

According to the present invention, antibodies having a relatively high affinity for hTfR, as well as having affinity for monkey TfR, show the following dissociation constant for human and monkey TfR, in particular when measured as described in Example 7:

(a) dissociation constant with hTfR: preferably not more than 1x10'1° M, more preferably not more than 2.5x10'11 M, even more preferably not more than 5x1°' ¹² M, and even more preferably not more than 1x10' ¹² M, and (b) a dissociation constant with monkey TfR: preferably not more than 1x10'9 M, more preferably not more than 5x10'1° M, and even more preferably not more than 1x10'1° M, for example, not more than 7.5x10'11 M.

For example, dissociation constants with monkey hTfR and TfR are no more than 1x10-10 M and no more than 1x10'9 M, no more than 1x10'11 M and no more than 5x10' ¹⁰ M, no more than 5x10' ¹² M and not more than 1x10' ¹⁰ M, not more than 5x10' ¹² M and not more than 7.5x10'11 M, not more than 1x10' ¹² M and not more than 1x10-10 M or not more than 1x10' ¹² M and no more than 7.5x10'11 M, respectively. In this context, although there is no particularly clear lower limit for the dissociation constant with human TfR, it may be, for example, 5x10'13 M or 1x10'13 M. Although there is no particularly clear lower limit for the dissociation constant with monkey TfR, it may be, for example, 1x10' ¹¹ M or 1x10' ¹² M. The same also applies if the antibody is a single chain antibody.

If the antibodies having a relatively high affinity for hTfR obtained by the above-mentioned method in which those cells that produce high affinity antibodies are selected are non-human animal antibodies, they can be converted into humanized antibodies.

A humanized antibody is an antibody obtained by using the amino acid sequence of a portion of the variable region (e.g., all or part of the CDRs) of a non-human animal antibody, and replacing the appropriate region of the human antibody with a sequence (which is implanted in a human antibody), while retaining specificity for the antigen. Examples of a humanized antibody include an antibody obtained by replacing three complementarity determining regions (CDRs) in an immunoglobulin light chain and three complementarity determining regions (CDRs) in an immunoglobulin heavy chain, both of which constitute a human antibody, with CDRs from a non-human mammal. Although there are no particular restrictions on the species from which the CDRs to be inserted into the human antibody are obtained, as long as it is a non-human mammal, it is preferably a mouse, a rat, a rabbit, a horse, and a non-human primate, more preferably a mouse and a rat, and still more preferably a mouse. However, it may be an antibody in which part of a human antibody is replaced by part of another human antibody.

Methods for producing a humanized antibody are well known in the art, and most commonly a method in which the amino acid sequence of complementarity determining regions (CDRs) in the variable region of a human antibody is replaced with a CDR of a non-human mammalian antibody, which was developed by Winter et al. (Verhoeyen M. Science. 239. 1534-1536 (1988)). It is also well known that in some cases the corresponding part of the human acceptor antibody is to be replaced not only by the CDR of the non-human mammalian antibody, but also by amino acid sequences located in regions outside the CDR, which play a role either in maintaining the structure of the CDR or in binding to an antigen in order to reproduce the activity originally possessed by the donor antibody (Queen C. Proc. Natl. Acad. Sci. USA. 86. 10029-10033 (1989)). In this case, the areas outside the CDR are called the framework.

- 19 042007 (FR) areas.

The variable regions of both the heavy chain and the light chain of an antibody contain four framework regions 1-4 (FR1-FR4). FR1 is the region adjacent to CDR1 on its N-terminal side and consists of the amino acid sequence from the N-terminus in each heavy chain and light chain peptide to the amino acid adjacent to the N-terminus of its CDR1. FR2 consists of the amino acid sequence between CDR1 and CDR2 in each peptide making up the heavy chain and light chain. FR3 consists of the amino acid sequence between CDR2 and CDR3 in each peptide making up the heavy chain and light chain. FR4 consists of an amino acid sequence from the amino acid near the C-terminus of CDR3 to the C-terminus of the variable region. However, according to the present invention, which is not limited to this, a region excluding 1-5 amino acids of the N-terminal side and/or 1-5 amino acids of the C-terminal side in each FR region mentioned above can be used as a framework region.

Making a humanized antibody involves methods of implanting the CDRs (and, by the way, their adjacent FRs) of a non-human mammal antibody in place of the CDRs (and, if applicable, their adjacent FRs) in the variable region of a human antibody. In such methods, the initial framework region of a human antibody variable region can be obtained from a public database of DNA and the like that contains germline antibody genes. For example, germline DNA sequences as well as human heavy chain and light chain variable region amino acid sequences can be selected from the VBase human germline database (available online at www.mrc-cpe.cam.ac.uk/vbase). In addition, they can be selected from published DNA sequences and amino acid sequences described in the literature, such as Kabat E.A. Sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson I.M. J. Mol. Biol. 227. 776-98 (1992); and CoxJPL. Eur. J Immunol. 24:827-836 (1994).

As mentioned above, in a humanized antibody, the non-human mammalian antibody regions to be implanted in the variable regions of the parent human antibody typically comprise the CDRs or CDRs themselves and their adjacent FR portion. However, such FRs implanted together with CDRs also play a role in either maintaining the structure of the CDR or binding to an antigen, thus having an essential function in determining the complementarity of an antibody, and the term CDR of the present invention therefore refers to areas that upon receipt of a humanized antibody taken or may be taken from the antibody of a non-human mammal and implanted in a humanized antibody. Thus, the region commonly considered to be the FR region according to the present invention is included in the CDR because it takes part either in maintaining the structure of the CDR or in binding to the antigen, and thus it is considered to have an essential function in determining the complementarity of the antigen.

The anti-hTfR antibody of the present invention, when administered to the body, for example by intravenous injection, effectively binds to hTfR located on capillary endothelial cells in the brain. hTfR-bound antibodies enter the brain through the blood-brain barrier via mechanisms such as endocytosis and transcytosis. Therefore, by binding to the anti-hTfR antibody of the present invention, those proteins, small molecule compounds, and the like to be involved in the brain can be effectively delivered to the brain through the blood-brain barrier. In addition, the anti-hTfR antibodies of the present invention, after passing through the blood-brain barrier, can reach the brain parenchyma and nerve-like cells in the hippocampus; cerebellar Purkinje cells and the like, or at least one of them. And they are also expected to reach the nerve-like cells in the striatum of the brain; and nerve-like cells in the substantia nigra of the midbrain. Therefore, one of these proteins, small molecule compounds, and the like, which can affect such tissues or cells, can be made to reach the tissues or cells by binding to an anti-hTfR antibody of the present invention.

The anti-hTfR antibody of the present invention can be an effective tool for ensuring the transfer of these compounds (proteins, small molecule compounds, and the like) from the blood to the brain and functioning there, otherwise these compounds cannot pass through the blood-brain barrier when administered intravenously and, therefore, they cannot or are unlikely to exhibit their physiological or pharmacological functions in the brain. In particular, the anti-hTfR antibodies of the present invention, after passing through the blood-brain barrier, can reach the brain parenchyma and nerve-like cells in the hippocampus; Purkinje cells and the like of the cerebellum or at least one of them. And they are also expected to reach the nerve-like cells in the striatum of the brain; as well as nerve-like cells in the substantia nigra of the midbrain. Therefore, it is possible to make these compounds function or enhance their function in these tissues or cells in the brain by administering these compounds in combination with anti-hTfR antibody molecules, parenterally, on

- 20 042007 example, intravenously.

For linking anti-hTfR antibodies to such compounds (proteins, small molecule compounds, and the like), a method is available for linking them together via a non-peptide linker or a peptide linker. As non-peptide linkers, polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymer, polymerized lipid, chitin and hyaluronic acid or their derivatives or combinations thereof can be used. A peptide linker is a peptide chain of 1-50 amino acids linked by peptide bonds, or a derivative thereof, the N-terminus and C-terminus of which must be covalently linked to either an anti-hTfR antibody or a compound such as a protein, a small molecule, and the like, respectively, for binding an anti-hTfR antibody to such a protein-like compound, a small molecule compound.

Specifically, a conjugate which is formed by linking an anti-hTfR antibody of the present invention to a desired other protein (A) via PEG as a non-peptidic linker is referred to as an anti-hTfR antibody-PEG protein. An anti-hTfR-PEG protein can be prepared by first coupling an anti-hTfR antibody to PEG to form an anti-hTfR-PEG antibody, and then coupling an anti-hTfR-PEG antibody to another protein (A). Alternatively, an anti-hTfR-PEG protein antibody can be prepared by first coupling another protein (A) to PEG to form a PEG protein, and then coupling the PEG protein to an anti-hTfR antibody. In order to bind PEG to an anti-hTfR antibody and another protein (A), a PEG is used that is modified with functional groups such as carbonate, carbonylimidazole, active carboxylic acid ester, azlactone, cyclic imidthione, isocyanate, isothiocyanate, imidate, aldehyde, etc. similar. Such a functional group introduced into PEG reacts primarily with amino groups in the anti-hTfR antibody and other protein (A) to covalently bind the PEG to the anti-hTfR antibody and other protein (A). Although there are no particular restrictions on the molecular weight and configuration used in this case, PEG, its average molecular weight (MW) is as follows: preferably MW=500-60000, more preferably MW=500-20000. For example, such a PEG having an average molecular weight of about 300, about 500, about 1000, about 2000, about 4,000, about 10,000, about 20,000, and the like. PEG is preferably used as a non-peptide linker. An anti-hTfR antibody can be linked to the desired small molecule compound in the same manner as described above.

For example, an anti-hTfR-PEG antibody can be prepared by mixing an anti-hTfR antibody with an aldehyde-functional polyethylene glycol (ALD-PEGALD) such that the molar ratio of ALD-PEG-ALD to the antibody is 11, 12.5, 15, 110, 120 and the like, and then adding a reducing agent such as NaCNBH ₃ to the mixture to allow the reaction to proceed. Then, by reacting the anti-hTfR-PEG antibody with another protein (A) in the presence of a reducing agent such as NaCNBH ₃ , an anti-hTfR-PEG protein antibody is obtained. Conversely, it is also possible to generate an anti-hTfRPEG protein antibody by first linking another protein (A) to an ALD-PEG-ALD to produce a PEG protein, and then linking the PEG protein to an anti-hTfR antibody.

The anti-hTfR antibody and another protein (A) can also be linked together via peptide bonds by linking the heavy chain or light chain of the anti-hTfR antibody at its C-terminal side or N-terminal side, either through a linker sequence or directly to the N-terminus or C -end of another protein (A), respectively. Thus, a fusion protein between an anti-hTfR antibody and another protein (A) can be obtained by inserting into a mammalian expression vector a DNA fragment in which the cDNA encoding the other protein (A) is located directly in the framework or through a DNA fragment encoding a linker sequence, on the 3' or 5' side of the cDNA encoding the heavy chain or light chain of the anti-hTfR antibody, and culturing mammalian cells into which the expression vector mentioned above has been introduced. When linking a DNA fragment encoding a different protein (A) to a heavy chain, a mammalian expression vector in which the cDNA fragment encodes an anti-hTfR antibody light chain is also introduced into the same host cells, whereas if a DNA fragment encoding a different protein (A) is coupled to the light chain, a mammalian expression vector in which the cDNA fragment encodes the heavy chain of an anti-hTfR antibody is also inserted into the same host cells. In the case where the anti-hTfR antibody is a single chain antibody, a combined fusion protein containing the anti-hTfR antibody and another protein (A) can be obtained by insertion into an expression vector (for eukaryotic cells such as mammals and yeast, or for prokaryotic cells such as as E. coli.), a DNA fragment that is formed by linking a cDNA encoding another protein (A) at its 5' end side or at its 3' end side, directly or through a DNA fragment encoding a linker sequence, with a cDNA encoding a single chain anti-hTfR antibody and allowing expression of the fusion protein in those cells into which the expression vector has been introduced.

In a fusion protein of the type in which another protein (A) is linked to an antibody light chain

- 21 042007 against hTfR on its C-terminal side, an antibody against human transferrin receptor contains an amino acid sequence containing all or part of the light chain variable region and an amino acid sequence containing all or part of the heavy chain variable region, and the other protein (A) is linked with the light chain of this anti-human transferrin receptor antibody on its C-terminal side. In this case, the light chain of the anti-hTfR antibody and the other protein (A) can be linked together directly or through a linker.

In a fusion protein of the type in which another protein (A) is linked to the heavy chain of an anti-hTfR antibody at its C-terminal side, the anti-human transferrin receptor antibody comprises an amino acid sequence containing all or part of the light chain variable region, and an amino acid sequence containing all or part of the heavy chain variable region, and the other protein (A) is linked to the heavy chain of this anti-human transferrin receptor antibody at its C-terminal side. In this case, the heavy chain of the anti-hTfR antibody and another protein (A) can be linked together directly or via a linker.

In a fusion protein of the type in which another protein (A) is linked to the light chain of an anti-hTfR antibody at its N-terminal side, the anti-human transferrin receptor antibody comprises an amino acid sequence containing all or part of the light chain variable region, and an amino acid sequence containing all or part of the heavy chain variable region, and the other protein (A) is linked to the light chain of this anti-human transferrin receptor antibody at its N-terminal side. In this case, the light chain of the anti-hTfR antibody and the other protein (A) can be linked together directly or through a linker.

In a fusion protein of the type in which another protein (A) is linked to the heavy chain of an anti-hTfR antibody at its N-terminal side, the anti-human transferrin receptor antibody comprises an amino acid sequence containing all or part of the light chain variable region, and an amino acid sequence containing all or part of the heavy chain variable region, and the other protein (A) is linked to the heavy chain of this anti-human transferrin receptor antibody at its N-terminal side. In this case, the heavy chain of the anti-hTfR antibody and another protein (A) can be linked together directly or via a linker.

In the above, the linker sequence placed between the anti-hTfR antibody and the other protein (A) may be a peptide chain consisting of preferably 1-50, more preferably 1-17, even more preferably 1-10, even more preferably 1 -5 amino acids, and according to the other protein (A) to be linked to the anti-hTfR antibody, the number of amino acids of the linker sequence can be adjusted to 1, 2, 3, 1-17, 1-10, 1040, 20-34 as needed , 23-31, 25-29, 27 etc. Although there is no particular limitation on the amino acid sequence of the linker sequence, since the anti-hTfR antibody linked to it retains affinity for hTfR, and the other protein (A) linked to the linker sequence also exhibits the protein's own physiological activity under physiological conditions, the linker may preferably be composed of glycine and serine. Examples of such linkers include a linker consisting of one amino acid, either glycine or serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence Gly-Gly-Gly-GlySer (SEQ ID NO: 3), amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence that contains 1-10 or 2-5 of any these consecutively linked amino acid sequences. They have sequences of 1-50, 2-17, 2-10, 10-40, 20-34, 23-31, 25-29, or 27 amino acids. For example, linkers containing the amino acid sequence Gly-Ser can preferably be used as linker sequences. In addition, a 27 amino acid linker sequence is preferably used, which consists of the Gly-Ser amino acid sequence followed by five consecutive copies of the Gly-Gly-Gly-Gly-Ser amino acid sequence (SEQ ID NO: 3). Moreover, it is also preferable to use a linker sequence containing 25 amino acids, which consists of consecutively linked five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

In the fusion protein of an anti-hTfR antibody and another protein (A), when the anti-hTfR antibody is a single chain antibody, an amino acid sequence containing all or part of the immunoglobulin light chain variable region and an amino acid sequence containing all or part of the immunoglobulin heavy chain variable region are linked , usually via a linker sequence. Since the affinity of an anti-hTfR antibody for hTfR is conserved, the amino acid sequence derived from the light chain can be linked at its C-terminal side to a linker sequence, which in turn is linked at its C-terminal side to the amino acid sequence derived from the heavy chain. , or conversely, an amino acid sequence derived from a heavy chain can be linked at its C-terminal side to a linker sequence which in turn is linked at its C-terminal side to an amino acid sequence derived from a light chain.

The linker sequence placed between the immunoglobulin light chain and heavy chain is a peptide chain consisting of preferably 2-50, more preferably 8-50, even more preferably 10-30, even more preferably 12-18 or 15-25 , and for example 15 or 25 amino acids. Although there is no particular limitation on the linker sequence, since an anti-hTfR antibody derived from both strands that are linked via a linker retains affinity for hTfR, and the other protein (A) associated with the antibody also exhibits the protein's own physiological activity under physiological conditions. , the linker preferably consists of glycine, or glycine and serine. Examples of such linkers include amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence GlyGly-Gly, amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), amino acid sequence Gly-Gly-Gly -Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence SerGly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence that contains 2-10 or 2-5 of any of these consecutively linked amino acid sequences. A preferred embodiment of such a linker sequence comprises 15 amino acids consisting of three consecutive copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

In the case where the anti-hTfR antibody is a single chain antibody, an example of specific embodiments of the fusion protein between the humanized anti-hTfR antibodies of the present invention and another protein (A) is a fusion protein consisting of another protein (A) that is linked at its C-terminal side and through a first 27 amino acid linker sequence consisting of the Gly-Ser amino acid sequence followed by five copies of the Gly-Gly-Gly-GlySer amino acid sequence (SEQ ID NO: 3) sequentially linked to a single chain antibody. An example of a preferred embodiment of single chain antibodies in this case is an antibody having the amino acid sequence of SEQ ID NO: 60, which consists of the amino acid sequence of the heavy chain variable region of an anti-hTfR antibody No. 3N, shown as SEQ ID NO: 65, which is linked at its C-terminus and by means of a first linker sequence of 15 amino acids consisting of three copies of the Gly-Gly-Gly-Gly-Ser amino acid sequence (SEQ ID NO: 3) linked in series to the light chain variable region of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO : 18, an antibody having the amino acid sequence of SEQ ID NO: 98, which consists of the amino acid sequence of the heavy chain of anti-hTfR Fab antibody No. 3N shown as SEQ ID NO: 61, which is linked at its C-terminus and via a linker sequence consisting of 32 amino acids, consisting of six consecutive copies the amino acid sequence of SEQ ID NO: 3 followed by the amino acid sequence of Gly-Gly, with the light chain variable region of the anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23.

When the anti-hTfR antibody is a single chain antibody, such a fusion protein can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA fragment containing a nucleotide sequence encoding the fusion protein, and then culturing the host cells.

In addition, according to the present invention, when a peptide chain contains a plurality of linker sequences, each of these linker sequences is designated for convenience from the N-terminal side as the first linker sequence, the second linker sequence, and so on.

In the case where the anti-hTfR antibody is a Fab, an example of specific embodiments of a fusion protein between humanized anti-hTfR antibodies and another protein (A) of the present invention is a fusion protein which consists of another protein (A) that is fused at its C-terminal side and through a 27 amino acid linker sequence consisting of Gly-Ser followed by five consecutive copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), with a region having a heavy chain variable region antibodies against hTfR, and C _H 1 region. Although a part of the hinge region can be included in this case in addition to the _CH 1 region, the hinge region does not contain a cysteine residue that would form a disulfide bond between the heavy chains.

In the case where the anti-hTfR antibody is a Fab, an example of a preferred heavy chain is the chain having the amino acid sequence of SEQ ID NO: 61. The amino acid sequence of SEQ ID NO: 61 is the heavy chain Fab of the humanized anti-hTfR antibody No. 3N having the amino acid sequence of SEQ ID NO: 66, and corresponds to the region at positions 1-22 6 from the N-terminal side of the amino acid sequence of SEQ ID NO: 66. In addition, the region at positions 1-118 from the N-terminus of SEQ ID NO: 61 corresponds to the variable region (SEQ ID NO: 65), the region at positions 119-216 corresponds to the CH1 region, and the region at positions 217-226 corresponds to the hinge region.

In the case where the anti-hTfR antibody is a Fab, the Fc region of another IgG can be additionally introduced into the fusion protein. By introducing an Fc region into the fusion protein, the stability of the fusion protein in the body, such as blood, can be improved. Such a fusion protein with an Fc region introduced into it is

- 23 042007 There is, for example, a protein in which a human IgG Fc region is linked directly or via a linker sequence to another protein (A) on its C-terminal side, and the Fab anti-human transferrin receptor heavy chain is linked directly or via a linker sequence. sequences with the human IgG Fc region on its C-terminal side.

The linker sequence between the other protein (A) and the human IgG Fc region preferably consists of 1-50 amino acids. In this context, the number of amino acids is adjusted as necessary to 1-17, 1-10, 10-40, 20-34, 23-31, 25-29, 27, etc. Although there are no particular restrictions on the amino acid sequence of the linker sequence, the linker may preferably be composed of glycine and serine. Examples of such linkers include a single amino acid linker, either glycine or serine, Gly-Ser amino acid sequence, Gly-Gly-Ser amino acid sequence, Gly-Gly-Gly-Gly-Ser amino acid sequence (SEQ ID NO: 3), the amino acid sequence of Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence of Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence that has 1-10 or 2- 5 of any of these consecutively linked amino acid sequences, and includes a sequence of not more than 50 amino acids, or a sequence of 2-17, 210, 10-40, 20-34, 23-31, 25-29, or 25 amino acids. For example, a linker sequence containing 25 amino acids is preferably used, which consists of five consecutive copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3). The same also applies to the linker sequence between the human IgG Fc region and the Fab heavy chain.

In addition, when a human IgG Fc region is introduced, the human IgG Fc region can be linked to either the heavy chain or the light chain of an anti-human transferrin receptor antibody. In addition, other antigen-binding fragments may be antibodies, including F(ab') ₂ , F(ab') and single chain antibody.

There is no particular restriction on the type of IgG human IgG Fc region to be administered, and it can be any of IgG1-IgG5. In addition, the Fc region of the human IgG to be administered may be the entire Fc region or may be part of it. A preferred embodiment of such a human IgG Fc region is an Fc region having the amino acid sequence of SEQ ID NO: 70, which is an entire human IgG1 Fc region. In addition, the amino acid sequence of a heavy chain Fab having a human IgG Fc region added thereto is an amino acid sequence having the amino acid sequence of SEQ ID NO: 71, in which a human IgG Fc region having the amino acid sequence of SEQ ID NO: 70 is linked by a linker sequence containing 25 amino acids, which consists of consecutively linked five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), with the N-terminal side of the amino acid sequence of the Fab heavy chain (SEQ ID NO: 61) humanized antibodies against hTfR 3N.

A fusion protein between an anti-hTfR antibody and another protein (A) can be modified to have an affinity for albumin. Affinity for albumin can be achieved by binding to a fusion protein of a compound, peptide, protein, and the like having an affinity for albumin. The fusion protein, which has been given an affinity for albumin, is at least partially bound to albumin and circulates in the blood. Albumin has the function of stabilizing its associated protein. Therefore, by introducing an affinity for albumin, it is possible to extend the blood half-life of a fusion protein administered to a living body, so that the pharmacological effect of the fusion protein can be enhanced. The introduction of albumin affinity is more effective when the anti-hTfR antibody is an antibody lacking an Fc region involved in antibody stability, such as a Fab.

Introduction of affinity for albumin is also effective when a fusion protein between an anti-hTfR antibody and another protein (A) shows immunogenicity when introduced into a living body. Because the fusion protein is bound to albumin, the presentation of the portion of the fusion protein that exhibits immunogenicity to immune cells is prevented, so that the immunogenicity is reduced.

When introduced into an albumin affinity fusion protein between an anti-hTfR antibody and another protein (A), the region into which the affinity is introduced can be any of the anti-hTfR antibody light chain, the anti-hTfR antibody heavy chain, the other protein (A), and the linker region. , or affinity can be entered in two or more of these areas.

As peptides or proteins having an affinity for albumin, among others, for example, peptides in which the albumin-binding domain of a protein derived from Streptococcus strain G418 (Alm T. Biotechnol J. 5. 605-17 (2010)) having the amino acid sequence of SEQ ID NO: 74 is modified so that it has alkali resistance. To link together a peptide or protein having an affinity for albumin (albumin affinity peptide) and a fusion protein between an anti-hTfR antibody and another protein (A) (anti-hTfR antibody fusion protein (A)), there is a way to link them together by a non-peptide linker; or a peptide linker. Polyethylene glycol, polypropylene glycol, copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides can be used as non-peptide linkers.

- 24 042007 dy, dextran, polyvinyl ether, biodegradable polymer, lipid polymer, chitin and hyaluronic acid or their derivatives, or combinations thereof. A peptide linker is a peptide chain consisting of 1-50 amino acids linked by peptide bonds or derivatives thereof, the N-terminus and the C-terminus of which are covalently linked to either an albumin affinity peptide or a fusion protein, respectively, to link together an albumin affinity peptide and fusion protein.

Specifically, a conjugate that is formed by linking an albumin affinity peptide to a fusion protein between an anti-hTfR antibody and another protein (A) via PEG as a non-peptidic linker is referred to as an albumin affinity peptide-PEG protein (A). The albumin affinity peptide-anti-hTfR protein fusion protein (A) can be prepared by coupling the albumin affinity peptide to PEG to form an albumin affinity peptide-PEG, and then coupling the albumin affinity peptide-PEG to the anti-hTfR-antibody fusion protein. protein (A). Alternatively, an albumin-affinity peptide-anti-hTfR-protein (A) fusion protein can be prepared by coupling the anti-hTfR-protein (A) fusion protein to PEG to form an anti-hTfR-protein (A) fusion protein and then binding the protein fusion of an anti-hTfR protein (A) PEG with an albumin affinity peptide. In order to bind PEG to an albumin affinity peptide and an anti-hTfR protein (A) fusion protein, PEG is used that is modified with functional groups such as carbonate, carbonylimidazole, active carboxylic acid ester, azlactone, cyclic imidthione, isocyanate, isothiocyanate, imidate, aldehyde and the like. Such a functional group introduced into PEG reacts primarily with amino groups in the albumin affinity peptide and anti-hTfR protein fusion protein (A) molecules to covalently bind the PEG to the albumin affinity peptide and anti-hTfR antibody fusion protein (A) . Although there are no particular restrictions on the molecular weight and configuration used in this case, PEG, its average molecular weight (MW) is as follows: preferably MW=500-60000, more preferably MW=500-20000. For example, such a PEG having an average molecular weight of about 300, about 500, about 1000, about 2000, about 4000, about 10,000, about 20,000, and the like. PEG is preferably used as the non-peptide linker.

For example, an albumin affinity peptide-PEG can be prepared by mixing an albumin affinity peptide with an aldehyde group-modified PEG (ALD-PEG-ALD) such that the molar ratio of the modified PEG to the albumin affinity peptide is 11, 12.5, 15 , 110, 120 and the like, and then adding a reducing agent such as NaCNBH ₃ to the mixture to allow the reaction to proceed. Then, by reacting the albumin affinity peptide-PEG with the anti-hTfR antibody fusion protein (A) in the presence of a reducing agent such as NaCNBH ₃ , an albumin affinity peptide-PE6 antibody fusion protein (A) is obtained. . In contrast, it is also possible to obtain an albumin-affinity peptide-PEG anti-hTfR antibody fusion protein (A) by first linking the anti-hTfR antibody protein (A) fusion protein to ALD-PEG-ALD to obtain an anti-hTfR antibody fusion protein (A )PEG and then binding the PEG-fusion protein to an albumin-affinity peptide.

It is also possible to merge the anti-hTfR antibody fusion protein (A) with an albumin affinity peptide. The fusion protein (anti-hTfR protein (A)-albumin affinity peptide fusion protein) can be prepared by inserting into a mammalian expression vector a DNA segment in which the cDNA encoding the albumin affinity peptide is inserted directly into the scaffold or via a DNA segment encoding a linker sequence at the 3' or 5' side of the cDNA encoding the heavy chain (including the fusion protein between the heavy chain and protein (A)) or the light chain (including the fusion protein between the light chain and protein (A)) of the antibody fusion protein against hTfR protein (A), and culturing mammalian cells into which the expression vector has been introduced. When a DNA fragment encoding an albumin affinity peptide is coupled to a heavy chain (or a fusion protein between a heavy chain and a protein (A)), a mammalian expression vector into which the cDNA segment encoding the fusion protein has been inserted is also introduced into the same host cells. between the light chain constituting the hTfR antibody and the protein (A) (or light chain), whereas when the DNA segment encoding the albumin affinity peptide is linked to the light chain (or the fusion protein between the light chain and protein (A)), a mammalian expression vector into which a cDNA segment encoding a fusion protein between the anti-hTfR antibody heavy chain and protein (A) (or heavy chain) has been inserted is also introduced into the same host cells. That is, an albumin affinity peptide can be linked to either the N-terminal or C-terminal side of the heavy chain (including the fusion protein between the heavy chain and the (A) protein) or light chain (including the fusion protein between the light chain and the (A) protein) of the fusion protein the anti-hTfR antibody protein (A), but when protein (A) is coupled to the heavy chain of the anti-hTfR antibody at the N-terminal side, the albumin affinity peptide is preferably coupled at the Terminal side of the anti-hTfR antibody, and it is particularly preferred to couple the albumin affinity peptide at C-terminal side of the heavy chain.

The anti-hTfR antibody fusion protein (A) can be fused to the albumin affinity peptide directly or via a linker sequence. In this case, the linker sequence

- 25 042007 value preferably consists of 1-50 amino acids. In this case, the number of amino acids is adjusted to 1-17, 1-10, 10-40, 20-34, 23-31, 25-29, 27, etc. as necessary. Although there is no limitation on the amino acid sequence of the linker sequence, the linker sequence is preferably composed of glycine and serine. Examples of linkers include linkers having sequences of 1-50, 2-17, 2-10, 10-40, 20-34, 23-31, 25-29, or 25 amino acids, the linker being one of the amino acids: glycine and serine, amino acid sequence: Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence that contains 1-10 or 2-5 of any of these consecutively linked amino acid sequences. For example, a linker sequence containing a total of 15 amino acids may preferably be used, which consists of three consecutively linked copies of the amino acid sequence: Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

The albumin binding affinity of the fusion protein of the anti-hTfR antibody (A) into which the albumin affinity peptide has been introduced is preferably not more than 1 x 10 ^-7 M, more preferably not more than 5 x 10 ^-7 M, even more preferably not more than 1 x 10 ^-8 M, even more preferably not more than 1 x 10 ^-9 M as measured by biolayer interferometry as described in Example 7.

The Fab antibody can be stabilized in blood even by a method other than the introduction of an Fc region or an albumin affinity peptide. For example, a Fab antibody can be stabilized by PEG modification of the Fab antibody itself or by fusion between the Fab antibody and another protein. Such a method is generally performed in the field of protein pharmaceuticals, and pegylated erythropoietin, interferon, etc. entered practical use in the form of pharmaceutical products. The Fab antibody can also be stabilized by introducing a mutation into the Fab antibody. For example, a Fab antibody can be stabilized by replacing the methionine with leucine at position 4 on the N-terminal side of the light chain. However, the method for introducing the mutation is not limited to this, and the mutation can be introduced into the heavy chain. In addition, the method for stabilizing the Fab antibody is not limited to the methods described above, and all well-known methods can be used.

Although there is no particular restriction on the other protein (A) to be linked to the anti-hTfR antibody, this protein, which can exercise its physiological activity in the body, and in particular, such a protein, which must enter the inside of the brain and exercise its function there , but due to its inability to pass through the blood-brain barrier under such conditions, it cannot be expected to function in the brain simply when administered intravenously. Examples of such proteins include lysosomal enzymes such as nerve growth factor (NGF), a-L-iduronidase (IDUA), iduronate-2-sulfatase (IDS), glucocerebrosidase (GBA), β-galactosidase, GM2 activator protein, β-hexosaminidase A , β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, amannosidase (LAMAN), β-mannosidase, galactosylceramidase (GALC), saposin C, arylsulfatase A (ARSA), a-L-fucosidase (FUCA1), aspartylglucosaminidase, a-N-acetylgalactosaminidase, acid sphingomyelinase (ASM), α-galactosidase A, β-glucuronidase (GUSB), heparan-N-sulfatase (SGSH), α-N-acetylglucosaminidase (NAGLU), acetyl-KoA:α-glucosaminide-N-acetyltransferase, N-acetylglucosamine- 6-sulfate-sulfatase, acid ceramidase (AC), amylo-1,6-glucosidase, sialidase, aspartylglucosaminidase, palmitoyl protein thioesterase 1 (PPT-1), tripeptidyl peptidase 1 (TPP-1), hyaluronidase 1, acid a β-glucosidase (GAA), CLN1 and CLN2 and the like.

Nerve growth factor (NGF) coupled to an anti-hTfR antibody can be used as a therapeutic agent for dementia in Alzheimer's disease; a-L-iduronidase (IDUA) coupled to an anti-hTfR antibody as a therapeutic agent for central nervous system disorders in Hurler syndrome or Hurler-Scheie syndrome; iduronate-2-sulfatase (IDS) coupled to an anti-hTfR antibody as a therapeutic agent for central nervous system disorders in Hunter's syndrome; glucocerebrosidase (GBA) as a therapeutic agent for central nervous system disorders in Gaucher's disease; β-galactosidase as a therapeutic agent for disorders of the central nervous system in GM1 gangliosidosis type 1-3; GM2 activator protein as a therapeutic agent for disorders of the central nervous system in 6M2 gangliosidosis, variant AB; β-hexosaminidase A as a therapeutic agent for central nervous system disorders in Sandhoff's disease and Tay-Sachs disease; β-hexosaminidase B as a therapeutic agent for central nervous system disorders in Sandhoff's disease; N-acetylglucosamine-1-phosphotransferase as a therapeutic agent for central nervous system disorders in I-cell disease; a-mannosidase (LAMAN) as a therapeutic agent for central nervous system disorders in a-mannosidosis; β-mannosidase as a therapeutic agent for central nervous system disorders in β-mannosidosis; galactosylceramidase (GALC) as a therapeutic agent for price disorders

- 26 042007 of the trawling nervous system in Krabbe's disease; saposin C as a therapeutic agent for disorders of the central nervous system in a storage disease like Gaucher's disease; arylsulfatase A (ARSA) as a therapeutic agent for central nervous system disorders in metachromatic white matter degeneration (metachromatic leukodystrophy); α-L-fucosidase (FUCA1) as a therapeutic agent for central nervous system disorders in flucosidosis; aspartylglucosaminidase as a therapeutic agent for central nervous system disorders in aspartylglucosaminuria; a-N-acetylgalactosaminidase as a therapeutic agent for central nervous system disorders in Schindler's disease and Kawasaki's disease; acid sphingomyelinase (ASM) as a therapeutic agent for central nervous system disorders in Niemann-Pick disease; α-galactosidase A as a therapeutic agent for central nervous system disorders in Fabry disease; β-glucuronidase (GUSB) as a therapeutic agent for central nervous system disorders in Sly's syndrome; heparan-N-sulfatase (SGSH), a-N-acetylglucosaminidase (NAGLU), acetyl-KoA:αglucosaminide-N-acetyltransferase and N-acetylglucosamine-6-sulfate-sulfatase as therapeutic agents for central nervous system disorders in Sanfilippo syndrome; acid ceramidase (AC) as a therapeutic agent for central nervous system disorders in Farber's disease; amyl-1,6-glucosidase as a therapeutic agent for disorders of the central nervous system in Cori's disease (Forbus-Corey's disease); sialidase as a therapeutic agent for central nervous system disorders in sialidase deficiency; palmitoyl protein thioesterase 1 (PPT-1) as a therapeutic agent for central nervous system disorders in neuronal ceroid lipofuscinosis or Santavuori-Haltia disease; tripeptidyl peptidase 1 (TPP-1) as a therapeutic agent for disorders of the central nervous system in neuronal ceroid lipofuscinosis or Jansky-Bilshovsky disease; hyaluronidase 1 as a therapeutic agent for central nervous system disorders in hyaluronidase deficiency; acid α-glucosidase (GAA) as therapeutics for central nervous system disorders in Pompe disease; CLN1 and CLN2 as therapeutic agents for central nervous system disorders in Batten's disease. In particular, the anti-hTfR antibodies of the present invention, after passing through the blood-brain barrier, reach the brain parenchyma and neuro-like cells of the cerebral hippocampus and Purkinje cells of the cerebellum, and are expected to further reach the nerve-like cells of the striatum of the brain and nerve-like cells of the nigra of the midbrain. Therefore, an anti-hTfR antibody can be fused to proteins whose function is to be exhibited in those tissues or cells to enhance the pharmacological effects of the proteins. However, this does not limit their medical use.

In addition, what is used as a therapeutic agent according to the present invention can be used to prevent the onset of a disease.

In addition, examples of proteins that can exert their pharmacological activity when bound to an anti-hTfR antibody include: lysosomal enzymes, ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin-3, neurotrophin-4/5, neurotrophin-6, neuregulin-1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β , interferon-γ, interleukin 6, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), cytokines, tumor necrosis factor receptor α (TNF-α receptor), PD-1, PD-L1, PD-L2 ligands, β-amyloid degrading enzymes, anti-amyloid β 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-based drugs.

It is possible to use lysosomal enzymes coupled to an anti-hTfR antibody as a therapeutic agent for central nervous system disorders in lysosomal storage diseases; CNTF as a therapeutic agent for amyotrophic lateral sclerosis; GDNF, neurotrophin-3 and neurotrophin-4/5 as therapeutic agents for cerebral ischemia; GDNF as a therapeutic agent for Parkinson's disease; neuregulin-1 as a therapeutic agent for schizophrenia; erythropoietin and darbepoetin as therapeutic agents for cerebral ischemia; bFGF and FGF2 as therapeutic agents for traumatic disorders of the central nervous system; for recovery after surgical operation of the brain and surgical operation of the spinal cord; amyloid-β degrading enzymes, anti-amyloid-β antibodies and anti-BACE antibodies as therapeutic agents for Alzheimer's disease; anti-EGFR antibodies, anti-PD-1 antibodies, anti-PDL1 antibodies, anti-PD-L2 antibodies, anti-HER2 antibodies and anti-CTLA-4 antibodies as therapeutic agents for tumors of the central nervous system, including brain tumor; and anti-TNFaR-hTfR antibody as therapeutic agents for cerebral ischemia and encephalitis.

- 27 042007

Possible candidates for another protein (A) to be fused to an anti-hTfR antibody typically include these therapeutic agents for diseases such as 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; tumors of the central nervous system, including a tumor of the brain; lysosomal storage diseases accompanied by encephalopathy; glycogenosis; muscular dystrophy; cerebral ischemia; encephalitis; prion diseases; traumatic disorders of the central nervous system. In addition, candidates for another protein (A) to be fused with an anti-hTfR antibody can also generally be a therapeutic agent for viral and bacterial diseases of the central nervous system. In addition, candidates for another protein (A) to be fused with an anti-hTfR antibody can also generally be pharmaceutical agents that can be used for recovery from brain surgery or spinal cord surgery.

In addition to the natural-type (wild-type) proteins mentioned above, the other protein (A) to be linked to the anti-hTfR antibody may also be one of their analogs in which one or more amino acids of these natural-type (wild-type) proteins are modified. , for example, are replaced with other amino acids or removed because they fully or partially share the functions of their respective parent proteins. When replacing one or more amino acids with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3. When deleting one or more amino acids, the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3. A combination of such substitution and deletion of amino acids can also be performed to obtain the desired analogues. In addition, amino acid sequences obtained by adding one or more amino acids inside or on the N-terminal side or on the C-terminal side of the amino acid sequence of natural-type (wild-type) proteins or their analogs are also included in the above proteins, insofar as they are completely or partially have the functions of their respective parent proteins. The number of amino acids to be added here is preferably 1-10, more preferably 1-5, even more preferably 1-3. It is also possible to obtain the desired analogues of the original proteins by combining the addition, substitution and deletion of amino acids.

In addition, in the case where a mutation is introduced into another protein (A) by adding one or more amino acids at its C-terminus or N-terminus, if the added amino acids are located between the protein and the anti-hTfR antibody when they are fused, the added amino acids form part of the linker .

Natural type human acidic α-glucosidase (hGAA) is a lysosomal enzyme consisting of an 883 amino acid sequence shown as SEQ ID NO: 55. However, natural type hGAA also includes an 896 amino acid sequence shown as SEQ ID NO: 56, in which 13 additional amino acids are added to the N-terminal side of the amino acid sequence of SEQ ID NO: 55.

An example of specific embodiments of the fusion proteins of the present invention between an anti-hTfR antibody and another protein (A) is of the type in which the heavy chain of the anti-hTfR antibody is linked at its C-terminus and via the amino acid sequence Gly-Ser as a linker sequence with natural hGAA type. Examples of this type of fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23, and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked by peptide bonds at its C-terminal side and through a linker GlySer sequences with hGAA. The protein having the amino acid sequence of SEQ ID NO: 66 is an anti-hTfR type IgG1 antibody, and the protein having the amino acid sequence of SEQ ID NO: 68 is an anti-hTfR type IgG4 antibody. A nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 66 is shown as, for example, SEQ ID NO: 67, and a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 68 is shown as, for example, SEQ ID NO :69.

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

According to the present invention, although the term human GAA or hGAA refers specifically to hGAA having the same amino acid sequence as natural hGAA

- 28 042007 type, it also includes those amino acid sequences that are obtained by introducing a mutation such as substitution, deletion, addition, and the like into hGAA of a natural type, since they have hGAA activity. When replacing one or more amino acids of the hGAA amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1-2. When deleting one or more amino acids of the hGAA amino acid sequence, the number of amino acids to be deleted is 1-10, more preferably 1-5, even more preferably 1-3, and even more preferably 1-2. It is also possible to introduce a combined mutation of such substitution and deletion of amino acids. When one or more amino acids are added to hGAA, they can be added inside or from the N-terminal side or C-terminal side of the hGAA amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3 , even more preferably 1-2. It is also possible to introduce a combined mutation of such addition, substitution and deletion. The amino acid sequence of the mutant hGAA has homology with the amino acid sequence of the original hGAA, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hGAA has the activity of hGAA, in this case, means that hGAA, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hGAA actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural type hGAA actually has. The same also applies if the hGAA fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and an hGAA can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA fragment containing the nucleotide sequence of SEQ ID NO: 59, which encodes the amino acid sequence of SEQ ID NO: 58 , and an expression vector having an inserted DNA fragment containing the nucleotide sequence of SEQ ID NO: 24 that encodes an anti-hTfR antibody light chain having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for Pompe disease, in particular a therapeutic agent for central nervous system disorders in Pompe disease. The fusion protein thus obtained is a fusion protein of a humanized anti-hTfR antibody of the IgG4 type and hGAA.

In addition, in the case where anti-hTfR or hGAA antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hGAA, the added amino acids form part of the linker.

Natural type human I2S is a lysosomal enzyme consisting of a sequence of 525 amino acids shown as SEQ ID NO: 50. against hTfR is fused at its C-terminus and via the Gly-Ser amino acid sequence as a linker sequence to natural type human I2S. Examples of this type of fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain is linked by peptide bonds at its C-terminal side and via a linker consisting of the amino acid sequence Gly-Ser to human I2S with the amino acid sequence of SEQ ID NO: 53. The protein having the amino acid sequence of SEQ ID NO: 53 is a fusion product of the heavy chain of an anti-hTfR type IgG1 antibody and hI2S, and the heavy chain fragment has the amino acid sequence of SEQ ID NO: 66. By substituting this fragment of the heavy chain amino acid sequence of SEQ ID NO: 68, you can also merge the antibody against hTfR type IgG4 with hI2S.

Human I2S (hI2S) has the activity of hydrolyzing sulfate bonds of heparan sulfate and dermatan sulfate related to glycosaminoglycan. Genetic disorders of this enzyme in patients with Hunter's syndrome lead to symptoms such as abnormal development of the skeleton, since the products of partial hydrolysis of heparan sulfate and dermatan sulfate accumulate in the tissues of the liver, spleen, and the like due to impaired metabolism of heparan sulfate and dermatan sulfate. In addition, patients with Hunter syndrome may present with disorders of the central nervous system. hI2S conjugated to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders associated with Hunter's syndrome.

According to the present invention, although the term human I2S or hI2S specifically refers to hI2S having the same amino acid sequence as natural type hI2S, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of natural type hI2S such a mutation, as substitution, deletion, addition and the like, as long as they have I2S activity. When replacing one or more amino acids

- 29 042007 amino acid sequence of hI2S with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1-2. When deleting one or more amino acids of the hI2S amino acid sequence, the number of amino acids to be deleted is 1-10, more preferably 1-5, even more preferably 1-3, and even more preferably 1-2. It is also possible to introduce a combined mutation of such substitution and deletion of amino acids. When one or more amino acids are added to hI2S, they can be added inside or on the N-terminal side or C-terminal side of the hI2S amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3 , even more preferably 1-2. It is also possible to introduce a combined mutation of such addition, substitution and deletion of an amino acid. The amino acid sequence of the mutant hI2S has homology with the amino acid sequence of the original hI2S, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hI2S has I2S activity in this case means that hI2S fused to an anti-hTfR antibody has an activity of at least 3% of the activity that naturally occurring hI2S actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural-type hI2S actually has. The same also applies if the hI2S fused to the anti-hTfR antibody is mutated.

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

In addition, in the case where anti-hTfR antibodies or human I2S are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and human I2S, the added amino acids are part linker.

Natural type human α-L-iduronidase (hIDUA) is a lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 75 or 76. hIDUA shown as SEQ ID NO: 76 refers to the type in which Ala-Pro is added at the N-terminal side of the amino acid sequence of SEQ ID NO: 75.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and through the amino acid sequence Gly-Ser as a linker sequence with natural-type hIDUA. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the Gly-Ser linker sequence. with hIDUA peptide bond. An anti-hTfR IgGl antibody has the amino acid sequence of SE ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Human a-L-iduronidase (hIDUA) is a lysosomal enzyme that hydrolyzes the iduronic acid bond present in the dermatan sulfate or heparan sulfate molecule. Hurler's syndrome, which is also called type I mucopolysaccharidosis, is a disease caused by accumulation of dermatan sulfate and the like in cells due to deficiency of α-L-iduronidase activity in the lysosome. Patients with Hurler syndrome may have an accompanying central nervous system disorder. hIDUA associated with an anti-hTfR antibody can be used as a therapeutic agent for the central nervous system disorders that accompany Hurler's syndrome.

According to the present invention, although the term human IDUA or hIDUA specifically refers to hIDUA having the same amino acid sequence as natural type hIDUA, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of natural type hIDUA such a mutation, as substitution, deletion, addition and the like, as long as they have hIDUA activity. When replacing one or more amino acids of the hIDUA amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more

- 30 042007 preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids of the hIDUA amino acid sequence, the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such a substitution and deletion of amino acids. When one or more amino acids are added to hIDUA, they can be added inside or on the N-terminal side or C-terminal side of the hIDUA amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3, still more preferably 1 or 2. It is also possible to introduce a combined mutation of such amino acid addition, substitution and deletion. The amino acid sequence of the mutant hIDUA has homology to the amino acid sequence of the original hIDUA, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hIDUA has the activity of hIDUA in this case means that hIDUA fused with an anti-hTfR antibody has an activity of not less than 3% of the activity that natural-type hIDUA actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural-type hIDUA actually has. The same also applies if the hIDUA fused to the anti-hTfR antibody is mutated.

A fusion protein between an hTfR antibody and hIDUA can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 90, and an expression vector having an inserted DNA segment, encoding the light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for Hurler's syndrome, in particular a therapeutic agent for disorders of the central nervous system accompanying Hurler's syndrome. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hIDUA.

Furthermore, in the case where anti-hTfR or hIDUA antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hIDUA, the added amino acids form part of the linker.

Natural type human palmitoyl protein thioesterase 1 (hPPT-1) is a lysosomal enzyme type consisting of the amino acid sequence of SEQ ID NO: 77.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and through the amino acid sequence Gly-Ser as a linker sequence with natural hPPT-1 type. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the linker sequence Gly-Ser with hPPT-1 peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Neuronal ceroid lipofuscinosis (including Santavuori-Haltia disease) is a disease caused by the accumulation of waxy lipofuscin in cells due to a deficiency in palmitoyl protein thioesterase 1 lysosome activity. Patients with neuronal ceroid lipofuscinosis may have an accompanying disorder of the central nervous system. hPPT-1 coupled to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders accompanying neuronal ceroid lipofuscinosis.

According to the present invention, while the term human PPT-1 or hPPT-1 specifically refers to hPPT-1 having the same amino acid sequence as natural-type hPPT-1, it also includes those amino acid sequences that are obtained by insertion into the amino acid sequence of hPPT-1 natural type mutation such as substitution, deletion, addition and the like, as long as they have hPPT-1 activity. When replacing one or more amino acids of the hPPT-1 amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids, the amino acid hPPT-1 sequence, the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such an amino acid substitution and deletion. When one or more amino acids are added to hPPT-1, they can be added orally or per N

- 31 042007 terminal side or C-terminal side of the amino acid sequence of hPPT-1, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. You can also enter a combined mutation of such addition, substitution and deletion of an amino acid. The amino acid sequence of the mutant hPPT-1 has homology with the amino acid sequence of the original hPPT-1, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hPPT-1 has the activity of hPPT-1, in this case, means that hPPT-1, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hPPT-1 actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural type hPPT-1 actually has. The same also applies if the hPPT-1 fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and hPPT-1 can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 100 and an expression vector having an inserted segment DNA encoding the light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for neuronal ceroid lipofuscinosis, in particular a therapeutic agent for disorders of the central nervous system accompanying neuronal ceroid lipofuscinosis. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hPPT-1.

In addition, in the case where anti-hTfR or hPPT-1 antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hPPT-1, the added amino acids form part of the linker.

Natural type human acid sphingomyelinase (haSM) is a type of lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 78.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and through the amino acid sequence Gly-Ser as a linker sequence with natural-type haSM. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the linker sequence Gly-Ser with a haSM peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Human acid sphingomyelinase (haSM) is a lysosomal enzyme that hydrolyses sphingomyelin to choline phosphate and ceramide. Niemann-Pick disease is classified into AF types according to the difference in cause and condition of the disease. Among them, types A and B are caused by a genetically determined deficiency of acid sphingomyelinase (ASM). Niemann-Pick disease is a disease caused by the accumulation of sphingomyelin in cells due to a deficiency in acid sphingomyelinase activity in the lysosome. Patients with Niemann-Pick disease may have an accompanying disorder of the central nervous system. haSM associated with an anti-hTfR antibody can be used as a therapeutic agent for disorders of the central nervous system accompanying Niemann-Pick disease.

According to the present invention, although the term human ASM or haSM specifically refers to a haSM having the same amino acid sequence as a natural type haSM, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of a natural type haSM such a mutation, as substitution, deletion, addition and the like, as long as they have haSM activity. When replacing one or more amino acids of the haSM amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids of the haSM amino acid sequence the number of amino acids to be deleted is 110, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such an amino acid substitution and deletion. When one or more amino acids are added to haSM, they can be added inside or at the N-terminal side or C-terminal side of the haSM amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more

- 32 042007 preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such addition, substitution and deletion of an amino acid. The amino acid sequence of the mutant haSM has homology to the amino acid sequence of the original haSM, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that haSM has the activity of haSM, in this case means that haSM, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type haSM actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that naturally occurring haSM actually has. The same also applies if the haSM fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and a haSM can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 106, and an expression vector having an inserted DNA segment, encoding a light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for Niemann-Pick disease, in particular a therapeutic agent for disorders of the central nervous system accompanying Niemann-Pick disease. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and haSM.

Furthermore, in the case where anti-hTfR or haSM antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and haSM, the added amino acids form part of the linker.

Natural type human arylsulfatase A (hARSA) refers to a type of lysosomal enzyme consisting of the amino acid sequence of SEQ ID NO: 79.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and through the amino acid sequence Gly-Ser as a linker sequence with natural type hARSA. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the Gly-Ser linker sequence. with hARSA peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Metachromatic white matter degeneration (metachromatic leukodystrophy) is a disease caused by accumulation of sulfatide and the like in cells due to deficiency of arylsulfatase A activity in the lysosome. Patients with metachromatic white matter degeneration may have an accompanying central nervous system disorder. hARSA coupled to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders accompanying metachromatic white matter degeneration.

According to the present invention, although the term human ARSA or hARSA specifically refers to hARSA having the same amino acid sequence as natural type hARSA, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of natural type hARSA such a mutation, as substitution, deletion, addition and the like as long as they have hARSA activity. When replacing one or more amino acids of the hARSA amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids, the hARSA amino acid sequence the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such an amino acid substitution and deletion. When adding one or more amino acids to hARSA, they can be added inside or on the N-terminal side or C-terminal side of the hARSA amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3 , even more preferably 1 or 2. It is also possible to introduce a combined mutation of such addition, substitution and deletion of an amino acid. The amino acid sequence of the mutant hARSA has homology with the amino acid sequence of the original hARSA, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hARSA has the activity of hARSA, in this case means that hARSA,

- 33 042007 fused with an anti-hTfR antibody has an activity not less than 3% of the activity that natural-type hARSA actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural-type hARSA actually has. The same also applies if the hARSA fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and hARSA can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 115, and an expression vector having an inserted DNA segment, encoding the light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for metachromatic white matter degeneration, in particular a therapeutic agent for disorders of the central nervous system accompanying metachromatic white matter degeneration. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hARSA.

Furthermore, in the case where anti-hTfR or hARSA antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hARSA, the added amino acids form part of the linker.

Natural type human heparan-I-sulfatase (hSGSH) is a type of lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 80.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and via the amino acid sequence Gly-Ser as a linker sequence with natural type hSGSH. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the linker sequence Gly-Ser with hSGSH peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Sanfilippo syndrome, also called mucopolysaccharidosis type II (MPS II type I), is a disease caused by the accumulation of heparan sulfate in cells due to a deficiency of heparan-N-sulfatase activity in the lysosome. It should be noted that Sanfilippo syndrome can be caused by a deficiency of other enzymes, such as a-N-acetylglucosaminidase. Patients with Sanfilippo syndrome may have an accompanying central nervous system disorder. hSGSH coupled to an anti-hTfR antibody can be used as a therapeutic agent for the central nervous system disorders that accompany Sanfilippo syndrome.

According to the present invention, although the term human SGSH or hSGSH specifically refers to hSGSH having the same amino acid sequence as natural type hSGSH, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of natural type hSGSH such a mutation, as substitution, deletion, addition and the like as long as they have hSGSH activity. When replacing one or more amino acids of the hSGSH amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids of the hSGSH amino acid sequence the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such an amino acid substitution and deletion. When one or more amino acids are added to hSGSH, they can be added inside or on the N-terminal side or C-terminal side of the hSGSH amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3, still more preferably 1 or 2. It is also possible to introduce a combined mutation of such amino acid addition, substitution and deletion. The amino acid sequence of the mutant hSGSH has homology with the amino acid sequence of the original hSGSH, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hSGSH has the activity of hSGSH, in this case, means that hSGSH, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hSGSH actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity actually possessed by hSGSH nat.

- 34 042007 standard type. The same also applies if the hSGSH fused to the anti-hTfR antibody is mutated.

A fusion protein between an hTfR antibody and hSGSH can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 124, and an expression vector having an inserted DNA segment, encoding a light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for Sanfilippo syndrome, in particular a therapeutic agent for central nervous system disorders accompanying Sanfilippo syndrome. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hSGSH.

Furthermore, in the case where anti-hTfR or hSGSH antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hSGSH, the added amino acids form part of the linker.

Natural type human glucocerebrosidase (hGBA) is a type of lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 81.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and via the Gly-Ser amino acid sequence as a linker sequence with natural-type hGBA. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the Gly-Ser linker sequence. with hGBA peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Human glucocerebrosidase (hGBA) is a lysosomal enzyme that hydrolyses the glycolipid glucocerebroside (glucosylceramide). Gaucher's disease is a disease caused by the accumulation of glucocerebroside in cells due to a deficiency of glucocerebrosidase activity in the lysosome. Patients with Gaucher disease may have an accompanying disorder of the central nervous system. hGBA bound to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders accompanying Gaucher's disease.

According to the present invention, although the term human GBA or hGBA specifically refers to hGBA having the same amino acid sequence as natural type hGBA, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of natural type hGBA such a mutation, as substitution, deletion, addition and the like, as long as they have hGBA activity. When replacing one or more amino acids of the hGBA amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids of the hGBA amino acid sequence the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such substitution and deletion of amino acids. When one or more amino acids are added to hGBA, they can be added inside or at the N-terminal side or C-terminal side of the hGBA amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3, still more preferably 1 or 2. It is also possible to introduce a combined mutation of such amino acid addition, substitution and deletion. The amino acid sequence of the mutant hGBA has homology with the amino acid sequence of the original hGBA, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hGBA has the activity of hGBA in this case means that hGBA, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hGBA actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural type hGBA actually has. The same also applies if the hGBA fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and an hGBA can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having

- 35 042007 containing an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 130 and an expression vector having an inserted DNA segment encoding an anti-hTfR antibody light chain having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for Gaucher's disease, in particular a therapeutic agent for disorders of the central nervous system accompanying Gaucher's disease. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hGBA.

In addition, in the case where anti-hTfR or hGBA antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hGBA, the added amino acids form part of the linker.

Natural type human tripeptidyl peptidase-1 (hTPP-1) is a type of lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 82.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and through the amino acid sequence Gly-Ser as a linker sequence with natural hTPP-1 type. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the linker sequence Gly-Ser with hTPP-1 peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SE ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Neuronal ceroid lipofuscinosis (including Santavuori-Haltia's disease) is a disease caused by accumulation of lipofuscin in cells due to deficiency of tripeptidyl peptidase 1 activity in the liposome. Patients with neuronal ceroid lipofuscinosis may have an accompanying disorder of the central nervous system. hTPP-1 coupled to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders accompanying neuronal ceroid lipofuscinosis.

According to the present invention, while the term human TPP-1 or hTPP-1 specifically refers to hTPP-1 having the same amino acid sequence as natural type hTPP-1, it also includes those amino acid sequences that are obtained by insertion into the amino acid sequence of hTPP-1 natural type of mutation such as substitution, deletion, addition and the like, as long as they have hTPP-1 activity. When replacing one or more amino acids of the hTPP-1 amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids, the amino acid hTPP-1 sequence, the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such a substitution and amino acid deletion. When one or more amino acids are added to hTPP-1, they can be added inside or on the N-terminal side or C-terminal side of the hTPP-1 amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1 -3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such amino acid addition, substitution and deletion. The amino acid sequence of the mutant hTPP-1 has homology with the amino acid sequence of the original hTPP-1, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hTPP-1 has the activity of hTPP-1, in this case means that hTPP-1, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hTPP-1 actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural type hTPP-1 actually has. The same also applies if the hTPP-1 fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and hTPP-1 can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 136 and an expression vector having an inserted segment DNA encoding the light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for neuronal ceroid lipofuscinosis, in particular a therapeutic agent for disorders of the central nervous system accompanying neuronal ceroid lipofuscinosis.

- 36 042007 nos. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hTPP-1.

In addition, in the case where anti-hTfR or hTPP-1 antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hTPP-1, the added amino acids form part of the linker.

Natural type human a-N-acetylglucosaminidase (hNAGLU) is a type of lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 83.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and via the Gly-Ser amino acid sequence as a linker sequence with natural-type hNAGLU. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the linker sequence Gly-Ser with hNAGLU peptide bond.

An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Sanfilippo syndrome is a disease caused by the accumulation of heparan sulfate in cells due to a deficiency in the activity of a-N-acetylglucosaminidase in the lysosome. Patients with Sanfilippo syndrome may have an accompanying central nervous system disorder. hNAGLU coupled to an anti-hTfR antibody can be used as a therapeutic agent for the central nervous system disorders that accompany Sanfilippo syndrome.

According to the present invention, although the term human NAGLU or hNAGLU specifically refers to an hNAGLU having the same amino acid sequence as a natural type hNAGLU, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of a natural type hNAGLU such a mutation, as substitution, deletion, addition and the like as long as they have hNAGLU activity. When replacing one or more amino acids of the hNAGLU amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids, the hNAGLU amino acid sequence the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such an amino acid substitution and deletion. When one or more amino acids are added to hNAGLU, they can be added inside or at the N-terminal side or C-terminal side of the hNAGLU amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3 , even more preferably 1 or 2. It is also possible to introduce a combined mutation of such addition, substitution and deletion of an amino acid. The amino acid sequence of the mutant hNAGLU has homology with the amino acid sequence of the original hNAGLU, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hNAGLU has the activity of hNAGLU, in this case, means that hNAGLU, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hNAGLU actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural-type hNAGLU actually has. The same also applies if the hNAGLU fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and hNAGLU can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 142, and an expression vector having an inserted DNA segment, encoding the light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for Sanfilippo syndrome, in particular a therapeutic agent for central nervous system disorders accompanying Sanfilippo syndrome. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hNAGLU.

In addition, in the case where anti-hTfR or hNAGLU antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hNAGLU, the added amino acids are

- 37 042007 part of the linker.

Natural type human β-glucuronidase (hGUSB) is a type of lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 84.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and via the Gly-Ser amino acid sequence as a linker sequence with natural type hGUSB. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the linker sequence Gly-Ser with hGUSB peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Sly's syndrome, also called mucopolysaccharidosis type VII (MPS type VII), is a disease caused by the accumulation of mucopolysaccharides in cells due to a deficiency of β-glucuronidase activity in the lysosome. Patients with Sly's syndrome may have an accompanying disorder of the central nervous system. hGUSB coupled to an anti-hTfR antibody can be used as a therapeutic agent for the central nervous system disorders that accompany Sly's syndrome.

According to the present invention, although the term human GUSB or hGUSB specifically refers to hGUSB having the same amino acid sequence as natural type hGUSB, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of natural type hGUSB such a mutation, as substitution, deletion, addition, and the like, as long as they have hGUSB activity. When replacing one or more amino acids of the hGUSB amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids of the hGUSB amino acid sequence the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such an amino acid substitution and deletion. When one or more amino acids are added to hGUSB, they can be added inside or at the N-terminal side or C-terminal side of the hGUSB amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3 , even more preferably 1 or 2. It is also possible to introduce a combined mutation of such addition, substitution and deletion of an amino acid. The amino acid sequence of the mutant hGUSB has a homology with the amino acid sequence of the original hGUSB, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hGUSB has the activity of hGUSB, in this case, means that hGUSB, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hGUSB actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural type hGUSB actually has. The same also applies if the hGUSB fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and hGUSB can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 150, and an expression vector having an inserted DNA segment, encoding a light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hGUSB. The fusion protein thus obtained can be used as a therapeutic agent for Sly's syndrome, in particular a therapeutic agent for disorders of the central nervous system accompanying Sly's syndrome.

Furthermore, in the case where anti-hTfR or hGUSB antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hGUSB, the added amino acids form part of the linker.

Natural type human galactosylceramidase (hGALC) is a type of lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 85.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused onto

- 38 042007 its C-terminus and through the Gly-Ser amino acid sequence as a linker sequence with natural type hGALC. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the Gly-Ser linker sequence. with hGALC peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Krabbe's disease is a disease caused by a deficiency in galactosylceramidase activity. Patients with Krabbe disease may have an accompanying disorder of the central nervous system. hGALC coupled to an anti-hTfR antibody can be used as a therapeutic agent for CNS disorders accompanying Krabbe's disease.

According to the present invention, although the term human GALC or hGALC specifically refers to hGALC having the same amino acid sequence as natural type hGALC, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of natural type hGALC such a mutation, as substitution, deletion, addition and the like, as long as they have hGALC activity. When replacing one or more amino acids of the hGALC amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids of the hGALC amino acid sequence the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such an amino acid substitution and deletion. When one or more amino acids are added to hGALC, they can be added inside or at the N-terminal side or C-terminal side of the hGALC amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3 , even more preferably 1 or 2. It is also possible to introduce a combined mutation of such addition, substitution and deletion of an amino acid. The amino acid sequence of the mutant hGALC has homology with the amino acid sequence of the original hGALC, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hGALC has the activity of hGALC, in this case, means that hGALC, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hGALC actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural type hGALC actually has. The same also applies if the hGALC fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and an hGALC can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 158, and an expression vector having an inserted DNA segment, encoding a light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for Krabbe's disease, in particular a therapeutic agent for disorders of the central nervous system accompanying Krabbe's disease. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hGALC.

In addition, in the case where anti-hTfR or hGALC antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hGALC, the added amino acids form part of the linker.

Natural type human acid ceramidase (hAC) is a type of lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 86.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and through the amino acid sequence Gly-Ser as a linker sequence with natural-type hAC. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the Gly-Ser linker sequence. with hAC peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Farber's disease is a disease caused by the accumulation of ceramide in cells

- 39 042007 due to deficiency of acid ceramidase activity in the lysosome. Patients with Farber's disease may have an accompanying disorder of the central nervous system. hAC associated with an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders accompanying Farber's disease.

According to the present invention, although the term human AS or hAC refers specifically to an hAC having the same amino acid sequence as a natural type hAC, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of a natural type hAC such a mutation, as substitution, deletion, addition and the like as long as they have hAC activity. When replacing one or more amino acids of the hAC amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids, the hAC amino acid sequence the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such an amino acid substitution and deletion. When one or more amino acids are added to hAC, they can be added inside or at the N-terminal side or C-terminal side of the hAC amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3 , even more preferably 1 or 2. It is also possible to introduce a combined mutation of such addition, substitution and deletion of an amino acid. The amino acid sequence of the mutant hAC has homology with the amino acid sequence of the original hAC, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hAC has the activity of hAC, in this case, means that hAC, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hAC actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural-type hAC actually has. The same also applies if the hAC fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and an hAC can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 166 and an expression vector having an inserted DNA segment, encoding a light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for Farber's disease, in particular a therapeutic agent for disorders of the central nervous system accompanying Farber's disease. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hAC.

Furthermore, in the case where anti-hTfR or hAC antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hAC, the added amino acids form part of the linker.

Natural type human α-L-fucosidase (hFUCA1) is a type of lysosomal enzyme composed of the amino acid sequence of SEQ ID NO: 87.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and via the Gly-Ser amino acid sequence as a linker sequence with natural type hFUCA1. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the Gly-Ser linker sequence. with hFUCA1 peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Flucosidosis is a disease caused by the accumulation of fucose-containing oligosaccharides in cells due to a deficiency in α-L-fucosidase activity in the lysosome. Patients with flucosidosis may have an accompanying disorder of the central nervous system. hFUCA1 associated with an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders accompanying flucosidosis.

According to the present invention, although the term human FUCA1 or hFUCA1 refers specifically to hFUCA1 having the same amino acid sequence as natural-type hFUCA1, it also includes those amino acid sequences obtained by

- 40 042007 introducing a mutation such as substitution, deletion, addition and the like into the amino acid sequence of hFUCAl of the natural type, since they have hFUCA1 activity. When replacing one or more amino acids of the hFUCA1 amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one or more amino acids, the hFUCA1 amino acid sequence the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such an amino acid substitution and deletion. When one or more amino acids are added to hFUCA1, they can be added inside or at the N-terminal side or C-terminal side of the hFUCA1 amino acid sequence, and the number of amino acids to be added is preferably 110, more preferably 1-5, even more preferably 1-3, still more preferably 1 or 2. It is also possible to introduce a combined mutation of such amino acid addition, substitution and deletion. The amino acid sequence of the mutant hFUCA1 has homology with the amino acid sequence of the original hFUCA1, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hFUCA1 has the activity of hFUCA1, in this case means that hFUCA1, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hFUCA1 actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that naturally occurring hFUCA1 type actually has. The same also applies if the hFUCA1 fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and hFUCA1 can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 174, and an expression vector having an inserted DNA segment, encoding the light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for flucosidosis, in particular a therapeutic agent for disorders of the central nervous system accompanying flucosidosis. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hFUCA1.

In addition, in the case where anti-hTfR or hFUCA1 antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hFUCA1, the added amino acids form part of the linker.

Natural type human α-mannosidase (hLAMAN) is a type of lysosomal enzyme consisting of the amino acid sequence of SEQ ID NO: 88.

A specific example of a fusion protein between an anti-hTfR antibody and another protein (A) according to the present invention is of the type in which the heavy chain of an anti-hTfR antibody is fused at its C-terminus and via the Gly-Ser amino acid sequence as a linker sequence with natural-type hLAMAN. Examples of such fusion proteins include a protein whose light chain consists of the amino acid sequence of SEQ ID NO: 23 and whose heavy chain consists of the amino acid sequence of SEQ ID NO: 66 or 68, and is linked at the C-terminal side and via the linker sequence Gly-Ser with hLAMAN peptide bond. An anti-hTfR IgG1 antibody has the amino acid sequence of SEQ ID NO: 66, and an anti-hTfR IgG4 antibody has the amino acid sequence of SEQ ID NO: 68.

Human α-mannosidase (hLAMAN) is a lysosomal enzyme that hydrolyses α-type mannose. α-mannosidosis is a disease caused by the accumulation of mannose-containing oligosaccharides in cells due to a deficiency of α-mannosidase activity in the lysosome. Patients with α-mannosidosis may have an accompanying central nervous system disorder. hLAMAN linked to an anti-hTfR antibody can be used as a therapeutic agent for central nervous system disorders accompanying α-mannosidosis.

According to the present invention, although the term human LAMAN or hLAMAN specifically refers to hLAMAN having the same amino acid sequence as natural type hLAMAN, it also includes those amino acid sequences that are obtained by introducing into the amino acid sequence of natural type hLAMAN such a mutation, as substitution, deletion, addition and the like as long as they have hLAMAN activity. When replacing one or more amino acids of the hLAMAN amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. When deleting one

- 41 042007 or more amino acids of the hLAMAN amino acid sequence, the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, even more preferably 1 or 2. It is also possible to introduce a combined mutation of such a substitution and amino acid deletion. When one or more amino acids are added to hLAMAN, they can be added inside or at the N-terminal side or C-terminal side of the hLAMAN amino acid sequence, and the number of amino acids to be added is preferably 1-10, more preferably 1-5, even more preferably 1-3 , even more preferably 1 or 2. It is also possible to introduce a combined mutation of such addition, substitution and deletion of an amino acid. The amino acid sequence of the mutant hLAMAN has homology with the amino acid sequence of the original hLAMAN, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

The statement that hLAMAN has the activity of hLAMAN, in this case means that hLAMAN, fused with an anti-hTfR antibody, has an activity of not less than 3% of the activity that natural-type hLAMAN actually has. However, the activity is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, even more preferably not less than 80% of the activity that natural type hLAMAN actually has. The same also applies if the hLAMAN fused to the anti-hTfR antibody is mutated.

A fusion protein between an anti-hTfR antibody and hLAMAN can be obtained, for example, by transforming host cells, such as mammalian cells, with an expression vector having an inserted DNA segment encoding the amino acid sequence of SEQ ID NO: 182, and an expression vector having an inserted DNA segment, encoding a light chain of an anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 23, and then culturing the host cells. The fusion protein thus obtained can be used as a therapeutic agent for α-mannosidosis, in particular a therapeutic agent for disorders of the central nervous system accompanying α-mannosidosis. The fusion protein thus obtained is a fusion protein between humanized antibodies against hTfR type IgG4 and hLAMAN.

In addition, in the case where anti-hTfR or hLAMAN antibodies are mutated by adding one or more amino acids thereto at their C-terminus or N-terminus, if the added amino acids are located between the anti-hTfR antibody and hLAMAN, the added amino acids form part of the linker.

As examples of fusion proteins between an anti-hTfR antibody and another protein (A), fusion proteins having as another protein (A) hGAA, hI2S, hIDUA, hPPT-1, haSM, hARSA, hSGSH, hGBA, hTTP-1, hNAGLU , hGUSB, hAC, hFUcal, and hLAMAN, respectively, are described above, but there is no particular restriction on the amino acid sequence of the heavy chain and light chain CDRs of an anti-hTfR antibody in preferred embodiments of the fusion proteins between the anti-hTfR antibody and such other protein (A), since the antibody has a specific affinity for hTfR.

However, the anti-TfR antibodies used in the present specification are those having the following dissociation constant, specifically as measured by the method described in Example 7: human TfR dissociation constant: preferably not more than 1x10 ^-10 M, more preferably not more than 1x10 ^-11 M, even more preferably not more than 5x10' ¹² M, even more preferably not more than 1x10 ^-12 M; and dissociation constant with monkey TfR: preferably not more than 1 x 10 ^-9 M, more preferably not more than 5 x 10 ^-10 M, even more preferably not more than 1 x 10 ^-10 M, for example, not more than 7.5 x 10 ^-11 M.

For example, dissociation constants with human TfR and TfR. monkeys are not more than 1x10 ^-10 M and not more than 1x 10 ^-9 M, not more than 1x10 ^-11 M and not more than 5x10 ^-10 M, not more than 5x10 ^-12 M and not more than 1x10 ^-10 M , no more than 5x10 ^-12 M and no more than 7.5x10 ^-11 M, no more than 1x10 ^-12 M and no more than 1x10 ^-10 M or no more than 1x10 ^-12 M and no more than 7.5x10 ^-11 M, respectively. In this context, although there is no particularly clear lower limit for the dissociation constant with human TfR, it may be, for example, 5x10 ^-13 M or 1x10 ^-13 M. Although there is no particularly clear lower limit for the dissociation constant with monkey TfR, it may be, for example, 1x10 ^-11 M or 1x10 ^-12 M. The same also applies if the antibody is a single chain antibody.

It is also possible to link a relatively short peptide chain to an anti-hTfR antibody in the same manner as linking another protein (A) to an anti-hTfR antibody. There are no particular restrictions on the peptide chain to be linked to an anti-hTfR antibody as long as the peptide chain has the required physiological activity. For example, there are peptide chains containing the amino acid sequence of a region of various proteins that exhibits physiological activity. Although there are no particular restrictions on the length of the peptide chain, they preferably consist of 2-200 amino acids, for example 5-50 amino acids.

When binding a small molecule compound to an anti-hTfR antibody, there are no particular limitations.

- 42 042007 in relation to candidate low molecular weight compounds, but they are such a low molecular weight compound that, although it is necessary to introduce inside the brain to manifest its function there, due to their inability to pass through the blood-brain barrier on their own, their function cannot be expected to manifest in the brain brain just when administered intravenously. Examples of such small molecule compounds include an anti-cancer drug such as cyclophosphamide, ifosfamide, melphalan, busulfan, thiotepa, nimustine, ranimustine, dacarbazine, procarbazine, temozolomide, carmustine, streptozocin, 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, vinblastine, vincristine, paclitaxel, docetaxin, doxorubicin C. Additional examples of a small molecule to be linked to an anti-hTfR antibody include siRNA, antisense DNA, and short peptides.

When binding between an anti-hTfR antibody and a small molecule, the small molecule may be bound to either only one of the light chain and the heavy chain, or it may be bound to both the light chain and the heavy chain, respectively. In addition, since it has affinity for hTfR, an anti-hTfR antibody may comprise an amino acid sequence containing all or part of the light chain variable region and/or an amino acid sequence containing all or part of the heavy chain variable region.

Candidates for small molecule compounds to be fused with anti-hTfR antibodies can typically be these therapeutic agents for diseases such as neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease; mental disorders such as schizophrenia, depression; multiple sclerosis; amyotrophic lateral sclerosis; a tumor of the central nervous system, including a tumor of the brain; lysosomal storage diseases with accompanying encephalopathy; glycogenosis; muscular dystrophy; cerebral ischemia; encephalitis; prion diseases; traumatic disorders of the central nervous system. In addition, therapeutic agents for viral and bacterial diseases of the central nervous system can also in general be candidates for small molecule compounds to be fused with anti-hTfR antibodies. In addition, those pharmaceuticals that can be used for recovery after brain surgery or spinal cord surgery can also generally be candidates for small molecule compounds to be fused.

If the anti-hTfR antibody is derived from a non-human animal, its administration to a human may entail a significant risk of antigen-antibody interaction, thereby provoking adverse side effects. By converting them into humanized antibodies, the antigenicity of antibodies from non-human animals can be reduced, and therefore, the provocation of side effects due to antigen-antibody interaction when administered to a human can be suppressed. In addition, it has been reported that humanized antibodies are more stable in the blood than mouse antibodies in monkey experiments, and it is expected that their therapeutic effect may therefore be correspondingly longer lasting. Provoking side effects due to antigen-antibody interaction can also be suppressed by using a human antibody as an anti-hTfR antibody.

Below, a detailed explanation will be given regarding the case where the anti-hTfR antibody is a humanized antibody or a human antibody. In the light chain of a human antibody, there are λ and k chains. The light chain constituting a human antibody can be either a λ or k chain. And in the human heavy chain, there are γ, μ, α, σ, and ε chains that correspond to IgG, IgM, IgA, IgD, and IgE, respectively. Although the heavy chain constituting the hTfR antibody can be any of the γ, μ, α, σ, and ε chains, the γ chain is preferred. In addition, the human heavy chain has γ1, γ2, γ3 and γ4 chains that correspond to IgG1, IgG2, IgG3 and IgG4, respectively. When the heavy chain constituting the anti-hTfR antibody is a γ chain, although the γ1, γ2, γ3 and γ4 chain may be any of the γ1, γ2, γ3 and γ4 chains, the γ1 or γ4 chain is preferred. In the case where the anti-hTfR antibody is a humanized antibody or a human antibody and IgG, the light chain of the human antibody can be either the λ chain or k chain, and although the heavy chain of the human antibody can be either γ1 or γ2 or γ3 or γ4 chain , γ1 or γ4 chain is preferred. For example, a preferred embodiment of an anti-hTfR antibody comprises a protein whose light chain is a λ chain and whose heavy chain is a γ1 chain.

In the case where the anti-hTfR antibody is a humanized antibody or a human antibody, the anti-hTfR antibody and another protein (A) can be linked together by linking the anti-hTfR antibody at the N-terminus (or C-terminus) of the heavy chain or light chain via a linker sequence or directly to the C-terminus (or N-terminus), respectively, of another protein (A) by peptide bonds. When another protein (A) binds to the heavy chain of the anti-hTfR antibody on its N-terminal side (or C-terminal side), the C-terminus (or N-terminus), respectively, of the other protein (A) binds

- 43 042007 zan with the N-terminus (or C-terminus) of the γ, μ, α, σ or ε chain of an anti-hTfR antibody via a linker sequence or directly, by peptide bonds. When the other protein (A) is bound to the light chain of the anti-hTfR antibody at its N-terminal side (or C-terminal side), the C-terminus (or N-terminus), respectively, of the other protein (A) is linked to the N-terminus (or C-terminus) to the λ chain and to the anti-hTfR antibody chain via a linker sequence or directly, by peptide bonds. However, in the case where the anti-hTfR antibody consists of a Fab region, or a Fab region and all or part of the hinge region (Fab, F(ab') ₂ and F(ab')), another protein (A) can be linked to its C-terminus (or N-terminus) and via a linker sequence or directly to the N-terminus (or C-terminus), respectively, of the heavy chain or light chain, which constitutes Fab, F(ab') ₂ and F(ab'), peptide bonds.

In a fusion protein obtained by linking another protein (A) to the light chain of an anti-hTfR antibody, which is a humanized antibody or a human antibody, at its C-terminal side or N-terminal side, the anti-human transferrin receptor antibody contains an amino acid sequence containing all or part of the light chain variable region; and an amino acid sequence containing all or part of the heavy chain variable region. The light chain of the anti-hTfR antibody and the other protein (A) in this case can be linked directly or via a linker.

In a fusion protein obtained by linking another protein (A) to the heavy chain of an anti-hTfR antibody, which is a humanized antibody or a human antibody, at its Terminal side or N-terminal side, the anti-human transferrin receptor antibody contains an amino acid sequence containing all or part of the light chain variable region; and an amino acid sequence containing all or part of the heavy chain variable region. The heavy chain of the anti-hTfR antibody and the other protein (A) in this case can be linked directly or via a linker.

When a linker sequence is placed between an anti-hTfR antibody or human antibody and another protein (A), the linker sequence is preferably a peptide chain of 1-50 amino acids, although the number of amino acids constituting such a linker sequence can be adjusted according to the other protein if necessary ( A) to be bound to an anti-hTfR antibody like 1-17, 1-10, 10-40, 20-34, 23-31, 25-29 and so on. Although there is no particular restriction on the specific amino acid sequence of such a linker sequence, since the anti-hTfR antibody and the other protein (A) linked by the linker sequence retain their respective functions (hTfR affinity and activity or function under physiological conditions), it is preferably composed of glycine or serine, for example, it consists of one amino acid, either glycine or serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence Gly-GlyGly-Gly-Ser (SEQ ID NO: 3), amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence that contains 1-10 or 2-5 of any these consecutively linked amino acid sequences. For example, a 27 amino acid linker sequence is preferably used which consists of the Gly-Ser amino acid sequence followed by five consecutive copies of the Gly-Gly-Gly-Gly-Ser amino acid sequence (SEQ ID NO: 3). In addition, it is also preferred to use a linker sequence containing 25 amino acids, which consists of consecutively linked five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

In addition, when stating in the present specification that another protein (A) fused to an anti-hTfR antibody or human antibody retains its activity or function under physiological conditions, or simply retains its activity, this means that compared to the intrinsic activity of the other protein (A) natural type activity or function is maintained not lower than 3%. However, such activity or function is preferably not less than 10%, more preferably not less than 20%, even more preferably not less than 50%, and even more preferably not less than 80% compared to the inherent activity of another natural protein (A). type. The same also applies when the other protein (A) fused to the anti-hTfR antibody is a mutant protein.

A further example of specific embodiments of a fusion protein between a humanized antibody or a human anti-hTfR antibody and another protein (A) of the present invention is a protein obtained by fusion of an anti-hTfR antibody heavy chain at its C-terminal side with another protein (A) via a A 27 amino acid linker sequence that consists of the Gly-Ser amino acid sequence followed by five consecutive copies of the Gly-GlyGly-Gly-Ser amino acid sequence (SEQ ID NO: 3).

In the case where the anti-hTfR antibody is a Fab, an example of specific embodiments of fusion proteins between humanized antibodies or human anti-hTfR antibody

- 44 042007 of the present invention and another protein (A) is a protein obtained by fusion of another protein (A) at its C-terminal side through linker sequences with a region consisting of the heavy chain variable region of an anti-hTfR antibody and its accompanying CH1 region, in which the linker sequence consists of 25 amino acids and consists of consecutively linked five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3). Although in this case it is also assumed that there is also a part of the hinge region in addition to the CH1 region, the hinge region does not contain a cysteine residue that would form a disulfide bond between the heavy chains.

The specific affinity of an anti-hTfR antibody for hTfR resides primarily in the amino acid sequences of the heavy chain and light chain CDRs of the anti-hTfR antibody. There are no particular restrictions on the amino acid sequences of these CDRs because, in addition to hTfR, the anti-hTfR antibody has specific affinity for monkey hTfR.

However, according to the present invention, a humanized antibody or human anti-hTfR antibody, which is an antibody having relatively high affinity for hTfR and having affinity for both human and monkey TfR at the same time, is an antibody whose dissociation constant with human TfR is preferably no more than 1x10'1 M, more preferably not more than 2.5x10'1 M, still more preferably not more than ^5x10'12 M, and even more preferably not more than ^1x10'12 M, and whose dissociation constant with monkey TfR is preferably not more than than 1x10'9 M, more preferably not more than ^5x10'10 M and even more preferably not more than 1x10'10 M, for example not more than 7.5x10'11 M, in particular when measured as described in Example 7.

For example, dissociation constants with human TfR and TfR. monkeys are not more than 1x10'1 M and not more than 1x10'9 M, not more than 1x10'11 M and not more than 5x10' ¹⁰ M, not more than 5x10' ¹² M and not more than 1x10' ¹⁰ M, not more than 5x10' ¹² M and not more than 7.5x10'11 M, not more than 1x10' ¹² M and not more than 1x10' ¹⁰ M or not more than lx10' ¹² M and not more than 7.5x10'11 M, respectively. In this context, although there is no particularly clear lower limit for the human TfR dissociation constant, it may be, for example, 5x10' ¹³ M or 1x10' ¹³ M. In addition, although there is no particularly clear lower limit for the monkey TfR dissociation constant, it may be , for example, lx 10' ¹¹ M or lx10' ¹² M and the like. The same also applies if the antibody is a single chain antibody.

Examples of preferred embodiments of an hTfR-affinity antibody include antibodies wherein, in the heavy chain variable region, (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 and (c) CDR3 contains the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

Examples of more specific embodiments of an hTfR-affinity antibody include antibodies wherein, in the heavy chain variable region, (a) CDR1 contains the amino acid sequence of SEQ ID NO: 62, (b) CDR2 contains the amino acid sequence of SEQ ID NO: 13, and (c ) CDR3 contains the amino acid sequence of SEQ ID NO: 15.

In the above preferred embodiments of the hTfR affinity antibody and more specific embodiments of the hTfR affinity antibody, preferred examples of the amino acid sequence of the antibody heavy chain framework region 3 include regions containing the amino acid sequence of SEQ ID NO: 64.

Examples of preferred light chain and heavy chain combinations of an hTfR affinity antibody include chains having amino acid sequences downstream in the variable regions: a light chain combination wherein:

(a) CDR1 contains the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, (b) CDR2 contains the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence of Lys-Val-Ser, a (c ) CDR3 contains the amino acid sequence of SEQ ID NO: 10, and a heavy chain in which (d) CDR1 contains the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, (e) CDR2 contains the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and (f) CDR3 contains the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

Examples of specific embodiments of light chain and heavy chain combinations of an hTfR affinity antibody include chains having the amino acid sequences below in the variable regions: a combination of a light chain containing the amino acid sequences shown as SEQ ID NO: 6 as CDR1, SEQ ID NO: 8 as CDR2 and SEQ ID NO: 10 as CDR3, respectively, and a heavy chain containing the amino acid sequences shown as SEQ ID NO: 62 as CDR1, SEQ ID NO: 13 as CDR2 and SEQ ID NO: 15 as CDR3, respectively.

- 45 042007

In the above-mentioned preferred light chain and heavy chain combinations of an hTfR-affinity antibody, and specific embodiments of light chain and heavy chain combinations of an hTfR-affinity antibody, preferred examples of the antibody heavy chain framework region 3 amino acid sequence include regions having the amino acid sequence of SEQ ID NO: 64.

Examples of preferred embodiments of hTfR-affinity humanized antibodies include antibodies having the amino acid sequences of the following: anti-hTfR antibodies whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, and whose heavy chain variable region contains the amino acid sequence of SEQ ID NO: 65.

In the light chain variable region amino acid sequences shown as SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, CDR1 contains the amino acid sequence SEQ ID NO: 6 or 7, CDR2 contains the amino acid sequence of SEQ ID NO: 8 or 9, and CDR3 contains the amino acid sequence of SEQ ID NO: 10. However, the term CDR as used above in relation to the light chain variable region amino acid sequences shown as SEQ ID NO: 17-22 is not limited to these specific sequences, but may also include a region containing the amino acid sequences of one of the CDRs, or include an amino acid sequence containing at least 3 consecutive amino acids of one of the CDRs mentioned above.

In the amino acid sequence of the heavy chain variable region shown as SEQ ID NO: 65:

(a) CDR1 contains the amino acid sequence of SEQ ID NO: 62 or 63, (b) CDR2 contains the amino acid sequence of SEQ ID NO: 13 or 14, and (c) CDR3 contains the amino acid sequence of SEQ ID NO: 15 or 16, and the amino acid sequence, shown as SEQ ID NO: 64 is also contained as frame region 3. However, the term CDR as used above in relation to the amino acid sequences of the heavy chain variable region shown as SEQ ID NO: 65 is not limited to these specific sequences, but may also include the region containing the amino acid sequences of one of the CDRs or include an amino acid sequence containing at least 3 consecutive amino acids of one of the CDRs mentioned above. The same also applies for wireframes.

Examples of more specific embodiments of a humanized antibody with hTfR affinity include:

an antibody that contains the amino acid sequence of SEQ ID NO: 18, in the variable region of the light chain and contains the amino acid sequence of SEQ ID NO: 65, in the variable region of the heavy chain, an antibody that contains the amino acid sequence of SEQ ID NO: 20, in the variable region of the light chain and contains the amino acid sequence of SEQ ID NO: 65, in the variable region of the heavy chain, an antibody that contains the amino acid sequence of SEQ ID NO: 21, in the variable region of the light chain and contains the amino acid sequence of SEQ ID NO: 65, in the variable region of the heavy chain, and an antibody , which contains the amino acid sequence of SEQ ID NO: 22, in the variable region of the light chain and contains the amino acid sequence of SEQ ID NO: 65, in the variable region of the heavy chain.

Examples of more specific embodiments of a humanized antibody with hTfR affinity include an antibody that contains the amino acid sequence of SEQ ID NO: 23 in the light chain and the amino acid sequence of SEQ ID NO: 66 in the heavy chain, an antibody that contains the amino acid sequence of SEQ ID NO: 25, in the light chain and the amino acid sequence of SEQ ID NO: 66, in the heavy chain, an antibody that contains the amino acid sequence of SEQ ID NO: 27, in the light chain and the amino acid sequence of SEQ ID NO: 66, in the heavy chain, an antibody that contains the amino acid sequence of SEQ ID NO: 29 in the light chain and the amino acid sequence of SEQ ID NO: 66 in the heavy chain, an antibody that contains the amino acid sequence of SEQ ID NO: 23 in the light chain and the amino acid sequence of SEQ ID NO: 68 in the heavy chain, an antibody that contains the amino acid sequence of SEQ ID NO: 25, in a light chain and amino acids the slot sequence of SEQ ID NO: 68 in the heavy chain, an antibody that contains the amino acid sequence of SEQ ID NO: 27 in the light chain and the amino acid sequence of SEQ ID NO: 68 in the heavy chain, and

- 46 042007 an antibody which contains the amino acid sequence of SEQ ID NO: 29 in the light chain and the amino acid sequence of SEQ ID NO: 68 in the heavy chain.

In the specific embodiments mentioned above, the humanized anti-hTfR antibody comprising the amino acid sequence of SEQ ID NO: 66 in the heavy chain is an IgG1 type antibody, and the antibody comprising the amino acid sequence of SEQ ID NO: 68 is an IgG4 type antibody. They both contain as variable region the amino acid sequence of SEQ ID NO: 65.

Examples of more specific embodiments of a humanized antibody that is a Fab having affinity for hTfR include:

an antibody that contains the amino acid sequence of SEQ ID NO: 23 in the light chain and contains the amino acid sequence of SEQ ID NO: 61 in the heavy chain of Fab, an antibody that contains the amino acid sequence of SEQ ID NO: 25 in the light chain and contains the amino acid sequence of SEQ ID NO: 61, in the heavy chain of a Fab, an antibody that contains the amino acid sequence of SEQ ID NO: 27, in the light chain and contains the amino acid sequence of SEQ ID NO: 61, in the heavy chain of a Fab, and an antibody that contains the amino acid sequence of SEQ ID NO: : 29, in the light chain and contains the amino acid sequence of SEQ ID NO: 61, in the heavy chain of Fab.

In the case where the anti-hTfR antibody is a Fab, specific examples of the antibody in which another Fc region is added to the heavy chain of the Fab include an antibody containing the amino acid sequence of SEQ ID NO: 23, in a light chain, and containing the amino acid sequence of SEQ ID NO: 71, in the heavy chain of a Fab with an Fc region added, the antibody contains the amino acid sequence of SEQ ID NO: 25; in the light chain, it contains the amino acid sequence of SEQ ID NO: 71; in the heavy chain of a Fab with an Fco region added, the antibody contains the amino acid sequence of SEQ ID NO: : 27, in the light chain and contains the amino acid sequence of SEQ ID NO: 71, in the heavy chain of a Fab with an Fc region added to it, and the antibody contains the amino acid sequence of SEQ ID NO: 29, in the light chain and contains the amino acid sequence of SEQ ID NO: 71, in the heavy chain of a Fab with an Fc region added to it.

By adding another Fc region mentioned above to the heavy chain of a Fab, for example, a heavy chain of a Fab with an Fc region added thereto can be linked directly or via a linker sequence to another protein (A) at its C-terminal side. In addition, a Fab heavy chain having the amino acid sequence of SEQ ID NO: 71 is a Fab heavy chain in which a human IgG Fco region having the amino acid sequence of SEQ ID NO: 70 is linked via a linker sequence of 25 amino acids, which consists of consecutively linked five copies of the Gly-Gly-Gly-Gly-Ser amino acid sequence (SEQ ID NO: 3), with the N-terminal side of the Fab heavy chain amino acid sequence (amino acid sequence: 61) of the humanized anti-hTfR 3N antibody.

The above have been exemplified by preferred embodiments of an hTfR affinity antibody. If necessary, the light chain and heavy chain of these anti-hTfR antibodies can be mutated by substitution, deletion, addition, and the like in the amino acid sequences of their variable regions in order to bring the affinity of the anti-hTfR antibody to hTfR to an appropriate level.

When replacing one or more amino acids of the light chain variable region amino acid sequence with other amino acids, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, and even more preferably 1-2. When deleting one or more amino acids of the light chain variable region amino acid sequence, the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, and even more preferably 1-2. It is also acceptable to introduce a combined mutation of such a substitution and deletion of amino acids.

When adding one or more amino acids with a light chain variable region, they can be added inside or on the N-terminal side or C-terminal side of the amino acid sequence of the light chain variable region, and in an amount of preferably 1-10, more preferably 1-5, even more preferably 1-3 and even more preferably 1-2. It is also acceptable to introduce a combined mutation of such addition, substitution and deletion of amino acids. Such a mutant light chain variable region amino acid sequence has homology with the amino acid sequence of the original light chain variable region, preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

In particular, when replacing one or more amino acids of the amino acid sequence of the corresponding CDRs or corresponding framework regions in the light chain with other amino acids

- 47 042007 the number of amino acids to be replaced is preferably 1-5, more preferably 13, even more preferably 1-2 and even more preferably 1. When one or more amino acids of the amino acid sequence of the respective CDRs or the respective framework regions are deleted, the number of amino acids to be deleted is preferably 1 -5, more preferably 1-3, even more preferably 1-2, and even more preferably 1. It is also acceptable to introduce a combined mutation of such a substitution and an amino acid deletion.

When one or more amino acids are added to the amino acid sequence of the corresponding CDRs or corresponding framework regions in the light chain, they are added inside or on the N-terminal side or C-terminal side of the amino acid sequence and in an amount of preferably 1-5, more preferably 1-3, even more preferably 1 -2 and even more preferably 1. It is also acceptable to introduce a combined mutation of such addition, substitution and deletion of amino acids. The amino acid sequence of each of such mutated CDRs or framework regions has homology with the amino acid sequence of the corresponding parental CDRs, preferably not less than 80%, more preferably not less than 90%, and even more preferably not less than 95%.

When replacing with other amino acids one or more amino acids of the amino acid sequence of SEQ ID NO: 65, which is a heavy chain variable region, the number of amino acids to be replaced is preferably 1-10, more preferably 1-5, even more preferably 1-3, and even more preferably 1 -2. When deleting one or more amino acids of the heavy chain variable region amino acid sequence, the number of amino acids to be deleted is preferably 1-10, more preferably 1-5, even more preferably 1-3, and even more preferably 1-2. It is also acceptable to introduce a combined mutation of such a substitution and deletion of amino acids.

When adding one or more amino acids to the amino acid sequence of SEQ ID NO: 65, which is a heavy chain variable region, they can be added inside or on the N-terminal side or C-terminal side of the amino acid sequence of the heavy chain variable region, and in an amount of preferably 1-10, more preferably 1-5, even more preferably 1-3 and even more preferably 1-2. It is also acceptable to introduce a combined mutation of such addition, substitution and deletion of amino acids. Such a mutated heavy chain variable region amino acid sequence has an amino acid sequence homology of the original heavy chain variable region preferably not less than 80%, more preferably not less than 90%, even more preferably not less than 95%.

In particular, when replacing one or more amino acids of the amino acid sequence of the corresponding CDRs or corresponding framework regions in the amino acid sequence of SEQ ID NO: 65 with other amino acids, the number of amino acids to be replaced is preferably 1-5, more preferably 1-3, even more preferably 1-2 and even more preferably 1. When deleting one or more amino acids of the amino acid sequence of the respective CDRs, the number of amino acids to be deleted is preferably 1-5, more preferably 1-3, even more preferably 1-2, and even more preferably 1. It is also acceptable to introduce a combination mutation of such amino acid substitutions and deletions.

When one or more amino acids are added to the amino acid sequence of the corresponding CDRs or corresponding framework regions in the amino acid sequence of SEQ ID NO: 65, they are added inside or on the N-terminal side or C-terminal side of the amino acid sequence and in an amount preferably 1-5, more preferably 1-3, even more preferably 1-2, and even more preferably 1. It is also acceptable to introduce a combined mutation of such addition, substitution and deletion of amino acids. The amino acid sequence of each of these mutant CDRs has homology with the amino acid sequence of the corresponding parental CDRs, preferably not less than 80%, more preferably not less than 90%, and even more preferably not less than 95%.

In addition, in the case where the amino acid sequence of SEQ ID NO: 65, which is the heavy chain variable region of the anti-hTfR antibody, is introduced with the above-described mutation such as substitution, deletion, addition, it is preferable that the amino acid methionine at position 5 from the N-terminal side the original CDR1, shown as SEQ ID NO: 62 or 63, and the amino acid leucine at position 17 on the N-terminal side of framework 3, shown as SEQ ID NO: 64, should be retained in the same position as the original. In addition, it is preferred that the amino acid sequences of CDR1 and heavy chain framework region 3 should also be retained in the same positions as the original ones.

Mutation can be introduced into both the light chain and heavy chain variable regions of an anti-hTfR antibody by combining the aforementioned mutation in the light chain variable region of the anti-hTfR antibody and the aforementioned mutation in the heavy chain variable region of the anti-hTfR antibody.

Examples of the above substitution of one or more amino acids in the amino acid sequence of the heavy chain and light chain variable regions of an anti-hTfR antibody include am

- 48 042007 no acids classified into the same groups, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having a hydroxy group (Ser, Thr).

In addition, in the case of introducing a mutation into an anti-hTfR antibody by adding one or more amino acids at its C-terminus or N-terminus, if, when they are fused, the added amino acids are located between the anti-hTfR antibody and another protein (A), the added amino acids form part of the linker .

In the above preferred embodiments of an antibody, including a humanized antibody having affinity for hTfR, there are no particular restrictions on the amino acid sequence of the heavy chain and light chain CDRs of the anti-hTfR antibody, as long as the antibody has specific affinity for monkey hTfR and TfR.

However, according to the present invention, a human antibody having a relatively high affinity for hTfR and having affinity for both human and monkey TfR at the same time is an antibody whose dissociation constant with human TfR is preferably not more than 1x10'1° M, more preferably not more than 2.5x10'11 M, even more preferably not more than 5x10' ¹² M and even more preferably not more than 1x10' ¹² M, and whose dissociation constant with monkey TfR is preferably not more than 1x10'9 M, more preferably not more than 5x10'1° M and even more preferably not more than 1x10'1° M, for example not more than 7.5x10'11 and M, in particular when measured as described in Example 7.

For example, dissociation constants with human TfR and TfR. monkeys are not more than 1x10' ¹⁰ M and not more than 1x10'9 M, not more than 1x10'11 M and not more than 5x10' ¹⁰ M, not more than 5x10' ¹² M and not more than 1x10' ¹⁰ M, not more than 5x10' ¹² M and not more than 7.5x10'11 M, not more than 1x10' ¹² M and not more than 1x10' ¹⁰ M or not more than 1x10' ¹² M and not more than 7.5x10'11 M, respectively. In this context, although there is no particularly clear lower limit for the human TfR dissociation constant, it may be, for example, 5x10' ¹³ M or 1x10' ¹³ M. In addition, although there is no particularly clear lower limit for the monkey TfR dissociation constant, it may be , such as 5x10'11 M, 1x10'11 M, 1x10' ¹² M and so on. The same applies when the antibody is a single chain antibody.

Specific embodiments of a fusion protein between a humanized antibody that has affinity for hTfR and another protein (A) described above include: fusion proteins wherein the other protein (A) is human acid α-glucosidase (hGAA), human iduronate- 2-sulfatase (hI2S), human a-L-iduronidase (hIDUA), human palmitoyl protein thioesterase 1 (hPPT-1), human acid sphingomyelinase (haSM), human arylsulfatase A (hARSA), human heparan-N-sulfatase (hSGSH) , human glucocerebrosidase (hGBA), human tripeptidyl peptidase 1 (hTPP-1), human α-N-acetylglucosaminidase (hNAGLU), human β-glucuronidase (hGUSB), human acid ceramidase (hAC), human a-L-fucosidase (hFUCA1) or α-mannosidase (hLAMAN).

Specific examples of a fusion protein when the other protein (A) is human acid α-glucosidase (hGAA) include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hGAA, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or N-terminal side and through a linker sequence with hGAA, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of the hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hGA A, and another region consisting of the light chain of hTfR, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 27, (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hGAA, and another region consisting of an hTfR light chain, the amino acid sequence of the heavy chain being shown as SEQ ID NO: 66 or 68 , and the amino acid sequence of the light chain is shown as SEQ ID NO: 29.

In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, amino acid sequence Gly-Ser, amino

- 49 042007 no acid sequence of Gly-Gly-Ser, amino acid sequence of SEQ ID NO: 3, amino acid sequence of SEQ ID NO: 4, amino acid sequence of SEQ ID NO: 5, and amino acid sequences consisting of 1-10 of them that are sequentially linked .

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hGAA is hGAA having the amino acid sequence of SEQ ID NO: 55 or 56, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is human acid α-glucosidase (hGAA) include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through the amino acid sequence Gly-Ser to hGAA, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 57, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, and (2) a protein that consists of: a region consisting of the hTfR heavy chain linked at its Terminal side and through the Gly-Ser amino acid sequence to hGAA, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 58 and the amino acid sequence of the latter being shown as SEQ ID NO: 23.

Antibody (1) is of the IgG1 type and antibody (2) is of the IgG4 type. In addition, hGAA is an hGAA having the amino acid sequence of SEQ ID NO: 55.

In the above (1), the amino acid sequence of the hTfR heavy chain, which is included in SEQ ID NO: 57, is the sequence shown as SEQ ID NO: 66. Namely, the fusion protein according to (1) above contains, as a humanized antibody, the amino acid sequence of light chain shown as SEQ ID NO: 23, and the amino acid sequence of the heavy chain shown as SEQ ID NO: 66. In the above (2), the amino acid sequence of the hTfR heavy chain, which is included in SEQ ID NO: 58, is the sequence shown as SEQ ID NO: 68. Namely, the fusion protein according to (2) above contains, as a humanized antibody, a light chain amino acid sequence shown as SEQ ID NO: 23 and a heavy chain amino acid sequence shown as SEQ ID NO: 68.

Specific examples of a fusion protein where the other protein (A) is human acid α-glucosidase (hGAA) and the humanized antibody is a Fab antibody include:

(1) a protein which consists of: a region consisting of an hTfR Fab heavy chain linked at its Terminal side or N-terminus and via a linker sequence to hGAA, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain The Fab is shown as SEQ ID NO: 61 and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of the hTfR Fab heavy chain linked at its Terminal side or N-terminus and through a linker sequence with hGAA, and another region consisting of the hTfR light chain, wherein the Fab heavy chain amino acid sequence is shown as SEQ ID NO: 61 and the light chain amino acid sequence is shown as SEQ ID NO: 25, (3) a protein that consists of: a region consisting of an hTfR Fab heavy chain linked at its Terminal side or N-terminus and via a linker sequence to hGAA, and another region consisting of a leg hTfR heavy chain, wherein the Fab heavy chain amino acid sequence is shown as SEQ ID NO: 61, and the light chain amino acid sequence is shown as SEQ ID NO: 27, (4) a protein that consists of: a region consisting of the hTfR Fab heavy chain, linked at its Terminal side or N-terminus and via a linker sequence to hGAA, and another region consisting of the hTfR light chain, the Fab heavy chain amino acid sequence shown as SEQ ID NO: 61 and the light chain amino acid sequence shown as SEQ ID NO: 29.

In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequences of SEQ ID NO: 5, and amino acid sequences consisting of 1-10 of them that are sequentially linked.

A more specific example of a fusion protein when the other protein (A) is human acid α-glucosidase (hGAA) and the humanized antibody is a Fab antibody includes: a protein that consists of a region consisting of an hTfR Fab heavy chain linked at its C-terminal side and through a linker sequence, and this linker sequence consists of three sequentially linked amino acid sequences, each of which is shown as SEQ ID NO: 3, with hGAA, and another section, consisting of the hTfR light chain, while the amino acid sequence of the first is shown as SEQ ID NO: 89, and the amino acid sequence of the latter

- 50 042007 is shown as SEQ ID NO: 23.

The fusion protein wherein the other protein (A) is human acid α-glucosidase (hGAA) has affinity for both human and monkey TfRs simultaneously, and is a protein whose dissociation constant with monkey TfR is preferably not more than 1x10'1 ° M, more preferably not more than 5x10'11 M, the dissociation constant of which with human TfR is preferably not more than 1x10'1° M, more preferably not more than 5x10'11 M, even more preferably not more than 1x10'11 M and even more preferably not more than 1x10 ^-12 M, as measured by the method described in example 7.

For example, dissociation constants with monkey TfR and human TfR are no more than 1x10' ¹⁰ M and no more than 1x10' ¹⁰ M, no more than 1x10' ¹⁰ M and no more than 1x10'11 M, no more than 1x10' ¹⁰ M and not more than ^1x10'12 M, not more than 5x10'11 M and not more than 1x10'11 M, not more than 5x10'11 M and not more than 1x10'11 M or not more than 5x10'11 M and not more than 1x10' ¹² M, respectively. In this context, although there is no particularly clear lower limit for the dissociation constant with the simian TfR, it could be, for example, 1x10'11 M, 1x10' ¹² M, or 1x10' ¹³ M. Although there is no particularly clear lower limit for the dissociation constant with the human TfR, it may be, for example, 1x10' ¹² M, 5x10' ¹³ M or 1x10' ¹³ M. The same also applies if the antibody is a single chain antibody.

Specific examples of a fusion protein when the other protein (A) is human iduronate-2sulfatase (hI2S) include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hI2S, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and through a linker sequence with hI2S, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hI2S, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 27, (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hI2S, and another region consisting of an hTfR light chain, the heavy chain amino acid sequence shown as SEQ ID NO: 66 or 68 , and the amino acid sequence of the light chain is shown as SEQ ID NO: 29.

In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequences of SEQ ID NO: 5, and amino acid sequences consisting of 1-10 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type.

More specific examples of a fusion protein when the other protein (A) is human iduronate-2-sulfatase (hI2S) include: a protein that consists of: a region consisting of an hTfR heavy chain linked at its C-terminal side and through an amino acid sequence Gly-Ser with hI2S and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 53 and the amino acid sequence of the latter being shown as SEQ ID NO: 23. In this case, the antibody is of the IgG1 type. The hTfR heavy chain amino acid sequence that is included in SEQ ID NO: 53 is the sequence shown as SEQ ID NO: 66. Namely, this fusion protein contains, as a humanized antibody, the light chain amino acid sequence shown as SEQ ID NO: 23, and the heavy chain amino acid sequence shown as SEQ ID NO: 66. The antibody may be of the IgG4 type, in which case the heavy chain amino acid sequence shown as SEQ ID NO: 66 may be replaced by the amino acid sequence SEQ ID NO: 68.

Specific examples of a fusion protein where the other protein (A) is human iduronate-2sulfatase (hI2S) and the humanized antibody is a Fab antibody include:

(1) a protein which consists of: a region consisting of an hTfR Fab heavy chain linked at its Terminal side or N-terminus and via a linker sequence to hI2S, and another

- 51 042007 a stack consisting of the hTfR light chain, wherein the Fab heavy chain amino acid sequence is shown as SEQ ID NO: 61, and the light chain amino acid sequence is shown as SEQ ID NO: 23, (2) a protein that consists of: a region, consisting of an hTfR Fab heavy chain linked at its Terminal side or N-terminus and via a linker sequence to hI2S, and another region consisting of an hTfR light chain, the amino acid sequence of the Fab heavy chain is shown as SEQ ID NO: 61, and the amino acid the light chain sequence is shown as SEQ ID NO: 25, (3) a protein which consists of: a region consisting of an hTfR Fab heavy chain linked at its Terminal side or N-terminus and via a linker sequence to hI2S, and another region consisting from the hTfR light chain, wherein the Fab heavy chain amino acid sequence is shown as SEQ ID NO: 61 and the light chain amino acid sequence is shown as SEQ ID NO: 27, (4) a protein which consists of: a region consisting of an hTfR Fab heavy chain connected at its Terminal side or N-terminus and via a linker sequence to hI2S, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain The Fab is shown as SEQ ID NO: 61 and the light chain amino acid sequence is shown as SEQ ID NO: 29.

In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequences of SEQ ID NO: 5, and amino acid sequences consisting of 1-10 of them that are sequentially linked.

Specific examples of a fusion protein when the other protein (A) is hIDUA include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hIDUA, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or N-terminal side and through a linker sequence with hIDUA, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its terminal side or N-terminal side and via a linker sequence with hIDUA, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hIDUA, and another region consisting of an hTfR light chain, the heavy chain amino acid sequence shown as SEQ ID NO: 66 or 68; sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence SE Q ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hIDUA is hIDUA having the amino acid sequence of SEQ ID NO: 75 or 76, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hIDUA include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through the amino acid sequence Gly-Ser to hIDUA, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 90, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein which consists of: a region consisting of the hTfR heavy chain linked at its Terminal side and through a linker containing a total of 42 amino acids, which consists of the Gly-Ser amino acid sequence followed by eight copies of the amino acid sequence of SEQ ID NO: 3, serially linked to hIDUA, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 91,

- 52 042007 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) a protein that consists of: a region consisting of the heavy chain of hTfR linked at its Terminal side and through a linker consisting of 20 consecutive copies of the amino acid sequence of SEQ ID NO: 3, with hIDUA, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 92 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (4) a protein which consists from: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and via a linker consisting of three copies of the amino acid sequence of SEQ ID NO: 3 linked in series to hIDUA, and another region consisting of an hTfR light chain, with in this, the amino acid sequence of the former is shown as SEQ ID NO: 93 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of five consecutive copies of the amino acid sequence of SEQ ID NO: 3 linked to hIDUA, and another region consisting of the hTfR light chain, with the amino acid the sequence of the former is shown as SEQ ID NO: 94 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (6) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker , consisting of sequentially linked ten copies of the amino acid sequence of SEQ ID NO: 3, with hIDUA, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 95, and the amino acid sequence of the latter is shown as SEQ ID NO : 23, (7) a protein that consists of: a region consisting of the hTfR heavy chain (Fab) linked on its Terminal side and via a linker, consisting consisting of 20 consecutive copies of the amino acid sequence of SEQ ID NO: 3, with hIDUA, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 96 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (8) a protein that consists of: a region consisting of hIDUA linked to an amino acid sequence on its C-terminal side, which consists of two consecutive copies of the hTfR heavy chain (Fab) having a linker on its C-terminal side , consisting of six consecutive copies of the amino acid sequence of SEQ ID NO: 3, and another section consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 97, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, and (9) a protein which consists of: a region consisting of hIDUA linked to a single chain humanized anti-hTf antibody R No. 3N(2), shown as SEQ ID NO: 98, on its C-terminal side and through a seven amino acid linker, which consists of the Gly-Gly amino acid sequence following the amino acid sequence of SEQ ID NO: 3, which is shown as SEQ ID NO: 99.

Fusion proteins (1)-(3) are proteins whose antibody is of the IgG1 type, and fusion proteins (4)-(9) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (3) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (3) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (4)-(9) are shown as SEQ ID NO: 61. A namely, the above-mentioned fusion proteins (4)-(9) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of the fusion protein when the other protein (A) is hPPT-1 include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hPPT-1, and another region consisting of an hTfR light chain, wherein the amino acid sequence the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N- the terminal side and through a linker sequence with hPPT-1, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence and with hPPT-1, and another region consisting of the hTfR light chain, with the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light

- 53 042007 chains are shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of the hTfR heavy chain linked at its Terminal side or N-terminal side and through a linker sequence to hPPT-1, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 29. In this case, it is preferred that the linker sequence is the sequence consisting from one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence SEQ ID NO: 3, the amino acid sequence SEQ ID NO: 4, the amino acid sequence SEQ ID NO: 5, and the amino acid sequences consisting of 1-10 or 1-20 of them that are connected in series.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hPPT-1 is hPPT having the amino acid sequence of SEQ ID NO: 77, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hPPT-1 include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through the amino acid sequence of Gly-Ser to hPPT-1, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 100, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through a linker consisting of five successively linked copies of the amino acid sequence of SEQ ID NO: 3, with hPPT-1, and another portion consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 101 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, ( 3) a protein that consists of: a region consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten consecutively linked copies of the amino acid sequence of SEQ ID NO: 3, with hPPT-1, and another portion consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 102 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, ( 4) a protein that consists of: a region consisting of an hTfR heavy chain connected on its Terminal side and via a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to hPPT-1, and another region consisting of hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 103 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: on its Terminal side and through a linker consisting of consecutively linked three copies of the amino acid sequence of SEQ ID NO: 3, with hPPT-1, and another section consisting of the hTfR light chain , wherein the amino acid sequence of the former is shown as SEQ ID NO: 104 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, and (6) a protein that consists of: a region consisting of hPPT-1 linked to an amino acid sequence on its The C-terminal side, which consists of two consecutive copies of the hTfR heavy chain (Fab), having on its C-terminal side a linker consisting of six consecutive copies of the amino acid sequence of SEQ ID NO: 3, and another section consisting of a light chain hTfR, with the amino acid sequence of the former shown as SEQ ID NO: 105 and the amino acid sequence of the latter shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5) and (6) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5) and (6) are shown as SEQ ID NO: 61. A namely, the fusion proteins (5) and (6) mentioned above have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is haSM include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to haSM, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of the hTfR heavy chain linked at its C-

- 54 042007 terminal side or N-terminal side and through a linker sequence with haSM, and another section consisting of the hTfR light chain, while the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to haSM, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked to its The terminal side or N-terminal side and through a linker sequence with haSM, and another region consisting of the hTfR light chain, while amino acids the heavy chain sequence is shown as SEQ ID NO: 66 or 68, and the light chain amino acid sequence is shown as SEQ ID NO: 29. In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, the amino acid sequence Gly- Ser, the amino acid sequence of Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them, which are connected in series.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, haSM is a haSM having the amino acid sequence of SEQ ID NO: 78, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is haSM include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and via the Gly-Ser amino acid sequence to haSM, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 106, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its sequence of SEQ ID NO: 3, with haSM, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 107, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) a protein, which consists of: a region consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten copi connected in series th amino acid sequence of SEQ ID NO: 3, with haSM, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 108 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (4) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side and through a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to haSM, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 109 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of consecutively linked three copies of the amino acid sequence of SEQ ID NO: 3, with haSM, and another section consisting of the hTfR light chain, while the amino acid sequence of the former is shown as SEQ ID NO: 110 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (6) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of five consecutive copies of the amino acid sequence of SEQ ID NO: 3, with haSM, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 111, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (7) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked at its Terminal side and through a linker consisting of ten consecutive copies of the amino acid sequence of SEQ ID NO: 3, with haSM, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 112 and the amino acid sequence the latter is shown as SEQ ID NO: 23, (8) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked at its Terminal side and via a linker consisting of 20 consecutive copies of the amino acid sequence of SEQ ID NO: 3 with haSM, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 113 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, and

- 55 042007 (9) a protein that consists of: a region consisting of haSM linked to an amino acid sequence on its C-terminal side, which consists of two consecutive copies of the hTfR heavy chain (Fab) having on its C-terminal side a linker consisting of six consecutive copies of the amino acid sequence of SEQ ID NO: 3 and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 114 and the amino acid sequence of the latter being shown as SEQ ID NO: 23 .

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5)-(9) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5) and (6) are shown as SEQ ID NO: 61. A namely, the fusion proteins (5) and (6) mentioned above have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hARSA include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hARSA, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or N-terminal side and via a linker sequence with hARSA, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its terminal side or N-terminal side and via a linker sequence with hARSA, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hARSA, and another region consisting of an hTfR light chain, with the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68; sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence SE Q ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hARSA is hARSA having the amino acid sequence of SEQ ID NO: 79, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hARSA include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and via the Gly-Ser amino acid sequence to hARSA, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 115, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its sequence of SEQ ID NO: 3, with hARSA, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 116, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) protein, which consists of: a region consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten co-linked in series pi of the amino acid sequence of SEQ ID NO: 3, with hARSA, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 117 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (4) a protein that consists of: a region consisting of the hTfR heavy chain linked at its terminal side and through a linker consisting of 20 amino acid copies linked in series

- 56 042007 of SEQ ID NO: 3, with hARSA, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 118, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, ( 5) a protein that consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and via a linker consisting of three copies of the amino acid sequence of SEQ ID NO: 3 linked in series to hARSA, and another region consisting from the hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 119 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (6) a protein which consists of: a region consisting of the hTfR heavy chain (Fab), linked on its Terminal side and through a linker consisting of five consecutive copies of the amino acid sequence of SEQ ID NO: 3, with hARSA, and another section consisting of the hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 120 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (7) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and via a linker consisting of ten consecutive copies of the amino acid sequence of SEQ ID NO: 3, to hARSA, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 121 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (8) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked at its Terminal side and via a linker consisting of 20 copies of the amino acid sequence linked in series of SEQ ID NO: 3, with hARSA, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 122, and the amino acid sequence The identity of the latter is shown as SEQ ID NO: 23, and (9) a protein which consists of: a region consisting of hARSA linked to an amino acid sequence on its C-terminal side which consists of two consecutive copies of the hTfR heavy chain (Fab) , having on its C-terminal side a linker consisting of six consecutive copies of the amino acid sequence of SEQ ID NO: 3, and another site consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 123, and the amino acid sequence the latter is shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5)-(9) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and the heavy chain of which contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5)-(9) are shown as SEQ ID NO: 61. A namely, the above-mentioned fusion proteins (5)-(9) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hSGSH include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hSGSH, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and through a linker sequence with hSGSH, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence with hSGSH, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hSGSH, and another region consisting of an hTfR light chain, the heavy chain amino acid sequence shown as SEQ ID NO: 66 or 68; follow

- 57 042007 the identity of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hSGSH is hSGSH having the amino acid sequence of SEQ ID NO: 80, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hSGSH include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through the amino acid sequence Gly-Ser to hSGSH, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 124, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its sequence of SEQ ID NO: 3, with hSGSH, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 125, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) protein, which consists of: a section consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten coherently linked pi of the amino acid sequence of SEQ ID NO: 3, with hSGSH, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 126 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (4) a protein that consists of: a region consisting of an hTfR heavy chain connected on its Terminal side and via a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to hSGSH, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 127 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of consecutively linked three copies of the amino acid sequence of SEQ ID NO: 3, with hSGSH, and another section consisting of the hTfR light chain, when in this, the amino acid sequence of the former is shown as SEQ ID NO: 128 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, and (6) a protein which consists of: a region consisting of hSGSH linked to an amino acid sequence at its C-terminal side , which consists of two consecutive copies of the hTfR heavy chain (Fab), having on its C-terminal side a linker consisting of six consecutive copies of the amino acid sequence of SEQ ID NO: 3, and another section consisting of the hTfR light chain, while the amino acid sequence of the former is shown as SEQ ID NO: 129 and the amino acid sequence of the latter is shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5) and (6) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5) and (6) are shown as SEQ ID NO: 61. A namely, the fusion proteins (5) and (6) mentioned above have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hGBA include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hGBA, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and through a linker sequence with hGBA, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hGBA, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light

- 58 042007 chain is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or

N-terminal side and via a linker sequence with hGBA, and another region consisting of the hTfR light chain, with the heavy chain amino acid sequence shown as SEQ ID NO: 66 or 68 and the light chain amino acid sequence shown as SEQ ID NO: 29. In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence of SEQ ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them, which are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hGBA is an hGBA having the amino acid sequence of SEQ ID NO: 81, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hGBA include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through the amino acid sequence Gly-Ser to hGBA, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 130, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its sequence of SEQ ID NO: 3, with hGBA, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 131, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) protein, which consists of: a region consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten copi connected in series th amino acid sequence of SEQ ID NO: 3, with hGBA, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 132, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (4) a protein that consists of: a region consisting of an hTfR heavy chain connected on its Terminal side and through a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to hGBA, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 133 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of consecutively linked three copies of the amino acid sequence of SEQ ID NO: 3, with hGBA, and another section consisting of the hTfR light chain, while the amino acid sequence of the former is shown as SEQ ID NO: 134 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, and (6) a protein which consists of: a region consisting of hGBA linked to an amino acid sequence at its C-terminal side, which consists of two consecutive copies of the hTfR heavy chain (Fab), having on its C-terminal side a linker consisting of six consecutive copies of the amino acid sequence of SEQ ID NO: 3, and another section consisting of the hTfR light chain, while the amino acid the sequence of the former is shown as SEQ ID NO: 135 and the amino acid sequence of the latter is shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5) and (6) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5) and (6) are shown as SEQ ID NO: 61. A namely, the fusion proteins (5) and (6) mentioned above have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hTPP-1 include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hTPP-1, and another region consisting of an hTfR light chain, wherein the amino acid sequence the heavy chain is shown as SEQ ID NO: 66 or 68 and the light chain amino acid sequence is shown as SEQ ID NO: 23,

- 59 042007 (2) a protein which consists of: a region consisting of the hTfR heavy chain connected on its Terminal side or N-terminal side and via a linker sequence to hTPP-1, and another region consisting of the hTfR light chain, with in this, the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) a protein which consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or the N-terminal side and through a linker sequence with hTPP-1, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO : 27, and (4) a protein that consists of: a region consisting of the hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence with hTPP-1, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 29. In this case, preferably, that the linker sequence is a sequence consisting of one glycine, one serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence SEQ ID NO: 5 and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hTPP-1 is hTPP-1 having the amino acid sequence of SEQ ID NO: 82, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hTPP-1 include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through the amino acid sequence of Gly-Ser to hTPP-1, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 136, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of the heavy chain of hTfR linked at its Terminal side and via a linker consisting of consecutively linked five copies of the amino acid sequence of SEQ ID NO: 3, with hTPP-1, and another portion consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 137 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, ( 3) a protein that consists of: a region consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten consecutively linked copies of the amino acid sequence of SEQ ID NO: 3, with hTPP-1, and another portion consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 138 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, ( 4) a protein that consists of: a region consisting of the hTfR heavy chain connected on its Terminal side and through a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to hTPP-1, and another region consisting of hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 139 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: on its Terminal side and through a linker consisting of consecutively linked three copies of the amino acid sequence of SEQ ID NO: 3, with hTPP-1, and another section consisting of the hTfR light chain , wherein the amino acid sequence of the former is shown as SEQ ID NO: 140, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, and (6) a protein that consists of: a region consisting of hTPP-1 linked to the amino acid sequence on its The C-terminal side, which consists of two consecutive copies of the hTfR heavy chain (Fab), having on its C-terminal side a linker consisting of six consecutive copies of the amino acid sequence of SEQ ID NO: 3, and another section consisting of a light chain hTfR, with the amino acid sequence of the former shown as SEQ ID NO: 141 and the amino acid sequence of the latter shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5) and (6) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5) and (6) are shown as SEQ ID NO: 61. A namely, the fusion proteins (5) and (6) mentioned above have

- 60 042007 humanized antibody, the light chain of which contains the amino acid sequence of SEQ ID NO: 23, and the heavy chain of which (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hNAGLU include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hNAGLU, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or N-terminal side and through a linker sequence with hNAGLU, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence and with hNAGLU, and another hTfR light chain region, wherein the heavy chain amino acid sequence is shown as SEQ ID NO: 66 or 68 and the light chain amino acid sequence is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hNAGLU, and another region consisting of an hTfR light chain, the amino acid sequence of the heavy chain being shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 29. In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence and SEQ ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hNAGLU is hNAGLU having the amino acid sequence of SEQ ID NO: 83, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hNAGLU include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and via the Gly-Ser amino acid sequence to hNAGLU, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 142, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of the hTfR heavy chain linked at its of SEQ ID NO: 3, with hNAGLU, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 143 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (3) a protein, which consists of: a region consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten consecutively linked copies of the amino acid sequence of SEQ ID NO: 3, with hNAGLU, and another portion consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 144 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (4) a protein that consists of: a region consisting of the hTfR heavy chain connected on its Terminal side and through a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to hNAGLU, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 145 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of consecutively linked three copies of the amino acid sequence of SEQ ID NO: 3, with hNAGLU, and another section consisting of the hTfR light chain , wherein the amino acid sequence of the former is shown as SEQ ID NO: 146 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (6) a protein that consists of: a region consisting of an hTfR heavy chain (Fab) linked at its Terminal side and through a linker consisting of consecutively linked five copies of the amino acid sequence of SEQ ID NO: 3, with hNAGLU, and another section consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 147, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (7) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked at its C

- 61 042007 to the terminal side and through a linker consisting of ten copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to hNAGLU, and another section consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 148, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, and (8) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked at its Terminal side and via a linker consisting of 20 consecutive copies of the amino acid sequence of SEQ ID NO: 3, with hNAGLU, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 149 and the amino acid sequence of the latter being shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5)-(8) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5)-(8) are shown as SEQ ID NO: 61. A namely, the above-mentioned fusion proteins (5)-(8) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hGUSB include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hGUSB, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or N-terminal side and via a linker sequence with hGUSB, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its terminal side or N-terminal side and via a linker sequence with hGUSB, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hGUSB, and another region consisting of an hTfR light chain, with the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68; sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence SE Q ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hGUSB is hGUSB having the amino acid sequence of SEQ ID NO: 84, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hGUSB include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and via the Gly-Ser amino acid sequence to hGUSB, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 150, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of the hTfR heavy chain linked at its sequence of SEQ ID NO: 3, with hGUSB, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 151, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) protein, which consists of: a section consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten coherently linked pium of the amino acid sequence of SEQ ID NO: 3, with hGUSB, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 152, and the amino acid

- 62 042007 the full sequence of the latter is shown as SEQ ID NO: 23, (4) a protein which consists of: a region consisting of the hTfR heavy chain linked at its Terminal side and via a linker consisting of 20 consecutive copies of the amino acid sequence linked SEQ ID NO: 3, with hGUSB, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 153 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (5) a protein that consists of : a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of three copies of the amino acid sequence of SEQ ID NO: 3 linked in series with hGUSB, and another region consisting of an hTfR light chain, while the amino acid sequence of the former is shown as SEQ ID NO: 154 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (6) a protein which consists of: a region , consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of five consecutive copies of the amino acid sequence of SEQ ID NO: 3, with hGUSB, and another section consisting of the hTfR light chain, with the amino acid sequence the former is shown as SEQ ID NO: 155 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (7) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and via a linker, consisting of ten consecutive copies of the amino acid sequence of SEQ ID NO: 3, with hGUSB, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 156 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, and (8) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of sequentially linked 20 copies of the amino acid sequence of SEQ ID NO: 3, with hGUSB, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 157, and the amino acid sequence of the latter is shown as SEQ ID NO: 23 .

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5)-(8) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5)-(8) are shown as SEQ ID NO: 61. A namely, the above-mentioned fusion proteins (5)-(8) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hGALC include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hGALC, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or N-terminal side and through a linker sequence with hGALC, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its terminal side or N-terminal side and via a linker sequence with hGALC, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hGALC, and another region consisting of an hTfR light chain, with the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68; sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence SE Q ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

- 63 042007

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hGALC is hGALC having the amino acid sequence of SEQ ID NO: 85, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hGALC include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through the amino acid sequence Gly-Ser to hGALC, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 158, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its sequence of SEQ ID NO: 3, with hGALC, and another region consisting of the light chain of hTfR, the amino acid sequence of the former is shown as SEQ ID NO: 159 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) a protein that consists of: a section consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten kopecks linked in series the second amino acid sequence of SEQ ID NO: 3, with hGALC, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 160 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (4) a protein that consists of: a region consisting of an hTfR heavy chain connected on its Terminal side and through a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to hGALC, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 161 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of consecutively linked three copies of the amino acid sequence of SEQ ID NO: 3, with hGALC, and another section consisting of the hTfR light chain, when in this, the amino acid sequence of the former is shown as SEQ ID NO: 162, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (6) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of five consecutive copies of the amino acid sequence of SEQ ID NO: 3, with hGALC, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 163, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (7) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked at its Terminal side and through a linker consisting of ten consecutive copies of the amino acid sequence of SEQ ID NO: 3 linked to hGALC , and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 164 and the amino acid sequence the latter is shown as SEQ ID NO: 23, and (8) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked at its Terminal side and through a linker consisting of 20 consecutive copies of the amino acid sequence of SEQ ID NO: 3, with hGALC, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 165 and the amino acid sequence of the latter being shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5)-(8) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5)-(8) are shown as SEQ ID NO: 61. A namely, the above-mentioned fusion proteins (5)-(8) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hAC include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hAC, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence with hAC, and another portion consisting of the hTfR light chain, wherein the amino acid sequence

- 64 042007 the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) a protein which consists of: a region consisting of the hTfR heavy chain linked on its Terminal side or N-terminal side and through a linker sequence with hAC, and another region consisting of the hTfR light chain, the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 27 , and (4) a protein that consists of: a region consisting of the hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hAC, and another region consisting of the hTfR light chain, wherein the amino acid sequence the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 29. In this case, it is preferred that the linker the sequence is a sequence consisting of one glycine, one serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence SEQ ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hAC is hAC having the amino acid sequence of SEQ ID NO: 86, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hAC include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through the amino acid sequence Gly-Ser to hAC, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 166, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its sequence of SEQ ID NO: 3, with hAC, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 167, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) protein, which consists of: a region consisting of the hTfR heavy chain linked at its terminal side and through a linker consisting of ten copies linked in series the amino acid sequence of SEQ ID NO: 3, with hAC, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 168 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (4) protein , which consists of: a region consisting of an hTfR heavy chain connected on its Terminal side and through a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to hAC, and another region consisting of an hTfR light chain, with in this, the amino acid sequence of the former is shown as SEQ ID NO: 169, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of consecutively linked three copies of the amino acid sequence of SEQ ID NO: 3, with hAC, and another site consisting of the hTfR light chain, while the amine the acid sequence of the former is shown as SEQ ID NO: 170 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (6) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of sequentially linked five copies of the amino acid sequence of SEQ ID NO: 3, with hAC, and another region consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 171, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (7) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked at its Terminal side and via a linker consisting of ten copies of the amino acid sequence of SEQ ID NO: 3 linked in series to hAC, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 172 and the amino acid sequence of the latter it is shown as SEQ ID NO: 23, and (8) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked at its Terminal side and via a linker consisting of 20 consecutive copies of the amino acid sequence of SEQ ID NO : 3, with hAC, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 173 and the amino acid sequence of the latter being shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5)-(8) are proteins whose antibody is of the Fab type.

- 65 042007

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and the heavy chain of which contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5)-(8) are shown as SEQ ID NO: 61. A namely, the above-mentioned fusion proteins (5)-(8) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hFUCA1 include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hFUCA1, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or N-terminal side and through a linker sequence with hFUCA1, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence and with hFUCA1 and another hTfR light chain region, wherein the heavy chain amino acid sequence is shown as SEQ ID NO: 66 or 68 and the light chain amino acid sequence is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hFUCA1, and another region consisting of an hTfR light chain, with the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 29. In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence and SEQ ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hFUCA1 is hFUCA1 having the amino acid sequence of SEQ ID NO: 87, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hFUCA1 include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and through the amino acid sequence Gly-Ser to hFUCA1, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 174, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its sequence of SEQ ID NO: 3, with hFUCA1, and another region consisting of the light chain of hTfR, the amino acid sequence of the former is shown as SEQ ID NO: 175, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) protein, which consists of: a region consisting of the hTfR heavy chain linked on its terminal side and through a linker consisting of ten consecutively linked copies of the amino acid sequence of SEQ ID NO: 3, with hFUCA1, and another portion consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 176 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (4) a protein that consists of: a region consisting of an hTfR heavy chain connected on its Terminal side and via a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 sequentially linked to hFUCA1, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the former is shown as SEQ ID NO: 177, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of consecutively linked three copies of the amino acid sequence of SEQ ID NO: 3, with hFUCA1, and another section consisting of the hTfR light chain , wherein the amino acid sequence of the former is shown as SEQ ID NO: 178 and the amino acid sequence of the latter is shown as SEQ ID NO: 23,

- 66 042007 (6) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked on its Terminal side and via a linker consisting of five consecutive copies of the amino acid sequence SEQ ID NO: 3 linked to hFUCA1, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 179 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (7) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked on its Terminal side and via a linker consisting of ten copies of the amino acid sequence of SEQ ID NO: 3, sequentially linked to hFUCA1, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO : 180, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, and (8) a protein that consists of: a region consisting of an hTfR heavy chain (Fab) linked to and its Terminal side and through a linker consisting of sequentially linked 20 copies of the amino acid sequence of SEQ ID NO: 3, with hFUCA1, and another site consisting of the hTfR light chain, while the amino acid sequence of the former is shown as SEQ ID NO: 181, and the amino acid the sequence of the latter is shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5)-(8) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5)-(8) are shown as SEQ ID NO: 61. A namely, the above-mentioned fusion proteins (5)-(8) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is hLAMAN include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hLAMAN, and another region consisting of an hTfR light chain, wherein the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked on its Terminal side or N-terminal side and via a linker sequence with hLAMAN, and another region consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) protein , which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence and with hLAMAN, and another hTfR light chain region, wherein the heavy chain amino acid sequence is shown as SEQ ID NO: 66 or 68 and the light chain amino acid sequence is shown as SEQ ID NO: 27, and (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to hLAMAN, and another region consisting of an hTfR light chain, with the amino acid sequence of the heavy chain shown as SEQ ID NO: 66 or 68, and the amino acid sequence of the light chain is shown as SEQ ID NO: 29. In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequence and SEQ ID NO: 5, and amino acid sequences consisting of 1-10 or 1-20 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type. In addition, hLAMAN is hLAMAN having the amino acid sequence of SEQ ID NO: 88, or mutants thereof.

More specific examples of a fusion protein when the other protein (A) is hLAMAN include:

(1) a protein which consists of: a region consisting of an hTfR heavy chain linked at its Terminal side and via the Gly-Ser amino acid sequence to hLAMAN, and another region consisting of an hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 182, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its sequence of SEQ ID NO: 3, with hLAMAN, and another region consisting of a light chain

- 67 042007 hTfR, wherein the amino acid sequence of the former is shown as SEQ ID NO: 183 and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (3) a protein which consists of: To the terminal side and through a linker consisting of ten copies of the amino acid sequence of SEQ ID NO: 3, sequentially linked to hLAMAN, and another section consisting of the hTfR light chain, the amino acid sequence of the former is shown as SEQ ID NO: 184, and the amino acid sequence of the latter shown as SEQ ID NO: 23, (4) a protein which consists of: a region consisting of the hTfR heavy chain linked at its Terminal side and via a linker consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3 linked in series to hLAMAN , and another region consisting of the hTfR light chain, with the amino acid sequence of the former shown as SEQ ID NO: 185, and the amino acid p the sequence of the latter is shown as SEQ ID NO: 23, (5) a protein which consists of: a region consisting of the hTfR heavy chain (Fab) linked at its Terminal side and via a linker consisting of three consecutive copies of the amino acid sequence of SEQ ID NO : 3, with hLAMAN, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 186 and the amino acid sequence of the latter being shown as SEQ ID NO: 23, (6) a protein that consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and via a linker consisting of five consecutive copies of the amino acid sequence of SEQ ID NO: 3 linked to hLAMAN, and another region consisting of an hTfR light chain, wherein the amino acid the sequence of the former is shown as SEQ ID NO: 187, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, (7) a protein which consists of: a region consisting of o from the hTfR heavy chain (Fab) linked on its Terminal side and through a linker consisting of ten copies of the amino acid sequence of SEQ ID NO: 3, sequentially linked to hLAMAN, and another section consisting of the hTfR light chain, while the amino acid sequence of the first shown as SEQ ID NO: 188, and the amino acid sequence of the latter is shown as SEQ ID NO: 23, and (8) a protein that consists of: a region consisting of an hTfR heavy chain (Fab) linked on its Terminal side and via a linker, consisting of 20 copies of the amino acid sequence of SEQ ID NO: 3, sequentially linked to hLAMAN, and another region consisting of the hTfR light chain, the amino acid sequence of the former being shown as SEQ ID NO: 189 and the amino acid sequence of the latter being shown as SEQ ID NO: 23.

Fusion proteins (1)-(4) are proteins whose antibody is of the IgG1 type, and fusion proteins (5)-(8) are proteins whose antibody is of the Fab type.

Here, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (1) to (4) are shown as SEQ ID NO: 68. Namely, the above fusion proteins (1) to (4) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23, and the heavy chain of which contains the amino acid sequence of SEQ ID NO: 68. In addition, the amino acid sequences of the hTfR heavy chains in the above fusion proteins (5)-(8) are shown as SEQ ID NO: 61. A namely, the above-mentioned fusion proteins (5)-(8) have a humanized antibody whose light chain contains the amino acid sequence of SEQ ID NO: 23 and whose heavy chain (Fab) contains the amino acid sequence of SEQ ID NO: 61.

Specific examples of a fusion protein when the other protein (A) is a human lysosomal enzyme include:

(1) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to a human lysosomal enzyme, and another region consisting of an hTfR light chain, wherein the amino acid sequence the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N- the terminal side and through a linker sequence with a human lysosomal enzyme, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 25, (3) a protein that consists of: a region consisting of the hTfR heavy chain linked on its Terminal side or N-those the rminal side and through a linker sequence with a human lysosomal enzyme, and another portion consisting of the hTfR light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 27, (4) a protein that consists of: a region consisting of an hTfR heavy chain linked at its Terminal side or N-terminal side and via a linker sequence to a human lysosomal enzyme, and another region consisting of an hTfR light chain, wherein

- 68 042007 The amino acid sequence of the heavy chain is shown as SEQ ID NO: 66 or 68 and the amino acid sequence of the light chain is shown as SEQ ID NO: 29.

In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequences of SEQ ID NO: 5, and amino acid sequences consisting of 1-10 of them that are sequentially linked.

In this case, the antibody having SEQ ID NO: 66 in the heavy chain is of the IgG1 type, and the antibody having SEQ ID NO: 68 is of the IgG4 type.

Specific examples of a fusion protein where the other protein (A) is a human lysosomal enzyme and the humanized antibody is a Fab antibody include:

(1) a protein which consists of: a region consisting of an hTfR Fab heavy chain linked at its Terminal side or N-terminus and via a linker sequence to a human lysosomal enzyme, and another region consisting of an hTfR light chain, wherein the amino acid sequence the heavy chain Fab is shown as SEQ ID NO: 61 and the amino acid sequence of the light chain is shown as SEQ ID NO: 23, (2) a protein that consists of: a region consisting of a heavy chain hTfR Fab linked at its Terminal side or N- end and through a linker sequence with a human lysosomal enzyme, and another portion consisting of the hTfR light chain, the Fab heavy chain amino acid sequence shown as SEQ ID NO: 61 and the light chain amino acid sequence shown as SEQ ID NO: 25, (3 ) a protein that consists of: a region consisting of the hTfR Fab heavy chain linked at its Terminal side or N-terminus and via a linker sequence with a human lysosomal enzyme, and another region consisting of the hTfR light chain, wherein the Fab heavy chain amino acid sequence is shown as SEQ ID NO: 61 and the light chain amino acid sequence is shown as SEQ ID NO: 27, (4) a protein that consists of: a region consisting of an hTfR Fab heavy chain linked at its Terminal side or N-terminus and via a linker sequence to a human lysosomal enzyme, and another region consisting of an hTfR light chain, with the amino acid sequence of the Fab heavy chain shown as SEQ ID NO: 61 and the amino acid sequence of the light chain is shown as SEQ ID NO: 29.

In this case, it is preferable that the linker sequence is a sequence consisting of one glycine, one serine, amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4, amino acid sequences of SEQ ID NO: 5, and amino acid sequences consisting of 1-10 of them that are sequentially linked.

A human lysosomal enzyme other than that described above can also be prepared as an anti-hTfR antibody fusion protein according to the same embodiments as those described for hGAA and hI2S. Examples of the human lysosomal enzyme include β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, β-mannosidase, galactosylceramidase, saposin C, aspartylglucosaminidase, α-galactosidase A, a-N- acetylglucosaminidase, acetyl-KoA:α-glucosaminide-N-acetyltransferase, N-acetylglucosamine-6-sulfate-sulfatase, amyl-1,6-glucosidase, sialidase, hyaluronidase 1, CLN1 and CLN2, although they are not limited to. Also, when a protein other than a lysosome is fused, a fusion protein can be obtained in the same manner.

The fusion protein wherein the other protein (A) is a human lysosomal enzyme has affinity for both human TfR and monkey TfR, and has the following dissociation constant when measured as described in Example 7: monkey TfR dissociation constant: preferably not more than 1x10 ' ¹⁰ M, more preferably not more than 5x10'11 M; and dissociation constant with human TfR: preferably not more than ^1x10'10 M, more preferably not more than 5x10'11 M, even more preferably not more than 1x10'11 M, even more preferably not more than ^1x10'12 M.

For example, dissociation constants with monkey TfR and human TfR are no more than 1x10' ¹⁰ M and no more than 1x10' ¹⁰ M, no more than 1x10' ¹⁰ M and no more than 1x10'11 M, no more than 1x10' ¹⁰ M and not more than ^1x10'12 M, not more than 5x10'11 M and not more than 1x10'11 M, not more than 5x10'11 M and not more than 1x10'11 M or not more than 5x10'11 M and not more than 1x10' ¹² M, respectively. In this context, although there is no particularly clear lower limit for the monkey TfR dissociation constant, it could be, for example, 1x10'11 M, ^1x10'12 M, or ^1x10'13 M. Although there is no particularly clear lower limit for the human TfR dissociation constant, it may be, for example, 1x10' ¹² M, 5x10' ¹³ M or 1x10' ¹³ M. The same also applies if the antibody is a single chain antibody.

- 69 042007

An anti-hTfR antibody of the present invention can be used to prepare a parenteral pharmaceutical for treating a disease state of the central nervous system by linking it to a physiologically active protein molecule or a pharmacologically active small molecule compound. And an anti-hTfR antibody conjugated to a molecule of a physiologically active protein or pharmacologically active small molecule compound can be used in a method of treating a patient with a disease state of the central nervous system, in which a therapeutically effective amount of a physiologically active protein or pharmacologically active small molecule compound is administered to a patient with a disease of the central nervous system parenterally (including intravenous injection such as intravenous infusion). An anti-hTfR antibody conjugated to a molecule of a physiologically active protein or a pharmacologically active small molecule compound, after parenteral administration, can not only enter the brain, but also reach other organs where hTfR is expressed. In addition, the pharmaceutical agent can be used to prevent the onset of a disease.

In particular, since the human acid α-glucosidase (hGAA) conjugate of the present invention, the anti-hTfR antibody of the present invention can allow hGAA to cross the blood-brain barrier and function in the brain, the antibody can be used to prepare a pharmaceutical agent for parenteral administration for the treatment of a disease state. central nervous system accompanying Pompe disease. In addition, an hGAA-conjugated hTfR antibody can be used in a method for treating a patient with a disease state of a central nervous system disorder accompanying Pompe disease, wherein a therapeutically effective amount of the antibody (including intravenous injection, such as intravenous infusion). After parenteral administration, an anti-hTfR antibody conjugated to hGAA can not only enter the brain but also reach other organs where hTfR is expressed. In addition, the pharmaceutical agent can be used to prevent the onset of a disease state.

In particular, since the human iduronate-2-sulfatase (hI2S) conjugate of the present invention, the anti-hTfR antibody can allow hI2S to cross the blood-brain barrier and function in the brain, the antibody can be used to prepare a parenteral pharmaceutical for the treatment of painful the state of the central nervous system that accompanies Hunter's syndrome. In addition, the hTfR antibody conjugated to hI2S can be used in a method of treating a patient with a disease state of a central nervous system disorder accompanying Hunter syndrome, wherein a therapeutically effective amount of the antibody is parenterally administered to a patient with Hunter syndrome (including an intravenous injection, such as intravenous infusion). After parenteral administration, an anti-hTfR antibody conjugated to hI2S can not only enter the brain, but also reach other organs where hTfR is expressed. In addition, the pharmaceutical agent can be used to prevent the onset of disorders.

In particular, since the human a-L-iduronidase (hIDUA) conjugate anti-hTfR antibody of the present invention can allow hIDUA to cross the blood-brain barrier and function in the brain, the antibody can be used to prepare a parenteral pharmaceutical for the treatment of central nervous system disease. nervous system that accompanies Hurler's syndrome. In addition, an hTfR antibody conjugated to hIDUA can be used in a method for treating a patient with a disease state of a central nervous system disorder accompanying Hurler's syndrome, wherein a therapeutically effective amount of the antibody is parenterally administered to a patient with Hurler's syndrome (including intravenous injection, such as intravenous infusion). After parenteral administration, an anti-hTfR antibody conjugated to hIDUA can not only enter the brain but also reach other organs where hTfR is expressed. The pharmaceutical agent can also be used to prevent such disease states.

Moreover, in particular, since the human lysosomal enzyme conjugate of the anti-hTfR antibody of the present invention can allow the human lysosomal enzyme to cross the blood-brain barrier and function in the brain, the antibody can be used to prepare a parenteral pharmaceutical for treating a disease state. central nervous system caused by human lysosomal enzyme deficiency. In addition, an anti-hTfR antibody conjugated to a human lysosomal enzyme can be used in a method of treating a patient with a central nervous system disease condition caused by human lysosomal enzyme deficiency, wherein a therapeutically effective amount of the antibody is parenterally administered to the patient (including intravenous injection such as intravenous infusion ). Anti-hTfR antibody conjugated with human lysosomal enzyme, after parenteral administration, can not only be ingested

- 70 042007 brain, but also reach other organs where the human lysosomal enzyme is expressed. In addition, the pharmaceutical agent can be used to prevent the onset of a disease state.

Proteins, a small molecule compound and the like which are conjugated with an anti-hTfR antibody of the present invention can be used as pharmaceuticals to exhibit their functions in the central nervous system (CNS) after parenteral administration. Such pharmaceutical agents can generally be administered to patients by intravenous injection such as intravenous injection, subcutaneous injection, intramuscular injection, and the like, although there is no particular restriction on the route of administration.

Proteins, small molecule compounds, and the like, which are conjugated with an anti-hTfR antibody of the present invention, can be supplied to healthcare facilities as pharmaceuticals in such forms as a lyophilized product or an aqueous preparation. In the case of an aqueous formulation, they can be supplied as formulations in which one of the pharmaceutical agents is pre-dissolved in a solution containing a stabilizer, a buffer and an isotonizing agent and sealed in vials or syringes. The type of preparation sealed in a syringe is commonly referred to as a prefilled preparation. The prefilled syringe form facilitates self-administration of the pharmaceutical agent by patients.

When the aqueous preparation is supplied, the concentration of the protein, small molecule and the like conjugated with the anti-hTfR antibody in the aqueous preparation is, for example, 1-4 mg/ml, although it needs to be adjusted according to the dosage if necessary. When there are no particular restrictions on the stabilizers to be contained in the aqueous preparation, since they are pharmaceutically available, non-ionic surfactants can be preferably used. Examples of such nonionic surfactants include polysorbate and poloxamer, any of which may be used alone or in combination. Among the polysorbates, polysorbate 20 and polysorbate 80 are preferably used. As the poloxamer, poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) glycol) is particularly preferred. In addition, the concentration of the non-ionic surfactant contained in the aqueous preparation is preferably 0.01-1 mg/ml, more preferably 0.01-0.5 mg/ml, and even more preferably 0.1-0.5 mg/ml. Amino acids such as histidine, arginine, methionine and glycine can also be used as stabilizers. When used as a stabilizer, the amino acid concentration in the aqueous formulation is preferably 0.1-40 mg/ml, more preferably 0.2-5 mg/ml, and even more preferably 0.5-4 mg/ml. Although there is no particular limitation on the buffer to be contained in the aqueous preparation, as long as it is pharmaceutically available, a phosphate buffer is preferred, and a sodium phosphate buffer is more preferred. When used as a buffer, the concentration of sodium phosphate is preferably 0.01-0.04 M. The pH of the buffer-adjusted aqueous preparation is preferably 5.5-7.2. Although there is no particular limitation on the isotonizing agent to be contained in the aqueous preparation as long as it is pharmaceutically available, sodium chloride or mannitol may preferably be used alone or in combination as the isotonizing agent.

Examples

Although the present invention is described in more detail below with reference to examples, these examples are not intended to limit the present invention. Examples 1-15 refer to the reference example (antibodies No. 3).

Example 1 Construction of an hTfR Expression Vector.

Using Quick Clone human spleen cDNA (Clontech Inc.) as a template and using primer hTfR5' (SEQ ID NO: 41) and primer hTfR3' (SEQ ID NO: 42), PCR was performed to amplify the gene fragment encoding the human transferrin receptor ( hTfR). The amplified fragment encoding hTfR was digested with MluI and NotI and then inserted between the MluI and NotI regions of the pCI-neo vector (Promega Inc.). The vector thus obtained was designated pCI-neo (hTfR). This vector was then cleaved with MluI and NotI to excise the hTfR-encoding gene fragment, and this fragment was inserted between the MluI and NotI regions of pE-mIRES-GS-puro, the expression vector disclosed in WO 2012/063799 to obtain the hTfR expression vector , pE-mIRES-GS-puro (hTfR).

Example 2 Preparation of recombinant hTfR.

pE-mIRES-GS-puro(hTfR) was electroporated into CHO-Ki cells and the cells were then selectively cultured in CD OptiCHO™ medium (Invitrogen Inc.) containing methionine sulfoximine (MSX) and puromycin to obtain recombinant hTfR expressing cells. . Expressing recombinant hTfR cells were cultured, and received recombinant hTfR.

Example 3 Immunization of mice with recombinant hTfR Mice were immunized with the recombinant hTfR obtained in Example 2 as an antigen. Immunization was carried out by means of intravenous

- 71 042007 injection or intraperitoneal injection of antigen into mice.

Example 4 Obtaining hybridoma cells.

Approximately one week after the last injection, the mice spleens were dissected and homogenized to isolate spleen cells. The spleen cells thus obtained were fused with cells of a mouse myeloma cell line (P3.X63.Ag8.653) by the polyethylene glycol method. After cell fusion, cells were suspended in RPMI 1640 medium supplemented with (1X) HAT (Life Technologies Inc.) and 10% ultra-low IgG fetal bovine serum (Life Technologies Inc.) and the cell suspension was distributed into 20 96-well plates, 200 µl/well. After culturing the cells for 10 days in a carbon dioxide incubator (37° C., 5% CO ₂ ), each well was examined under a microscope, and the wells that contained one colony were selected.

When the cells in each well almost reached confluence, the culture supernatant was collected as hybridoma culture supernatant and subjected to the next screening process.

Example 5 Screening for a High Affinity Antibody-Producing Cell Line.

Recombinant hTfR solution (Sino Biologies Inc.) was diluted with 50 mM sodium phosphate buffer (pH 9.5-9.6) to 5 μg/ml to obtain a solid phase solution. After adding 50 μl of solid phase solution (substrate: polystyrene, manufactured by Nunc Inc.) to each well of a Nunc MaxiSorp™ 96-well flat-bottomed plate, the plate was allowed to stand for one hour at room temperature, allowing recombinant hTfR to adhere and immobilize on the plate. The solid phase solution was discarded, each well was washed three times with 250 µl of wash solution (PBS containing PBST: 0.05% Tween20), then 200 µl of blocking solution (PBS containing 1% BSA) was added to each well, and the plate was allowed to stand for one hour at room temperature.

The blocking solution was discarded and each well was washed three times with 250 μl PBS-T. 50 μl of hybridoma culture supernatant was added to each well, and the plate was allowed to stand for one hour at room temperature, allowing the mouse anti-hTfR antibodies contained in the culture supernatant to bind to the recombinant hTfR. At the same time, 50 μl supernatant of a hybridoma culture that did not produce mouse anti-hTfR antibodies was added to some wells as a control. In addition, 50 μl of hybridoma culture medium was added to the wells as mock wells, except for those wells in which the culture supernatant was added. The measurement was carried out in the mode n=2. The solution was then discarded and each well was washed three times with 250 μl PBS-T.

100 μl of HRP-labelled goat anti-mouse immunoglobulin solution (Promega Inc.) was added to each of the above wells, and the plate was allowed to stand for 30 minutes at room temperature. The solution was then discarded and each well was washed three times with 250 μl PBST. 50 μl of a chromogenic substrate solution, a TMB-stabilized substrate for horseradish peroxidase (Promega Inc.) was added to each well, and the wells were allowed to stand for 10-20 minutes at room temperature. Then, after adding 100 μl of stopping solution (2N sulfuric acid), the absorbance of each well was measured on a plate reader at 450 nm. For each of the culture supernatant and control, respectively, the averages of two wells were taken, and from each of the averages, the corresponding average of the two mock wells placed corresponding to each of the culture supernatant and control was subtracted to provide a measurement.

Fourteen hybridoma cell types corresponding to culture supernatants added to wells that showed higher measurements were selected as cell lines (high affinity antibody producing cell line) that produce antibodies with high affinity for hTfR (high affinity anti hTfR antibodies). These fourteen types of cell lines were designated clonal line 1 - clonal line 14. From these cell lines, clonal line 3 was selected and used in the experiments below. In addition, the anti-hTfR antibodies generated by clonal line 3 were referred to as anti-hTfR antibodies #3.

Example 6 Analysis of the amino acid sequence of the variable region of high affinity anti-hTfR antibodies.

From clonal line 3 selected in Example 5, cDNA was obtained, using which the genes encoding the light chain and heavy chain of the antibody were amplified as a template. By translation of the nucleotide sequence of the amplified genes, the corresponding amino acid sequences of the light chain and heavy chain variable regions of the cell line generated anti-hTfR antibody No. 3 were determined.

The anti-hTfR antibody No. 3 was found to contain the amino acid sequence of SEQ ID NO: 48 as the light chain variable region and the amino acid sequence of SEQ ID NO: 49 as the heavy chain variable region. It was found that the variable region of the light chain contains the amino acid sequence of SEQ ID NO: 6 or 7, as CDR1; SEQ ID NO: 8 or 9 as CDR2 and SEQ ID NO: 10 as CDR3; and the heavy chain variable region contains the amino acid sequence SEQ ID NO: 11 or 12 as CDR1, SEQ ID NO: 13 or 14 as CDR2, and SEQ ID NO: 15 or 16 as CDR3. However, it was also considered that CDRs are not limited to CDRs that consist of these amino acid sequences, but they can also be regions or amino acid sequences that contain any of the above.

- 72 042007 sequences above, or amino acid sequences consisting of at least three consecutive amino acids containing part of the sequences mentioned above.

In table. 1 collects all SEQ ID NOs of the respective amino acid sequences contained in the light chain variable region CDR1-CDR3 and the heavy chain variable region CDR1-CDR3 of the anti-hTfR antibody #3. 1, these amino acid sequences are shown by way of example only and do not limit the amino acid sequence of each CDR to the amino acid sequences in Table 1. 1, but it was considered that CDRs are not limited to CDRs that consist of these amino acid sequences, but they can also be regions of either amino acid sequences that contain any of the sequences mentioned above, or amino acid sequences consisting of at least three consecutive amino acids containing part of the above sequences.

Table 1. Corresponding amino acid sequence numbers contained in the CDR1CDR3 of the light chain and heavy chain variable regions of anti-hTfR antibody #3

Antibody No. light chain variable region heavy chain variable region CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 3 6, 7 8, 9 10 11, 12 13, 14 15, 16

Example 7 Measurement of the affinity of an anti-hTfR antibody for human and monkey TfR.

The affinity of the anti-hTfR antibody for human and monkey TfR was measured on an Octet RED96 (ForteBio Inc., a division of Pall Corporation), a system for analyzing interactions between biomolecules using biolayer interferometry (BLI). The basic principles of biolayer interferometry are briefly explained below. When a layer of a biomolecule immobilized on the surface of a sensor tip is irradiated with light at a certain wavelength, the light is reflected from two surfaces, one at the biomolecule and the other at the inner, reference layer, producing interfering light waves. The molecule in the measured sample is bound to the biomolecule on the surface of the sensor tip and thus increases the thickness of the layers on the sensor tip, resulting in a shift between interfering waves. By measuring the fluctuations of this shift between interfering waves, it is possible in real time to determine the number of molecules associated with a layer of biomolecules immobilized on the surface of the sensor tip and their kinetic analysis. The measurement was carried out in general according to the instructions for use supplied with the Octet RED96. As the human TfR, a recombinant human TfR (human r TfR: Sino Biological Inc.) was used, which had the amino acid sequence of the extracellular region hTfR, i. a cysteine residue at position 89 from the N-terminal side to the phenylalanine at the C-terminus, amino acid sequence of SEQ ID NO: 1, with a histidine tag attached to the N-terminus. As the monkey TfR, a recombinant monkey TfR (r monkey TfR: Sino Biological Inc.) was used, which had the amino acid sequence of the extracellular region of the crabeater monkey TfR, i. a cysteine residue at position 89 from the N-terminal side to a phenylalanine at the C-terminus, amino acid sequence of SEQ ID NO: 2, with a histidine tag attached to the N-terminus.

Clonal line 3, selected in Example 5, was diluted with RPMI 1640 supplemented with (1X) HAT (Life Technologies Inc.) and 10% ultra low IgG fetal bovine serum (Life Technologies Inc.) in order to adjust cell density to approximately 2x10 ⁵ cells/ml. 200 ml of each cell suspension was added to a 1 L conical flask and cultured for 6-7 days in a humid environment at 37° C., 5% CO ₂ and 95% air, with shaking at approximately 70 rpm. To collect the culture supernatant, the culture medium was centrifuged and then filtered through a 0.22 μm filter (Millipore Inc.). The culture supernatant thus collected was loaded onto a Protein G column (column volume: 1 ml, GE Healthcare Inc.), which was equilibrated in advance with three column volumes of 20 mM Tris buffer (pH 8.0) containing 150 mM NaCl. After washing the column with 5 column volumes of the same buffer, adsorbed antibodies were eluted with 4 column volumes of 50 mM glycine buffer (pH 2.8) containing 150 mM NaCl, and the eluted fractions were collected. The eluted fractions were adjusted to pH 7.0 by adding 1M Tris buffer (pH 8.0). In the experiments described below, they were used as purified products of antibodies against hTfR No. 3.

The purified anti-hTfR antibody product #3 was subjected to 2-fold dilution steps with HBS-P+ (10 mM HEPES containing 150 mM NaCl, 50 μM EDTA and 0.05% P20 surfactant) to obtain antibody solutions of 7 different concentrations, 0, 78125-50 nm (0.117-7.5 µg/ml). The antibody solution was used as the sample solution. Monkey and human r TfR, respectively, were diluted with HBS-P+ to obtain 25 μg/ml solutions, which were used as human r TfR ECD solution (Histag) and monkey r TfR-ECD solution (Histag), respectively.

Each of the above sample solutions were added via 2-fold dilution steps, 200 μl/well, to a 96 well plate, black (Greiner Bio-One Inc.). Each of the above solutions of human r TfR-ECD (Histag) and monkey r TfR-ECD (Histag) solutions were added, 200

- 73 042007 µl/well, in the given wells. HBS-P+, 200 μl/well was added to the appropriate wells for baseline, dissociation and washing. 10 mM glycine-HCl (pH 1.7), 200 μl/well, was added to regeneration wells. In wells for activation was added 0.5 mm solution of NiCl ₂ , 200 μl/well. The plate and biosensor (Biosensor/Ni-NTA: ForteBio Inc., a division of Pall Corporation) were installed in the prescribed positions of the Octet RED96.

Octet RED96 was used under the conditions shown in Table 1. 2 below to collect data from which the association rate constant (kon) and the rate constant dissociation (koff) of anti-hTfR antibodies with human r TfR and monkey r TfR, and the dissociation constant (K _D ) was calculated. The measurement was carried out at 25-30°C.

Table 2 Octet RED96 Operating Conditions

Stage Contact time (sec) Speed (rpm) Threshold level 1 Baseline 1 60 1000 - 2 Loading 600 1000 1.5-2.0 3 Baseline 2 60 1000 - 4 Association 180 1000 - 5 Dissociation 540 1000 - 6 Regeneration 5 1000 - 7 Washing 5 1000 - Steps 6-7 were repeated 6-7 times 8 Activation 60 1000 - Steps 1-8 were repeated until all samples were measured.

Tab. 3 shows the results of measuring the association rate constant (kon), the dissociation rate constant (koff) of antibodies against hTfR #3, and the dissociation constant (k _D ) with human TfR and monkey TfR.

Table 3. Affinity of antibodies against hTfR No. 3 for human TfR and monkey TfR

kon (M ² s ² ) koff (with ^-1 ) K _D (M) Human TfR 6.53x10 ⁵ <1, 0x1~ ⁷ <1, 0x10~ ¹² TfR Monkeys 3.89x10 ⁵ <1, 0x1~ ⁷ <1, 0x10~ ¹²

As a result of measuring the affinity of anti-hTfR antibodies with human TfR, the dissociation constant of antibodies against hTfR No. 3 with human TfR was no more than 1x10-12 M, and the dissociation constant for monkey TfR was no more than 1x10 ¹² M. ^The result shows that the anti-hTfR antibody No. 3 is an antibody having high affinity not only for human TfR but also for monkey TfR.

Example 7-2. Evaluation of the transfer of anti-hTfR antibodies to the brain using mice.

BBB transmission to the brain was then assessed for anti-hTfR antibodies No. 3 by using hTfR knockout mice (hTfR-KI mice) in which the gene encoding the extracellular region of the mouse transferrin receptor was replaced with the gene encoding the extracellular region of the human transferrin receptor. hTfR-KI mice were obtained by the method generally described below. In addition, the purified products obtained in Example 7 were used as antibodies against hTfR No. 3.

A DNA fragment having the nucleotide sequence of SEQ ID NO: 45 was chemically synthesized, in which the neomycin resistance gene flanked by loxP sequences was placed on the 3' side of the cDNA encoding a chimeric hTfR, the intracellular region of which consisted of the amino acid sequence of mouse TfR, and the extracellular region consisted of the amino acid sequence of the human hTfR sequence. This DNA fragment was inserted into a targeting vector having as the 5'-arm sequence the nucleotide sequence of SEQ ID NO: 46 as the 3'-arm sequence and the nucleotide sequence of SEQ ID NO: 47 as the 3'-arm sequence, and the construct was introduced into mouse ES cells via electroporation. Mouse ES cells into which the gene had been introduced were selectively cultured in neomycin medium to select those mouse ES cells in which the targeting vector was introduced into the chromosome through homologous recombination. The recombinant mouse ES cells thus obtained were injected into 8-cell stage ICR mouse embryos (host embryos), and the embryos thus obtained were implanted into pseudo-pregnant mice (recipient mice) which were obtained by mating with mice subjected to vasoligation. The resulting offspring (chimeric mice) were examined for the color of their coat, and those mice were selected that had a higher proportion of white wool in their total hairline, i.e. those mice in which ES cells contributed more to the development of individual organisms. Each of these chimeric mice were mated with ICR mice to produce F1 mice. White-haired F1 mice were selected, DNA isolated from their tail tissue was analyzed, and those mice in which the mouse transferrin receptor gene had been replaced with a chimeric hTfR on their chromosomes were referred to as hTfR-KI mice.

The purified anti-hTfR #3 antibody product was fluorescently labeled with fluorescein isothiocyanate (FITC) using a Fluorescein Labeling Kit-NH2 (Dojindo Laboratories) according to the instructions provided. Received a solution of PBS containing fluorescently labeled FITC antibodies. The PBS antibody solution was intravenously injected with hTfR-KI mice (male, aged 10-12 weeks) at a dosage of anti-hTfR antibodies of 3 mg/kg. As a control, a PBS solution containing mouse IgG1 (Sigma Inc.) fluorescently labeled with FITC in the same manner as described above was intravenously injected with hTfR-KI mice (male, aged 10-12 weeks) at a dose of 3 mg/kg. Approximately eight hours after intravenous injection, the whole body was washed with physiological saline, and the cerebrum (part including the cerebrum and cerebellum) was obtained. The thus excised brain was weighed (wet weight) and then the brain tissues were homogenized with T-PER (Thermo Fisher Scientific Inc.) containing a mixture of protease inhibitors (Sigma Inc.). The homogenate was centrifuged, the supernatant was collected, and the amount of fluorescently labeled FITC antibodies contained in the supernatant was measured as follows. First, 10 µl of anti-FITC antibody (Bethyl Inc.) was added to each well of a highly binding plate (Meso Scale Diagnostics Inc.) and allowed to stand for one hour in order to immobilize them on the plate. The plate was then blocked by adding 150 μl of SuperBlock blocking buffer in PBS (Thermo Fisher Scientific Inc.) to each well and shaking the plate for one hour. Then, 25 µl of brain tissue homogenate supernatant was added to each well, and the plate was shaken for one hour. Then, 25 μl of anti-mouse antibody (Goat) SULFO-TAG (Meso Scale Diagnostics Inc.) was added to each well, and shaking was continued for one hour. Then, 150 µl of Reading Buffer T (Meso Scale Diagnostics Inc.) was added to each well, and the luminescence value from each well was read on a Sector™ Imager 6000 reader. against hTfR in brain tissues) was calculated by obtaining a standard curve based on measurements of standard samples containing known concentrations of fluorescently labeled FITC antibodies against hTfR, and then interpolating the measurement of each of the samples with reference to the standard. The results are shown in table. 4.

The concentration of antibodies against hTfR No. 3 in the brain tissues was approximately 27.8 times greater than the concentration in the control. The result means that anti-hTfR #3 antibodies pass into the brain, actively passing through the BBB.

Table 4. Concentration of antibodies against hTfR in brain tissues

Antibodies No. brain tissues (µg/g wet weight) Relative value to control Control 0.003 1 3 0.0833 27.8

Example 8 Pharmacokinetic analysis of anti-hTfR antibodies in a monkey.

Anti-hTfR antibody No. 3 was administered intravenously once to a male crabeater monkey at a dosage of 5.0 mg/kg, and 8 hours after administration, the whole body was washed with physiological saline. As a negative control, a monkey that did not receive anti-hTfR antibodies was subjected to a whole body wash in the same manner. After washing, the brain tissue, including the medulla oblongata, was excised. Using brain tissue, the concentration of antibodies against hTfR was measured, and immunohistochemical staining was performed. The anti-hTfR No. 3 antibodies used were the purification products of the antibodies described in Example 7.

Measurement of the concentration of anti-hTfR antibodies in brain tissues was carried out largely following the procedure described below. The collected brain tissues were divided into cerebrum, cerebellum, hippocampus and medulla oblongata, and they were respectively homogenized with RIPA buffer (Wako Pure Chemical Industries Inc.) containing a mixture of protease inhibitors (Sigma-Aldrich Inc.) and centrifuged to collect the supernatant . Affinity purified goat anti-mouse IgG Fcy pAb (Jackson ImmunoResearch Inc.), 10 μl each, was added to the wells of a high binding plate (Meso Scale Diagnostics Inc.) and the plate was allowed to stand for one hour to immobilize the antibodies. The plate was then blocked by adding 150 μl of SuperBlock blocking buffer in PBS (Thermo Fisher Scientific Inc.) to each well and shaking for one hour. Then, 25 µl of brain tissue homogenate supernatant was added to each well, and the plate was shaken for one hour. Then, 25 µl of affinity purified goat anti-mouse IgG Fab-Biotin (Jackson ImmunoResearch Inc.) was added to each well and shaking was continued for one hour. Then, 25 μl of streptavidin SULFO-Tag (Meso Scale Diagnostics Inc.) was added to each well, and shaking was continued for 30 minutes. 150 µl of Reading Buffer T (Meso Scale) was added to each well.

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Diagnostics Inc.), and the luminescence value from each well was read on a Sector™ Imager 6000 reader (Meso Scale Diagnostics). The amount of anti-hTfR antibodies contained per gram of brain (wet weight) (concentration of anti-hTfR antibodies in brain tissue) was calculated by obtaining a standard curve based on the measurement of standard samples containing known concentrations of anti-hTfR antibodies, and then interpolating the measurement of each of the samples with reference to the standard.

The result of measuring the concentration of antibodies against hTfR in brain tissues is shown in table. 5. Anti-hTfR #3 antibodies were observed to accumulate in all parts from the cerebrum, cerebellum, hippocampus and medulla oblongata. These results demonstrate that anti-hTfR #3 antibodies had the ability to cross the blood-brain barrier and accumulate in brain tissues, and show that by binding these antibodies to a pharmaceutical agent to be delivered to function in brain tissues, efficient accumulation of these pharmaceuticals in brain tissue.

Table 5. Concentration of antibodies against hTfR in brain tissues (µg/g wet weight)

Antibody No. big brain cerebellum hippocampus cervical region spinal cord 3 0.72 0.6 0.33 0.31

Immunohistochemical staining of antibodies against hTfR in these brain tissues was performed generally following the procedures described below. The harvested tissues were flash frozen to -80° C. in a Tissue-Tek Cryo 3DM (Sakura Finetek Inc.) to obtain frozen tissue blocks. Frozen blocks were cut into 4 μm sections, which were placed on MAS-coated glass slides (Matsunami Glass Inc.). Tissue sections were reacted with 4% paraformaldehyde (Wako Pure Chemical Industries Inc.) for 5 minutes at 4°C and fixed on glass slides. The tissue sections were then reacted with a methanol solution containing 0.3% hydrogen peroxide (Wako Pure Chemical Industries Inc.) for 30 minutes. For inactivation of own peroxidases. The slides were then blocked by reaction with SuperBlock blocking buffer in PBS for 30 minutes. At room temperature. The tissue sections were then reacted with anti-mouse IgG heavy and light chain antibodies (Bethyl Laboratories Inc.) for one hour at room temperature. Tissue sections were allowed to change color with DAB substrate (3,3'-diaminobenzidine, Vector Laboratories Inc.), counterstained with Mayer's hematoxylin solution (Merck Inc.), dehydrated, cleaned, stacked and viewed under a microscope.

In FIG. 1 shows the result of immunohistochemical staining of antibodies against hTfR in the cerebral cortex. In the cerebral cortex of monkeys injected with anti-hTfR No. 3 antibodies, specific staining of blood vessels was observed (FIG. 1b). In addition, a specific extensive staining of the area of the brain parenchyma outside the blood vessels was also observed. In contrast, no staining was observed in the cerebral cortex of the control monkey, which was not injected with anti-hTfR antibodies, indicating that there was almost no background staining (Fig. 1a).

In FIG. 2 shows the result of immunohistochemical staining of antibodies against hTfR in the hippocampus. In the cerebral cortex of monkeys injected with anti-hTfR No. 3 antibodies, specific staining of blood vessels was observed (FIG. 2b). In addition, specific staining of nerve-like cells was also observed, and specific and extensive staining of the brain parenchyma region outside the blood vessels was also observed. On the other hand, no staining was observed in the control hippocampus without administration of anti-hTfR antibodies, indicating that there was almost no background staining (Fig. 2a).

In FIG. 3 shows the result of immunohistochemical staining of antibodies against hTfR in the cerebellum. In the cerebral cortex of monkeys injected with anti-hTfR No. 3 antibodies, specific staining of blood vessels was observed (FIG. 3b). In addition, specific staining of Purkinje cells was also observed. In contrast, no staining was observed in the control cerebellum without administration of anti-hTfR antibodies, indicating that there was almost no background staining (Fig. 3a).

From the above results of immunohistochemical staining of the cerebrum, hippocampus, and cerebellum, it was found that antibodies against hTfR #3 can bind to hTfRs located on the endothelium of cerebral blood vessels, and after binding to hTfR, they pass through the blood-brain barrier and pass into the brain parenchyma. , and further from the brain parenchyma to nerve-like cells in the hippocampus, and are taken up by Purkinje cells in the cerebellum.

Example 9 Preparation of Humanized Anti-hTfR Antibodies.

The amino acid sequence contained in the variable regions of the light chain and the heavy chain of the anti-hTfR antibody No. 3 was humanized. Thus, a humanized light chain variable region having one of the amino acid sequences shown as SEQ ID NO: 17 - SEQ ID NO: 22 was obtained, and a humanized heavy chain variable region having one of the amino acid sequences shown as SEQ ID NO: 31 - SEQ ID NO: 36.

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Example 10 Generation of Genes Encoding Humanized Anti-hTfR Antibodies.

For the above-mentioned anti-hTfR antibodies No. 3, DNA fragments were artificially synthesized that contained a gene encoding a full-length light chain and a heavy chain having the light chain and heavy chain variable regions of a humanized anti-hTfR antibody, respectively. In this case, the MluI sequences and the sequence encoding the leader peptide were added, in this order from the 5' end, on the 5' side of the gene encoding the full-length light chain, and the NotI sequence was added on the 3' side. And the MluI sequences and the leader peptide coding sequence were added in this order from the 5' end, on the 5' side of the full-length heavy chain coding gene, and the NotI sequence was added on the 3' side. The leader peptide introduced above is designed to function as a secretion signal when the light chain and heavy chain of the humanized antibody are expressed in mammalian cells as host cells in order to secrete the light chain and the heavy chain from the cells.

For the light chain of anti-hTfR antibody No. 3, a DNA fragment (SEQ ID NO: 24) was synthesized, which contained a gene encoding a full-length light chain (light chain of a humanized anti-hTfR antibody No. 3) consisting of the amino acid sequence of SEQ ID NO: 23, which had in the variable region, the amino acid sequence of SEQ ID NO: 18.

With respect to the light chain of anti-hTfR antibody No. 3, a DNA fragment (SEQ ID NO: 26) encoding the full-length amino acid sequence of the light chain (light chain of humanized anti-hTfR antibody No. 3-2) was also synthesized, consisting of the amino acid sequence of SEQ ID NO: 25 , which had in the variable region the amino acid sequence of SEQ ID NO: 20;

DNA fragment (SEQ ID NO: 28) encoding the full-length amino acid sequence of the light chain (light chain of humanized anti-hTfR antibody No. 3-3) consisting of the amino acid sequence of SEQ ID NO: 27, which had in the variable region the amino acid sequence of SEQ ID NO: 21;

a DNA fragment (SEQ ID NO: 30) encoding the full-length amino acid sequence of the light chain (the light chain of the humanized anti-hTfR antibody No. 3-4) consisting of the amino acid sequence of SEQ ID NO: 29, which had in the variable region the amino acid sequence of SEQ ID NO: 22.

For the heavy chain of anti-hTfR antibody No. 3, a DNA fragment (SEQ ID NO: 38) was synthesized that encoded a full-length heavy chain (heavy chain of humanized anti-hTfR antibody No. 3) consisting of the amino acid sequence of SEQ ID NO: 37, which had in the variable region amino acid sequence of SEQ ID NO: 32. The heavy chain of the humanized anti-hTfR antibody encoded by the DNA fragment shown as SEQ ID NO: 38 is IgG1.

In addition, for the heavy chain of anti-hTfR antibody No. 3, a DNA fragment was also synthesized (SEQ ID NO: 40) encoding the full-length amino acid sequence of the heavy chain (IgG4 heavy chain of humanized anti-hTfR antibody No. 3) consisting of the amino acid sequence of SEQ NO: 39, which had in the variable region the amino acid sequence of SEQ ID NO: 32.

The heavy chain of the humanized anti-hTfR antibody encoded by the DNA fragment shown as SEQ ID NO: 40 is IgG4.

Example 11 Construction of an expression vector for a humanized anti-hTfR antibody.

The pEF/myc/nuc vector (Invitrogen Inc.) was digested with KpnI and NcoI to excise the region containing the EF-loc promoter and its first intron, and this was a blunt-ended T4 DNA polymerase. The region containing the CMV enhancer/promoter and intron was removed from pCI-neo (Invitrogen Inc.) by digesting it with BglII and EcoRI, and the remaining fragment thus remaining was blunt-ended T4 DNA polymerase. To obtain the pE-neo vector, the region mentioned above containing the EF-1a promoter and its first intron was inserted into it. This vector, pE-peo, was digested with SfiI and BstXI to remove the approximately 1 kb region containing the neomycin resistance gene. PCR was performed using pcDNA3,1/Hygro(+)(Invitrogen) as template and using primer Hyg-Sfi5' (SEQ ID NO: 43) and primer Hyg-BstX3' (SEQ ID NO: 44) to amplify the hydromycin gene . To obtain the pE-hygr vector, the hydromycin gene thus amplified was digested with SfiI and BstXI and inserted into the pE-peo vector mentioned above, from which the neomycin resistance gene had been removed.

The pE-hygr and pE-neo vectors were digested with both MluI and NotI. A DNA fragment (SEQ ID NO: 24) encoding the humanized anti-hTfR antibody No. 3 light chain and a DNA fragment (SEQ ID NO: 38) encoding the antibody heavy chain, both synthesized in Example 10, were digested with MluI and NotI, and the fragments thus obtained were inserted into the pE-hygr vector and the pE-neo vector, respectively, between their MluI and NotI regions. The vectors thus obtained were used as the expression vector for the light chain of the humanized anti-hTfR No. 3 antibody, pE-hygr(LC3), and as the expression vector for the heavy chain of the antibody, pE-neo(HC3), in the experiments described below.

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In addition, with respect to the light chain of anti-hTfR antibody No. 3, the following fragments were synthesized in Example 10, namely:

a DNA fragment (SEQ ID NO: 26) encoding the light chain of a humanized anti-hTfR antibody No. 3-2, a DNA fragment (SEQ ID NO: 28) encoding a light chain of a humanized anti-hTfR antibody No. 3-3, and a DNA fragment (SEQ ID NO: 30) encoding the light chain of the humanized anti-hTfR antibody #3-4 was digested with MluI and NotI and inserted into the pE-hygr vector between its MluI and NotI regions to obtain pE-hygr (LC3-2), an expression vector for light chain of a humanized anti-hTfR antibody #3-2, pE-hygr (LC3-3), an expression vector for the light chain of a humanized anti-hTfR antibody #3-3, and pE-hygr (LC3-4), an expression vector for a humanized light chain antibodies against hTfR No. 3-4, respectively.

In addition, in the same manner as described above for the heavy chain of anti-hTfR antibody No. 3, a DNA fragment (SEQ ID NO: 40) encoding the humanized anti-hTfR antibody IgG4 heavy chain No. 3 synthesized in Example 10 was digested with MluI and NotI and inserted into the pE-peo vector between its MluI and NotI regions to generate pE-neo (HC3-IgG4), an expression vector for the humanized anti-hTfR antibody IgG4 heavy chain #3.

Example 12. Obtaining cells for the expression of humanized antibodies against hTfR.

CHO cells (CHO-K1: purchased from the American Type Culture Collection) were transformed with pE-hygr(LC3), a light chain expression vector, and pE-neo(HC3), a heavy chain expression vector, both of which were obtained in example 11 as follows, using GenePulser (BioRad Inc.). The transformation of cells as a whole was carried out as follows. CHO-Ki cells, 5x105, were seeded in a 3.5 cm culture dish containing CD OptiCHO™ medium (Life Technologies Inc.) and cultured overnight at 37°C, 5% CO ₂ . Medium was replaced with Opti-MEM™ I medium (Life Technologies Inc.) and cells were suspended at a density of 5x106 cells/ml. One hundredth µl of cell suspension was taken, to which 5 µl of each of pE-hygr (LC3) solution and pEneo (HC3) plasmid DNA solution were added, both diluted with Opti-MEM™ I medium to 100 µg/ml. These plasmids were introduced into cells by electroporation using a GenePulser (Bio-Rad Inc.). The cells were then cultured overnight at 37° C., 5% CO ₂ and subjected to selection culture in D OptiCHO™ medium supplemented with 0.5 mg/ml hygromycin and 0.8 mg/ml G418.

Then, the cells selected above by selection culture were plated in 96-well plates so as to seed no more than one cell per well by limiting dilution. The cells were then cultured for about 10 days so that monoclonal colonies were formed. The corresponding culture supernatants of wells in which a monoclonal colony had formed were collected, the amount of humanized antibodies contained in the culture supernatants was determined by ELISA, and cell lines with high expression of humanized antibodies were selected.

The above-mentioned ELISA was carried out in general as follows. To each well of 96-well microtiter plates (Nunc Inc.) was added 100 μl of a solution of goat anti-human IgG polyclonal antibodies diluted in 0.05 M sodium bicarbonate buffer (pH 9.6) to 4 μg/ml, and the plate was allowed to stand for at least one hour at room temperature in order to allow the adsorption of antibodies to the plates. Then, after washing the wells three times with PBS-T, 200 μl of Starting Block Buffer (PBS) (Thermo Fisher Scientific Inc.) was added to each well and the plates were allowed to stand for 30 minutes at room temperature. After washing each well with PBS-T, culture supernatant or standard human IgG product was added three times to each well, which was diluted with PBS supplemented with 0.5% BSA and 0.05% Tween20 (PBS-BT) to the desired concentrations, in an amount of 100 μl, and the plates were left to stand for at least one hour at room temperature. After washing the plates three times with PBS-T, 100 μl of a solution of HRP labeled anti-human IgG polyclonal antibodies that had been diluted with PBS-BT was added to each well, and the plates were allowed to stand for at least one hour at room temperature. After washing the wells three times with PBS-T, 0.4 mg/ml of ophenylenediamine in citrate-phosphate buffer (pH 5.0) was added to each well in an amount of 100 μl, and the wells were left to stand for 8-20 minutes at room temperature. Then, 1 mol/L sulfuric acid was added to each well in an amount of 100 μL to complete the reaction, and the absorbance for each well at 4-90 nm was measured using a 96-well plate reader. Cells corresponding to the wells that exhibited the above measurements were assigned to a highly expressing cell line for humanized anti-hTfR antibody #1.

- 78 042007 exact line.

In addition, in the same manner, CHO cells were transformed with the light chain expression vector pE-hygr (LC3-2) and the heavy chain expression vector pE-peo(HC3), both obtained in Example 11, and a cell line with high expression for humanized antibodies against hTfR No. 3-2. They were designated antibody-expressing No. 3-2 cell line.

In addition, in the same manner, CHO cells were transformed with the light chain expression vector pE-hygr (LC3-3) and the heavy chain expression vector pE-neo (HC3), both of which were obtained in Example 11, and a cell line with high expression for humanized antibodies against hTfR No. 3-3. They were designated antibody-expressing No. 3-3 cell line.

In addition, in the same manner, CHO cells were transformed with the light chain expression vector pE-hygr (LC3-4) and the heavy chain expression vector pE-neo (HC3), both of which were obtained in Example 11, and a cell line with high expression for humanized antibodies against hTfR No. 3-4. They were designated antibody-expressing No. 3-4 cell line.

In addition, in the same manner, CHO cells were transformed with the light chain expression vector pE-hygr(LC3) and the heavy chain expression vector pE-neo(HC3-IgG4), both obtained in Example 11, and a cell line with high expression for humanized antibodies against hTfR No. 3 (IgG4). They were designated antibody-expressing No. 3 (IgG4) cell line.

In addition, in the same manner, CHO cells were transformed with pE-hygr (LC3-2) light chain expression vector and pE-neo (HC3-IgG4) heavy chain expression vector, both were obtained in Example 11, and a cell line was obtained. with high expression for humanized antibodies against hTfR No. 3-2 (IgG4). They were designated expressing antibodies No. 3-2 (IgG4) cell line.

Example 13 Purification of Humanized Anti-hTfR Antibodies.

The No. 3 antibody expressing cell line, No. 3-2 antibody expressing cell line, No. 3-3 antibody expressing cell line, and No. 3-4 antibody expressing cell line obtained in Example 12, respectively, were diluted with CD OptiCHO™ medium to a density of approximately 2x105 cells/ml. Cell suspensions, 200 ml, were added to a 1 L conical flask and cultured for 6-7 days in humid environment at 37° C., 5% CO ₂ , 95% air with shaking at approximately 70 rpm. To obtain a culture supernatant, each culture supernatant was collected by centrifugation and filtered through a 0.22 μm filter (Millipore Inc.). Five volumes of 20 mM Tris buffer (pH 8.0) containing 150 mM NaC were added to each culture supernatant thus obtained, and loaded onto a Protein A column (column volume: 1 ml, Bio-Rad Inc.), which was pre-equilibrated with three column volumes of 20 mM Tris buffer (pH 8.0) containing 150 mM NaCl. The column was then washed with five column volumes of the same buffer, and the adsorbed humanized antibodies were eluted with four column volumes of 50 mM glycine buffer (pH 2.8) containing 150 mM NaCl, and the eluted fraction was collected. The eluted fractions were added and neutralized with 1 M Tris buffer (pH 8.0) and used as a preparation of purified antibodies.

In the above, purified antibodies from the culture supernatant of the antibody-expressing cell line #3 were designated humanized anti-hTfR antibodies #3. Purified antibodies from the culture supernatant of the antibody-expressing cell line #3-2 were designated humanized anti-hTfR antibodies #3-2. Purified antibodies from the culture supernatant of the antibody-expressing cell line #3-3 were designated humanized anti-hTfR antibodies #3-3. Purified antibodies from the culture supernatant of the antibody-expressing cell line #3-4 were designated humanized anti-hTfR antibodies #3-4.

In addition, the No. 3 (IgG4) expressing antibody cell line and No. 3-2 (IgG4) expressing antibody cell line obtained in Example 12 were also cultured in the same manner as described above, and purified humanized antibodies against hTfR #3 (IgG4) and humanized anti-hTfR #3-2 (IgG4), respectively. These two antibodies were used in a pharmacokinetic analysis using the monkeys described in Example 15.

Example 14 Measurement of the affinity of humanized anti-hTfR antibodies for human TfR and monkey TfR.

The affinity of the humanized anti-hTfR antibodies prepared in Example 13 for human and monkey TfR was measured by the method described in Example 7. Table. 6 shows the measurement result of association rate constant (kon), dissociation rate constant (koff), and dissociation constant (k _D ) of humanized anti-hTfR antibodies No. 3-3-4 (corresponding to No. 3-3-4, respectively, in the table) with TfR person.

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Table 6 Affinity of humanized anti-hTfR antibodies with human TfR

Antibody No. Kon (M-4c-4) 3 1.19*10 ⁶ 3-2 6.06* ¹⁰⁵ 3-3 6.00* ¹⁰⁵ 3-4 1.01*10 ⁶

koff (c ¹ ) k _D (M) <1.0*1(G ⁷ <1.0*10~ ¹² 1.45*10~ ⁵ <2.39*10-44 1.25*10~ ⁵ <2.09*10-41 1.0*10~ ⁷ <1.0*10~ ¹²

In table. 7 shows the result of measuring association rate constant (kon), dissociation rate constant (koff), and dissociation constant (kD) of humanized anti-hTfR antibodies No. 3-3-4 (corresponding to No. 3-3-4, respectively, in the table) with monkey TfR .

Table 7

Antibody No. Kon (M ² c 4) koff (c ^-1 ) k _D (M) 3 6.03* ¹⁰⁵ 6.76*10~ ⁴ 1, 12* ^10-9 3-2 4.95* ¹⁰⁵ 8.76* ^10-4 1.77* ^10-9 3-3 4.88* ¹⁰⁵ 9.32*10-4 1.91* ^10-9 3-4 5.19* ¹⁰⁵ 1.35*10-4 2.6O*1O- ¹⁰

The result of measuring the affinity of humanized anti-hTfR antibodies no. 3-3-4 to human TfR showed that the dissociation constant between humanized anti-hTfR antibodies no. 3 and 3-4 and human TfR was less than 1 x ^10-12 M (Table 6). And the dissociation constant between humanized antibodies against hTfR #3-2 and 3-3 and human TfR was 2.39x10-11 M and 2.09x10-11 M, respectively. At the same time, the dissociation constant between pre-humanized anti-hTfR antibodies (antibody no. 3) corresponding to these antibodies and human TfR was: ^1x10-12 M (Table 3). These results demonstrate that the high affinity of these pre-humanized anti-hTfR antibodies to human TfR was maintained after humanization of the antibodies.

Then looking at the result of measuring the affinity of the humanized anti-hTfR antibody to the monkey TfR for the humanized anti-hTfR antibody #3-3-4, although the dissociation constant of the anti-hTfR antibody #3, their corresponding pre-humanized antibody, with the monkey TfR was less than 1x10- ¹² M, their dissociation constant after humanization was from 2.60x10-10 M to 1.91x10-9 M, indicating a decrease in affinity for monkey TfR. With respect to humanized anti-hTfR antibodies #3, although a decrease in affinity for monkey TfR was observed, the result indicates that the high affinity in pre-humanization of anti-hTfR antibodies to monkey TfR was not lost after humanization, but was generally maintained.

Example 15 Pharmacokinetic analysis of humanized anti-hTfR antibodies in a monkey.

Using monkeys, pharmacokinetic analyzes of four antibodies were performed: humanized anti-hTfR antibody #3, humanized anti-hTfR antibody #3-2, humanized anti-hTfR antibody #3 (IgG4), and humanized anti-hTfR antibody #3-2 (IgG4). In addition, the heavy chain of the humanized anti-hTfR #3 antibody was IgG1, although in the humanized anti-hTfR #3 (IgG4) antibody, the heavy chain of the humanized anti-hTfR #3 antibody was converted to IgG4, leaving its variable region intact. In addition, the heavy chain of the humanized anti-hTfR antibody No. 3-2 was IgG1, although in the humanized anti-hTfR antibody No. 3-2 (IgG4), the heavy chain of the humanized anti-hTfR antibody No. 3-2 was converted to IgG4, with its variable region remaining intact. . These four antibodies were respectively administered once intravenously to male crabeater monkeys at a dosage of 5.0 mg/kg, and a sample of their peripheral blood was taken before administration, at 2 minutes, 30 minutes, 2 hours, 4 hours and 8 hours after administration. , and then they were subjected to washing of the whole body. As a negative control, trastuzumab (Herceptin™, Chugai Pharmaceutical Co., Ltd.), a humanized antibody against HER2 protein, was administered intravenously once in the same manner to one monkey, and a sample of its peripheral blood was taken before administration, after 2 minutes, 30 minutes, 2 hours, 4 hours and 8 hours after administration, and then she was subjected to whole body washing. After washing, tissues of the brain and spinal cord were excised, including the medulla oblongata and other tissues (liver, heart, spleen, and bone marrow). Using these brain and spinal cord tissues and other tissues, the concentration of humanized anti-hTfR antibody was measured, and immunohistochemical staining was performed.

Measurement of the concentration of humanized antibodies against hTfR in tissues and peripheral blood was carried out largely following the procedure described below. In addition, with regard to the brain, the obtained tissues were separated into the cerebral cortex, cerebellum, hippocampus and medulla oblongata, and then the concentration of humanized anti-hTfR antibodies was measured. Appropriate tissues thus obtained were homogenized with RIPA buffer (Wako Pure Chemical Industries Inc.) containing a mixture of protease inhibitors (Sigma-Aldrich Inc.), centrifuged, and the supernatant was collected. Serum was isolated from the peripheral blood mentioned above. Goat Biotinylated Anti-Carr Human IgG Light Chain (Immuno-Biological Laboratories Co, Ltd.), Sul Antibodies

- 80 042007 anti-human IgG (H+L) fo-tag (Bethyl Inc.) and brain tissue homogenate were added to streptavidin plate (Meso Scale DiagNotics Inc.), blocked with SuperBlock blocking buffer in PBS (Thermo Fisher Scientific Inc.), and the tablet was shaken for one hour for immobilization. 150 μl of reading buffer T (Meso Scale DiagNotics Inc.) was added to each well, and the amount of luminescence from each well was measured on a Sector™ Imager 6000 reader. The amount of antibodies contained in each tissue and peripheral blood was calculated by obtaining a standard curve based on the measurement standard samples containing known concentrations of anti-hTfR antibodies, and then interpolating the measurement of each of the samples with reference to the standard. The concentration measurement was repeated three times for each sample.

In table. 9 shows the result of measuring the concentration of humanized anti-hTfR antibodies in the tissues of the brain and spinal cord.

Table 8 Concentration of Humanized Anti-hTfR Antibodies in Brain Tissues (µg/g ww)

Antibody No. bark cerebellum hippocampus oblong dorsal head brain brain brain 3 0.6710.12 0.6110.02 0.4910.02 0.5910.10 0.4610.17 3-2 1.0510.07 0.7210.04 0.7210.07 0.6910.03 0.4610.02 3(IgG4) 0.6510.05 0.5910.03 0.5610.02 0.5910.02 0.4610.07 3-2(IgG4) 0.7610.02 0.5710.07 0.6210.05 0.7310.16 0.4810.03 Negative control 0.008210.00 32 0.009010.0067 0.005310.0009 0.01110.003 0.1510.04

It was observed that all antibodies, ie. humanized anti-hTfR antibody #3, humanized anti-hTfR antibody #3-2, humanized anti-hTfR antibody #3 (IgG4), and humanized anti-hTfR antibody #3-2 (IgG4), accumulate in cortex, cerebellum, hippocampus, medulla oblongata and spinal cord (Table 8). The corresponding accumulated amount was as follows:

humanized anti-hTfR #3 antibodies are approximately 82-fold greater in the cerebral cortex, approximately 68-fold in the cerebellum, approximately 92-fold in the hippocampus, approximately 54-fold in the medulla oblongata, and approximately 3.1-fold in the spinal cord compared with the negative control, trastuzumab (Herceptin™), humanized anti-hTfR #3-2 antibodies are approximately 128-fold greater in the cerebral cortex, approximately 80-fold in the cerebellum, approximately 136-fold in the hippocampus, approximately 63-fold in the medulla oblongata , approximately 3.1-fold in the spinal cord compared to the negative control, trastuzumab, humanized anti-hTfR #3 (IgG4) antibodies are approximately 79-fold greater in the cerebral cortex, approximately 66-fold in the cerebellum, approximately 106-fold in hippocampus, approximately 54 times in the medulla oblongata, approximately 3.1 times in the spinal cord compared with the negative control, trastuzumab, and in humanized a antibody against hTfR #3-2 (IgG4) is approximately 93-fold greater in the cerebral cortex, approximately 63-fold in the cerebellum, approximately 117-fold in the hippocampus, approximately 66-fold in the medulla oblongata, approximately 3.2-fold in the spinal cord compared with the negative control, trastuzumab (Table 1). 9).

These results show that these four humanized anti-hTfR antibodies have a property that allows them to pass through the blood-brain barrier and accumulate in brain tissues, and that it is now possible to efficiently accumulate there pharmaceutical agents to be targeted in brain tissues by binding such pharmaceuticals with one of these antibodies.

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Table 9 Amount of Humanized Anti-hTfR Antibodies Accumulating in Brain Tissues (Factors vs. Negative Control)

Antibody No. cortex cerebellum hippocampus oblong brain spinal cord 3 82 68 92 54 3.1 3-2 128 80 136 63 3.1 3(IgG4) 79 66 106 54 3.1 3-2(IgG4) 93 63 117 66 3.2 Negative control 1 1 1 1 1

Then in FIG. 4 shows the result of measuring the concentration of humanized antibodies against hTfR in tissues of the liver, heart, spleen and bone marrow. It was observed that four humanized anti-hTfR antibodies, as well as a negative control, trastuzumab, accumulated in the liver and spleen, and their accumulated amount was equal to the four humanized anti-hTfR antibodies and trastuzumab. In the heart, humanized anti-hTfR antibodies tended to accumulate more than trastuzumab, the negative control, but were only approximately 1.5-2.8 times the negative control. In bone marrow, humanized anti-hTfR antibodies tended to accumulate markedly more than trastuzumab, negative control, and were 3.5 to 16 times more than negative controls, thought to be the reason for this accumulation of humanized anti-hTfR antibodies in bone marrow is that TfRs are expressed at high levels in the bone marrow, the hematopoietic organ, and therefore more humanized anti-hTfR antibodies accumulate by binding to TfR than in the negative control. These data show that the four humanized anti-hTfR antibodies have a property that allows them to specifically accumulate in the cerebrum, cerebellum, hippocampus and medulla oblongata, which constitute the central nervous system, and that it is now possible to ensure efficient accumulation of the pharmaceutical agents there that should be involved. in brain tissues, by binding such pharmaceutical agents to one of these antibodies.

Then Table. 9-2 shows the result of a pharmacokinetic measurement of humanized anti-hTfR antibodies in blood. As a pharmacokinetic measure of the negative control, trastuzumab, blood concentrations of four humanized anti-hTfR antibodies remained at high levels, greater than 60 µg/mL, even eight hours after administration, indicating that they are stable in blood (Table 9-2).

Table 9-2. Pharmacokinetics of humanized anti-hTfR antibodies in blood (µg/ml blood)

No. of antibody Time after administration 2 minutes 30 min 2 hour 4 hour 8 hour 3 173 147 128 117 97.5 3-2 124 99.5 78.5 76.5 61 3(IgG4) 141 113 99 95 83 3-2(IgG4) 132 111 98.5 99 95.5 Negative control 124 92.5 96 75.5 60.5

Immunohistochemical staining of humanized anti-hTfR antibodies in brain tissue was performed using the method described in Example 8. However, instead of anti-mouse IgG heavy and light chain antibodies (Bethyl Laboratories Inc.), anti-human IgG heavy and light chain antibodies (Bethyl Laboratories Inc.) were used. .).

In FIG. 5 shows the result of immunohistochemical staining of humanized anti-hTfR antibodies in the cerebral cortex. Specific staining of blood vessels and nerve-like cells was observed in the cerebral cortex of monkeys injected with 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) (Fig. 5b-e, respectively). In the cerebral cortex of monkeys injected with humanized antibodies against hTfR no. 3-2, in particular (Fig. 5c), specific extensive staining of the area of the brain parenchyma outside the blood vessels was also observed. In addition, no staining was observed in the cerebral cortex of monkeys treated with Herceptin as a control, indicating that what was observed in FIG. 5b tissue staining was specific for humanized anti-hTfR antibodies (FIG. 5a).

In FIG. 6 shows the result of immunohistochemical staining of humanized anti-hTfR antibodies in the hippocampus. Specific staining of blood vessels and nerve-like

- 82 042007 cells were observed in the hippocampus of monkeys injected with humanized anti-hTfR No. 3, humanized anti-hTfR No. 3-2, humanized anti-hTfR No. 3 (IgG4), and humanized anti-hTfR No. 3-2 (IgG4) (Fig. 6b-e, respectively). Furthermore, no staining was observed in the hippocampus of monkeys treated with Herceptin as a control, indicating that what was observed in FIG. 6b tissue staining was specific for humanized anti-hTfR antibodies (FIG. 6a).

In FIG. 7 shows the result of immunohistochemical staining of humanized anti-hTfR antibodies in the cerebellum. Specific staining of blood vessels and Purkinje cells was observed in the cerebellum of monkeys injected with 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 ) (Fig. 7b-e, respectively). In addition, no staining was observed in the cerebellum of monkeys treated with Herceptin as a control, indicating that what was observed in FIG. 7b tissue staining was specific for humanized anti-hTfR antibodies (FIG. 7a).

In FIG. 8 shows the result of immunohistochemical staining of humanized anti-hTfR antibodies in the medulla oblongata. Specific staining of blood vessels and nerve-like cells was observed in the medulla oblongata of monkeys injected with humanized anti-hTfR antibody #3, humanized anti-hTfR antibody #3-2, humanized anti-hTfR antibody #3 (IgG4), and humanized anti-hTfR antibody #3-2 ( IgG4) (Fig. 8b-e, respectively). In addition, no staining was observed in the medulla oblongata of monkeys treated with Herceptin as a control, indicating that what was observed in FIG. 8b tissue staining was specific for humanized anti-hTfR antibodies (FIG. 8a).

From the result of immunohistochemical staining of the cerebrum and cerebellum in Example 8 above, it was shown that anti-hTfR antibody No. 3, a pre-humanized mouse antibody, can bind to hTfR located on the endothelium of blood vessels in the brain, and after binding to hTfR, pass through the blood-brain barrier in the brain parenchyma and further absorbed by the brain parenchyma and nerve-like cells in the hippocampus and Purkinje cells in the cerebellum.

From the result of immunohistochemical staining of the cerebrum, hippocampus, cerebellum and medulla oblongata in Example 15, it was found that the tested four humanized anti-hTfR antibodies obtained by humanizing the anti-hTfR antibody No. 3 subjected to the experiment can bind to hTfRs located on the endothelium of the blood vessels of the brain brain, and after binding to hTfR, pass through the blood-brain barrier and pass into the brain parenchyma, and in addition, be taken up by nerve-like cells in the cerebral cortex; in the brain parenchyma and nerve-like cells in the hippocampus; into Purkinje cells in the cerebellum; and in nerve-like cells in the medulla oblongata.

Example 16 Introduction of Mutation into the Heavy Chain of Humanized Anti-hTfR Antibody No. 3

In the heavy chain variable region of the humanized anti-hTfR antibody No. 3, shown as SEQ ID NO: 37, when CDR1 was CDR1, shown as SEQ ID NO: 12, one amino acid in the amino acid sequence of CDR1 was replaced with another amino acid, in order to obtain the heavy chain of the humanized antibody, in while one amino acid in the framework region 3 amino acid sequence was replaced with another amino acid. By replacing the two amino acids constituting the heavy chain amino acid sequence of the humanized anti-hTfR antibody No. 3 set forth in SEQ ID NO: 37, the heavy chain of this new antibody contains the amino acid sequence of SEQ ID NO: 62 or 63 in CDR1 and contains the amino acid sequence of SEQ ID NO: 64, in framework region 3. The heavy chain of this novel antibody (heavy chain of humanized anti-hTfR antibody No. 3N) contains the amino acid sequence of SEQ ID NO: 66, and the variable region in the amino acid sequence contains the amino acid sequence of SEQ ID NO: 65.

In table. 10 shows the alignment between CDR1 shown as SEQ ID NO: 12 in the heavy chain of humanized anti-hTfR antibody #3 and CDR1 shown as SEQ ID NO: 62 in the heavy chain of humanized anti-hTfR antibody # 3N. in alignment, the amino acid threonine at position 5 on the N-terminal side was replaced by methionine.

In table. 11 shows the alignment between framework 3 (SEQ ID NO: 73) in the heavy chain of humanized anti-hTfR antibody #3 and framework region 3 (SEQ ID NO: 64) in the heavy chain of humanized anti-hTfR antibody # 3N. in alignment, the tryptophan amino acid at position 17 on the N-terminal side was replaced with leucine.

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Table 10 CDR1 amino acid sequence alignment

CDR1

Humanized anti-hTfR antibody No. 3 GYSFTNYW

Humanized anti-hTfR antibody No. 3N GYSFMNYW* presents identical amino acids.

[0403] [Table 11]

Table 11 Framework Amino Acid Sequence Alignment

FR3

Humanized anti-hTfR antibody No. 3 QVTISADKSISTAYLQWSSLKASDTAMYYC

Humanized anti-hTfR antibody #3N QVTISADKSISTAYLQLSSLKASDTAMYYC* presents identical amino acids.

Example 17. Preparation of cells for the expression of humanized antibodies against hTfR No. 3N.

A DNA fragment (SEQ ID NO: 67) was artificially synthesized, which contained a gene encoding an antibody heavy chain consisting of the amino acid sequence of SEQ ID NO: 66. The MluI sequence and the sequence encoding the leader peptide in this order from the 5' end, and on its 3' side the NotI sequence. The DNA thus synthesized was inserted into the pE-neo vector by the method described in Example 11, and the thus obtained vector was used as an expression vector for the heavy chain of the humanized anti-hTfR antibody No. 3N, pE-neo(HC3)N. To obtain a humanized anti-hTfR antibody No. 3N cell line, CHO cells were transformed by the method described in Example 12 using this pE-neo(HC3)N and the light chain expression vector, pE-hygr(LC3), obtained in Example 11.

Example 18 Purification of Humanized Anti-hTfR No. 3N Antibodies.

A purified humanized anti-hTfR antibody No. 3N product was prepared by the method described in Example 13 using the humanized anti-hTfR antibody No. 3N cell line obtained in Example 17. 10 and tab. 11, in the amino acid sequence of the heavy chain of the humanized anti-hTfR antibody No. 3. On the other hand, the light chains of the humanized anti-hTfR antibody No. 3N and the humanized anti-hTfR antibody No. 3 have identical amino acid sequences.

Example 19 Affinity comparison for human TfR and monkey TfR between humanized anti-hTfR antibody #3 and humanized anti-hTfR antibody #3N.

The affinity of the humanized anti-hTfR antibodies No. 3 prepared in Example 13 and the humanized anti-hTfR antibodies No. 3N prepared in Example 17 for human TfR and human TfR was measured by the method described in Example 7. In table. 12 shows the results of measuring the association rate constant (kon), the dissociation rate constant (koff) of each antibody, and the dissociation constant (kD) with human TfR. In addition, the measurement showing the affinity of humanized antibodies against hTfR No. 3 to human TfR was different from the measurement shown in table. 6, but constituted an experimental error.

Table 12 Affinity of Humanized Anti-hTfR Antibodies for Human TfR

The affinity of the humanized anti-hTfR No. 3 antibodies obtained in Example 13 and the humanized anti-hTfR antibodies No. 3N obtained in Example 17 for human TfR and monkey TfR was measured by the method described in Example 7. In table. 13 shows the results of measuring association rate constant (kon), dissociation rate constant (koff) of each antibody, and dissociation constant (kD) with monkey TfR. In addition, the measurement showing the affinity of humanized antibodies against hTfR No. 3 to monkey TfR was different from the measurement shown in table. 7, but constituted an experimental error.

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Table 13 Affinity of Humanized Anti-hTfR Antibodies for Monkey TfR

kon (M ^-s ^x ) koff (s') K _D (M) Humanized antibody against hTfR 3 6.09xl0 ^{5 8.06xl0-4} l, ^32xl0-9 Humanized antibody against hTfR 3N 3.86xl0 ⁵ 1.96xl0 ^-5 5.07χ10- ^η

When comparing KD with monkey TfR between humanized anti-hTfR #3N and humanized anti-hTfR #3, the former was 5.07x10-11 M and the latter was ^1.32x10-9 M; thus, the _KD of humanized anti-hTfR No. 3N with monkey TfR was approximately 1/30 of that of humanized anti-hTfR No. 3. On the other hand, when comparing the Kd with human TfR between humanized anti-hTfR No. No. 3, both were smaller than ^1x10-12 M; thus, both antibodies had high affinity for human TfR. Also, both antibodies had higher affinity for human TfR than affinity for monkey TfR.

As part of the non-clinical trials required to develop humanized anti-hTfR antibodies as pharmaceuticals, pharmacological tests are often performed using monkeys. Since the results of these pharmacological tests using monkeys are used to decide the validity of human clinical trials, it is preferable that behavior in monkeys would be closer to that in humans. As shown in the above results, the humanized anti-hTfR antibody #3N exhibits approximately 30-fold higher affinity for monkey TfR than the humanized anti-hTfR antibody #3, and therefore the behavior of the humanized anti-hTfR antibody #3N in monkeys can be considered more similar. to its behavior in humans. Thus, by using humanized antibodies against hTfR #3N, results are obtained that are more representative of behavior in humans in tests using monkeys. Thus, tests using monkeys provide more useful results for the decision to conduct clinical trials using humans.

Example 20 Comparison of transfer to monkey brain tissue between humanized anti-hTfR antibody #3 and humanized anti-hTfR antibody #3N.

Each of the humanized anti-hTfR antibody No. 3 prepared in Example 13 and the humanized anti-hTfR antibody No. 3N prepared in Example 17 were intravenously administered once to a male crabeater monkey at a dosage of 5.0 mg/kg, and 8 hours after administration the whole body was washed. After washing, tissues of the brain and spinal cord, including the medulla oblongata, were excised. The brain tissue thus excised was divided into the cerebral cortex, cerebellum, hippocampus, and medulla oblongata.

Measurement of the concentration of humanized anti-hTfR antibodies contained in each tissue was carried out in accordance with the measurement method described in Example 15.

The result of measuring the concentration of anti-hTfR antibodies in the brain tissues is shown in Table 14. When comparing the concentration in the cerebrum, cerebellum and hippocampus between humanized anti-hTfR antibodies No. 3N and humanized anti-hTfR antibodies No. 3, the concentration of humanized anti-hTfR antibodies No. 3N was approximately 1 ,42 times in the cerebrum, approximately 1.56 times in the cerebellum and approximately 1.29 times in the hippocampus higher than the concentration of humanized antibodies against hTfR # 3. In the medulla oblongata, their concentrations were almost equal. Also in the cervical spine, the concentration of humanized anti-hTfR #3N antibodies was approximately 1.47-fold higher than the concentration of humanized anti-hTfR #3 antibodies. These results demonstrate that humanized anti-hTfR #3N antibodies compared to humanized anti-hTfR #3 antibodies had the property of more efficient passage through the blood-brain barrier and accumulation in the tissues of the cerebrum, cerebellum, hippocampus, and the like. Namely, the results show that by linking these antibodies to a pharmaceutical that needs to be delivered to function in brain tissues, humanized anti-hTfR #3N antibodies had the property of allowing these pharmaceuticals to efficiently accumulate in brain tissues.

Table 14 Concentration of antibodies against hTfR in brain tissue (µg/g wet weight)

Antibodies No. Big brain Cerebellum hippocampus Oblong brain cervical spinal cord 3 0.670 0.610 0.490 0.589 0.589 3N 0.953 0.950 0.631 0.586 0.672

Example 20-2. Obtaining cells for the expression of hFc-humanized antibodies against hTfR.

A DNA fragment (SEQ ID NO: 72) was artificially synthesized, which contained a gene encoding

- 85 042007 amino acid sequence of the Fab heavy chain of a humanized anti-hTfR 3N antibody with an added human IgG Fc region, consisting of the amino acid sequence of SEQ ID NO: 71. To this DNA fragment was added on its 5' side the MluI sequence and the sequence encoding the leader peptide, in this order from the 5' end, and on its 3' side the NotI sequence. The DNA thus synthesized was inserted into the pE-peo vector by the method described in Example 11, and the vector thus obtained was designated pE-neo(Fc-Fab HC(3N)). To obtain an Fc-Fab(3N) expressing cell line, CHO cells were transformed by the method described in Example 12 using this pE-neo(Fc-Fab HC(3N)) and the light chain expression vector, pEhygr(LC3), obtained in Example eleven.

Example 20-3. Preparation of hFc-Humanized Anti-hTfR Antibodies Purified Fc-Fab (3N) product was obtained by the method described in Example 13 using an Fc-Fab(3N) expressing cell line.

Example 20-4. Affinity comparison of hFc-humanized anti-hTfR antibodies between human TfR and monkey TfR.

The affinity of the purified Fc-Fab (3N) product prepared in Example 20-3 for human TfR and monkey TfR was measured using the method described in Example 7. The results are shown in table. 14-2. FcFab (3N) showed high affinity (K _D <1.0x10 ^-12 ) for human TfR. These results show that Fc-Fab (3N) has a property that allows efficient accumulation in brain tissues of pharmaceuticals to be targeted in brain tissues by binding such pharmaceuticals to one of these antibodies.

Table 14-2. Affinity of hFc-Humanized Anti-hTfR Antibodies for Human TfR and Monkey TfR

kon (M ^-1 s ^-1 ) koff (s ¹ ) K _D (M) Human TfR 6.67x10 ⁵ <1, OxlO~ ⁷ <1, OxlO~ ¹² TfR Monkeys 2.43x10 ⁵ 2.09x10 ~ ⁴ 8, 61xlO~ ¹⁰

Example 20-5. Measurement of the transfer of hFc-humanized anti-hTfR antibodies in monkey brain tissue Fc-Fab (3N) obtained in Example 20-3 was administered intravenously once to a male crabeater monkey at a dosage of 5.0 mg/kg, and 8 hours after injections were carried out washing of the whole body. After washing, tissues of the brain and spinal cord, including the medulla oblongata, were excised. The brain tissue thus excised was divided into the cerebral cortex, cerebellum, hippocampus, and medulla oblongata.

Measurement of the concentration contained in each tissue humanized antibodies against hTfR was performed in accordance with the measurement method described in example 15.

The result of measuring the concentration of antibodies against hTfR in brain tissues is shown in table. 14-3. Fc-Fab (3N) concentrations in the cerebrum, cerebellum, and hippocampus were 0.80 µg/g wet weight, 0.80 µg/g wet weight, and 1.05 µg/g, respectively. Concentrations in the medulla oblongata and spinal cord were 0.68 µg/g wet weight and 0.67 µg/g wet weight, respectively. These results show that anti-hTfR antibodies can pass through the BBB even when humanized anti-hTfR antibodies, which are Fab, are linked at their N-terminal side to the Fc region. When a conjugate in which the desired substance (protein or physiologically active substance) is bound to a Fab is administered intravenously and is unstable, the Fab is linked at its N-terminal side to the Fc region, whereby such a conjugate can be stabilized in the body, for example , to increase the half-life of the conjugate in the blood, while maintaining the property of passing through the BBB.

Table 14-3. Concentration of hFc-humanized antibodies against hTfR in brain tissues (µg/g wet weight)

Big brain Cerebellum hippocampus Oblong brain cervical spinal cord hF c-humanized antibodies against hTfR 0.80 0.80 1.05 0.67 0.68

Example 21 Preparation of cells expressing the hI2S-humanized anti-hTfR antibody fusion protein.

A DNA fragment having the nucleotide sequence of SEQ ID NO: 52 was artificially synthesized, which contained a gene encoding a protein in which the heavy chain of a humanized anti-hTfR antibody No. 3 having the amino acid sequence of SEQ ID NO: 51 was linked on its Terminal side and through a linker sequence (Gly Ser) with hI2S having the amino acid sequence of SEQ ID NO: 50. This DNA fragment encoded a protein in which the heavy chain of a humanized anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 247 was linked via a linker sequence (Gly Ser) with hI2S. This DNA fragment had on its 5' side the sequence MluI and the sequence encoding the leader peptide,

- 86 042007 acting as a secretion signal in this order from the 5' end, and on its 3' side the NotI sequence. To obtain pE-neo(HC-I2S-1), the DNA fragment was digested with MluI and NotI and inserted into the pE-neo vector between its MluI and NotI.

A DNA fragment having the nucleotide sequence of SEQ ID NO: 54 was artificially synthesized, which contained a gene encoding a protein in which the heavy chain of a humanized anti-hTfR antibody No. 3N having the amino acid sequence of SEQ ID NO: 66 was linked on its Terminal side and through a linker sequence (Gly Ser) with hI2S having the amino acid sequence of SEQ ID NO: 50. This DNA fragment encoding a protein in which the heavy chain of a humanized anti-hTfR antibody having the amino acid sequence of SEQ ID NO: 53 was linked via a linker sequence (Gly Ser ) with hI2S. This DNA fragment had on its 5' side the MluI sequence and the sequence encoding the leader peptide acting as a secretion signal in that order from the 5' end, and on its 3' side the NotI sequence. To obtain pE-neo(HC-I2S-2), the DNA fragment was digested with MluI and NotI and inserted into the pE-neo vector between its MluI and NotI.

To obtain a cell line expressing a fusion protein between hI2S and a humanized anti-hTfR antibody, CHO cells (Cho-Κι: purchased from the American Type Culture Collection) were transformed with pE-neo(HC-I2S-3) and pE-hygr(LC3) which were obtained in Example 11. This cell line was designated hI2S anti-hTfR expressing cell line 3. The fusion protein between hI2S and the humanized anti-hTfR antibody expressed by the cell lines was designated anti-hTfR 3 I2S.

To obtain a cell line expressing a fusion protein between hI2S and a humanized anti-hTfR antibody, CHO cells were transformed with pE-neo(HC-I2S-3N) and pE-hygr(LC3). This cell line was designated the hI2S anti-hTfR expressing cell line 3N. The cell-expressed fusion protein between hI2S and the humanized anti-hTfR antibody was designated I2S anti-hTfR 3N.

Example 22 Preparation of anti-hTfR I2S antibodies Anti-hTfR I2S antibodies were prepared using the following method, the hI2S anti-hTfR antibody-expressing cell lines 3 and 3N obtained in Example 21 were diluted with CD OptiCHO™ medium to a density of approximately 2x105 cells/ml, respectively. Cell suspensions, 200 ml, were added to appropriate 1 L conical flasks and cultured for 6-7 days in humid ambient conditions of 37° C., 5% CO ₂ , 95% air with shaking at approximately 70 rpm. To obtain a culture supernatant, each culture supernatant was collected by centrifugation and filtered through a 0.22 μm filter (Millipore Inc.). Five column volumes of 20 mM Tris buffer (pH 8.0) containing 150 ml NaCl were added to each culture supernatant thus obtained, and loaded onto a Protein A column (column volume: 1 ml, Bio-Rad Inc.), which had been equilibrated in advance three column volumes of 20 mM Tris buffer (pH 8.0) containing 150 mM NaCl. The column was then washed with five column volumes of the same buffer and adsorbed anti-hTfR I2S antibodies were eluted with four column volumes of 50 mM glycine buffer (pH 2.8) containing 150 mM NaCl. To obtain purified products, I2S anti-hTfR 3 and I2S anti-hTfR 3N eluate containing I2S anti-hTfR antibodies was adjusted to pH 7.0 with 1 M Tris buffer (pH 8.0), and then the buffer was changed PBS buffer using an Amicon Ultra 30 kDa membrane (Millipore Inc.).

Example 23 Measurement of the affinity of anti-hTfR I2S antibodies to human TfR and monkey TfR.

The affinity of the anti-hTfR I2S antibodies to human TfR and monkey TfR obtained in Example 22 was measured according to the method described in Example 7. In table. 15 shows the results of measuring the association rate constant (kon), the dissociation rate constant (koff) of I2S anti-hTfR 3 and I2S anti-hTfR 3N antibodies, and the dissociation constant (k _D ) with human TfR. In table. 16 shows the results of measuring the association rate constant (kon), dissociation rate constant (koff) of anti-hTfR 3 I2S antibodies and anti-hTfR 3N I2S antibodies, and dissociation constant (k _D ) with monkey TfR.

I2S antibodies against hTfR 3N showed an affinity for human TfR approximately 5 times greater than I2S antibodies against hTfR (Table 15). In addition, anti-hTfR 3N I2S antibodies showed significantly higher affinity for monkey TfR than anti-hTfR I2S antibodies (Table 16). Anti-hTfR 3N I2S antibodies showed higher affinity for human TfR and monkey TfR than anti-hTfR 3 I2S antibodies.

Table 15 Anti-hTfR I2S affinity for TfR

kon (M ^x s ^x ) koff (s ^x ) K _D (M) 123-antibody against hTfR 3 ^8.29x10s 1.04xl0~ ⁴ l,26xl0~ ¹⁰ 123-antibody against hTfR 3N 6.92xl0 ⁵ 1.90xl0~ ⁵ 2.75xl0~ ⁿ

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Table 16 Affinity of anti-hTfR K3 antibodies to monkey TfR

kon (M ² s ² ) koff ( s ² ) K _D (M) 123-antibody against hTfR 3 ^3.77x10s 4.10xl0~ ⁴ l, ^09xl0-9 123-antibody against hTfR 3N 3.83xl0 ⁵ <1, OOxlO- ⁷ <1, OOxlO- ¹²

Example 24 Comparison of transfer to brain tissue in monkey between anti-hTfR 3 I2S antibodies and anti-hTfR 3N I2S antibodies.

Each of the anti-hTfR 3 I2S antibody and the anti-hTfR 3N K3 antibody obtained in Example 22 was intravenously administered once to a male cynomolgus monkey at a dosage of 5.0 mg/kg, and a whole body wash was performed 8 hours after administration. After washing, tissues of the brain and spinal cord, including the medulla oblongata, were excised. The brain tissue thus excised was divided into the cerebral cortex, cerebellum, hippocampus, and medulla oblongata.

Measurement of the concentration of anti-hTfR I23 antibodies contained in each tissue was carried out essentially following the procedure described in Example 20. The result of measuring the concentration of KZantibodies against hTfR in brain tissues is shown in Table. 17. When comparing the concentration in the cerebrum, cerebellum, hippocampus, medulla oblongata and cervical spine between 123 anti-hTfR 3N antibody and 123 anti-hTfR 3 antibody, the concentration of 123 anti-hTfR 3N antibody was approximately 2.12 times in the cerebrum, approximately 1.97-fold in the cerebellum, approximately 2.41-fold in the hippocampus, 1.94-fold in the medulla oblongata, and 1.63-fold in the cervical spine are higher than the concentration of anti-hTfR 3 123 antibodies. This result demonstrates that that 123 anti-hTfR 3N can cross the blood-brain barrier more efficiently than 123 anti-hTfR 3.

Table 17 Concentration of 123 anti-hTfR antibodies in brain tissue (µg/g ww)

Big brain Cerebellum hippocampus Oblong brain cervical spinal cord 128-antibody against hTfR 3 0.224 0.249 0.183 0.229 0.188 128-antibody against hTfR 3N 0.475 0.491 0.441 0.450 0.306

Example 25 Generation of Cells for Expression of the hGAA-Humanized Anti-hTfR Fusion Protein.

A DNA fragment having the nucleotide sequence of 3EQ ID NO: 59 was artificially synthesized, which contained a gene encoding a protein in which the heavy chain of a humanized anti-hTfR 3N antibody having the amino acid sequence of 3EQ ID NO: 68 was linked on its Terminal side and through a linker sequence (Gly 3er) with hGAA having the amino acid sequence 3EQ ID NO: 55. This DNA fragment encoding a protein in which the heavy chain (IgG4) of a humanized anti-hTfR 3N antibody having the amino acid sequence 3EQ ID NO: 58 was linked to hGAA via linker sequence (Gly 3er). This DNA fragment had on its 5' side the MluI sequence and the sequence encoding the leader peptide acting as a secretion signal in that order from the 5' end, and on its 3' side the NotI sequence. To obtain pE-neo(HC-GAA-3N (IgG4)) the DNA fragment was digested with MluI and NotI and inserted into the pE-neo vector between its MluI and NotI.

To obtain a cell line expressing a fusion protein between hGAA and a humanized anti-hTfR antibody, CHO cells (CHO-K1: purchased from the American Type Culture Collection) were transformed with pE-neo(HC-GAA-3N(IgG4)) and pE-hygr (LC3) prepared in Example 11. This cell line was designated the 3N(IgG4) hGAA-expressing anti-hTfR cell line. The cell-expressed fusion protein between hGAA and the humanized anti-hTfR antibody was designated anti-hTfR GAA 3N(IgG4).

Example 26 Preparation of anti-hTfR 3N GAA antibodies (IgG4).

GAA anti-hTfR 3N (IgG4) antibodies were generated essentially following the procedure described in Example 22 using the hGAA anti-hTfR antibody-expressing 3N (IgG4) cell line prepared in Example 25.

Example 27 Measurement of the affinity of hGAA antibodies against hTfR 3N (IgG4) for human TfR and monkey TfR.

The affinity of the anti-hTfR 3N (IgG4) antibodies obtained in Example 26h6AA for human TfR and monkey TfR was measured according to the method described in Example 23. Also described herein are the results of measuring the affinity of the G3-GAA Fab obtained in Example 31 below for human TfR and monkey TfR.

- 88 042007 hGAA antibodies against hTfR 3N (IgG4) showed very high affinity for both human TfR and monkey TfR. The results are shown in table. 17-2. The GS-GAA fab also showed high affinity for both human TfR and monkey TfR, but the affinity was lower than that of hGAA antibodies against hTfR 3N (IgG4).

Table 17-2. Affinity of hGAA antibodies against hTfR 3N (IgG4) and

Fab GS-GAA to human TfR and monkey TfR

kon (1/Ms) koff (1/s) K _D (M) hGAA antibody against hTfR 3N (IgG4) Human TfR 4.86xl0 ⁵ <1.00xl0~ ⁷ <1, OOxlO- ¹² TfR Monkeys 2.03xl0 ⁵ <1.00xl0~ ⁷ <1, OOxlO- ¹² Fab GS-GAA Human TfR 4.14xl0 ⁵ 1.67xl0~ ^{4 4.04xl0-10} TfR Monkeys 3.29xl0 ^{5 5.01xl0-3} l, ^52xl0-8

Example 28 Evaluation of the transfer of anti-hTfR 3N (IgG4) hGAA antibodies into monkey brain tissue.

Transfer obtained in example 26 hGAA antibodies against hTfR 3N (IgG4) in the brain tissue can be assessed according to the method described in example 24. Specifically, hGAA antibody against hTfR 3N (IgG4) was intravenously administered once to a male crabeater monkey at dosages of 5.0 mg/kg and 20 mg/kg. As a control, a male crabeater monkey was administered once intravenously at a dosage of 5.0 mg/kg and 20 mg/kg of hGAA marketed as a therapeutic agent for Pompe disease. Three monkeys were injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg, and one monkey was injected with other antibodies. each of the monkeys injected with hGAA anti-hTfR 3N (IgG4) at a dosage of 20 mg/kg was subjected to a whole body wash with physiological saline (Otsuka Pharmaceutical Co., Ltd.) 4 hours, 8 hours and 24 hours after administration. Other monkeys were subjected to whole body washing 8 hours after injection.

After washing, brain tissues were excised, including the medulla oblongata, spinal cord tissues, and skeletal muscle tissues. The brain tissues were divided into the cerebral cortex, cerebellum, hippocampus, medulla oblongata and pons. For skeletal muscle tissue, the rectus femoris, extensor digitorum longus, soleus, triceps brachii, psoas major, diaphragm, and tongue muscles were excised. One male crabeater monkey, which was not injected with a pharmaceutical agent, was subjected to a whole-body wash with physiological saline (Otsuka Pharmaceutical Co., Ltd.), and the cortex and rectus femoris were dissected. To collect the supernatant, excised tissues were homogenized with T-PER (Thermo Fisher Scientific Inc.) containing a mixture of protease inhibitors (Sigma Inc.) and centrifuged. Using the obtained supernatant, the concentrations of hGAA antibodies against hTfR 3N (IgG4) and hGAA were measured by the following ECL method, and immunohistochemical staining was performed.

Measurement of protein concentration by means of the ECL method.

To generate SULFO-labeled anti-hGAA antibodies, rabbit anti-hGAA polyclonal antibodies were labeled with SULFO with NHS ester MSD GOLD Sulfo-TAG (Meso Scale Discovery Inc.). In addition, to obtain biotin-labeled anti-hGAA antibodies, rabbit polyclonal anti-hGAA antibodies were labeled with biotin using EZ-Link NHS-PEG ₄ -Biotin (Thermo Fisher Scientific Inc.). To obtain a reactive antibody sample, a solution of a mixture of equal amounts of SULFO-labeled anti-hGAA antibodies and biotin-labeled anti-hGAA antibodies was prepared, the supernatant obtained as described above was added to this solution, and the resulting mixture was incubated at room temperature for 1 hour. A 1% BSA/PBS solution was added to each well of a 96-well Streptavidin Gold plate (Meso Scale DiagNotics Inc.), which is a streptavidin-coated plate, and the plate was allowed to stand for 1 hour to block the plate.

After blocking, each well of the plate was washed with 200 μl of PBS-T (Sigma Inc.), and then a reactive antibody sample was added to each well and incubated at room temperature for 1 hour. After incubation, each well of the plate was washed with PBS-T, then a mixed liquid of equal amounts of 4X reading buffer T (Meso scale DiagNotics Inc.) and water for injection (Otsuka Pharmaceutical Co., Ltd.) was added, and the luminescence value from each well was measured using Sector™ Imager 6000 (Meso Scale DiagNotics Inc.). The amount of antibodies contained in one gram of brain (wet weight) was calculated by obtaining a standard curve based on the measurement of standard samples containing known concentrations of hGAA antibodies against hTfR 3N (IgG4) or hGAA, and interpolating the measurement of each sample with reference to the standard curve to determine the amounts of hGAA antibodies against hTfR 3N (IgG4) and hGAA.

The results of measuring concentrations by the ELC method are shown in table. 18. First, the results for brain tissues will be discussed. In a monkey administered with hGAA anti-hTfR 3N (IgG4), concentrations of administered hGAA anti-hTfR 3N (IgG4) at 5 mg/kg and 20 mg/kg increased in a concentration-dependent manner to 0.740 μg/g in terms of crude weight and 1.42 µg/g in terms of wet weight, respectively, in the cerebral cortex 8 hours after administration

- 89 042007 The same results were obtained for the cerebellum, hippocampus, medulla oblongata and pons. On the other hand, in an hGAA-treated monkey, the concentrations of administered hGAA at 5 mg/kg and 20 mg/kg showed nearly equal values of 0.414 µg/g wet weight and 0.435 µg/g wet weight, respectively. , in the cerebral cortex 8 hours after administration. The same results were obtained for the cerebellum, hippocampus, medulla oblongata and pons. Considering that the value of intrinsic GAA concentration in the cerebral cortex having a certain amount of hGAA in the monkey not injected with the pharmaceutical agent was 0.425 µg/g in terms of wet weight, it is clear that little intravenous hGAA enters the brain. On the other hand, it is clear that intravenously administered hGAA antibody against hTfR 3N (IgG4) enters the brain by passing through the BBB.

Next, the results for skeletal muscle tissues will be discussed. In a monkey injected with each of hGAA anti-hTfR 3N (IgG4) and hGAA at 5 mg/kg, the concentration of hGAA anti-hTfR 3N (IgG4) and hGAA in the rectus femoris muscle 8 hours after administration was 0.311 µg/g in wet weight for hGAA-anti-hTfR 3N (IgG4) and 0.251 µg/g wet weight for hGAA. Namely, the concentration of hGAA antibody against hTfR 3N (IgG4) was 1.23 times the concentration of hGAA. The same results were obtained when these antibodies were administered at a dosage of 20 mg/kg. In addition, the same results were obtained for the extensor digitorum longus, soleus, triceps brachii, psoas major, diaphragm, and tongue muscles. Namely, it appears that intravenously administered hGAA antibodies against hTfR 3N (IgG4) enter the skeletal muscle tissue in greater amounts than the amount of intravenously administered hGAA.

Table 18. Concentration of hGAA antibodies against hTfR 3N (IgG4) in brain tissue and skeletal muscle (µg/g wet weight)

Substance injected hGAA antibody against hTfR 3N (IgG4) hGAA Own GAA Dose 5 mg/kg 20 mg/kg 5 mg/kg 20 mg/kg Time elapsed after administration (h) 4 8 24 8 8 8 Main brain The cortex of the brain brain 1.07 0.740 1.00 1.42 0.414 0.435 0.425 Cerebellum 0.776 0.625 0.509 1.16 0.275 0.279 - hippocampus 1.34 0.966 1D4 1.68 0.471 0.467 - Oblong brain 0.886 0.678 0.869 1.22 0.371 0.394 - Bridge of the brain 0.904 0.627 0.730 1.12 0.403 0.371 - Skeletal muscles Rectus femoris 0.445 0.311 0.458 0.425 0.251 0.377 0.140 The muscle of the long extensor of the fingers 0.463 0.293 0.379 0.554 0.261 0.449 - soleus 0.527 0.361 0.377 0.485 0.277 0.320 - muscle Triceps brachii 0.315 0.311 0.250 0.482 0.231 0.314 - psoas major 0.301 0.241 0.275 0.383 0.238 0.225 - Diaphragm 0.289 0.212 0.238 0.339 0.199 0.381 - tongue muscle 0.882 0.631 0.794 1.10 0.316 0.915 -

Immunohistochemical staining.

Immunohistochemical staining of hGAA antibodies against hTfR 3N (IgG4) and hGAA in brain tissues was performed following the procedures generally described below. Brain tissues obtained as described above were flash frozen to -80°C in Tissue-Tek Cryo 3DM (Sakura Finetek Inc.), the thus obtained frozen block was cut into 4 μm sections using a LEICA CM 1860 UV cryostat (Leica Biosystems Nussloch Inc.) and then the tissue sections were mounted on MAS-coated glass slides (Matsunami Glass Inc.). Tissue sections were reacted with 4% paraformaldehyde (Wako

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Pure Chemical Industries Inc.) at 4°C for 5 minutes and attached to glass slides. After that, tissue sections were reacted with a methanol solution containing 0.3% hydrogen peroxide (Wako Pure Chemical Industries Inc.) for 30 minutes to inactivate its own peroxidase. The slides were then blocked by reacting with SuperBlock blocking buffer in PBS at room temperature for 30 minutes. The tissue sections were then reacted with rabbit anti-hGAA polyclonal antibodies as primary antibodies at 4° C. for 16 hours. The tissue sections were then treated with CSAII rabbit linker (Agilent Inc.) and a biotin-free CSAII tyramide signal amplification system kit (Agilent Inc.). The tissue sections were then reacted with horseradish peroxidase-conjugated antibodies against fluorescein at room temperature for 15 minutes. Tissue sections were allowed to change color with DAB substrate (3,3'-diaminobenzidine, Vector Laboratories Inc.), counterstained with Mayer's hematoxylin solution (Merck Inc.), dehydrated, cleaned, stacked and viewed under a microscope. It should be noted that immunohistochemical staining was performed only on the cerebellum of monkeys that were injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg, and that were injected with hGAA at a dosage of 20 mg/kg. Therefore, the time elapsed after administration is 8 hours for both antibodies.

In FIG. 9 shows the results of immunohistochemical staining of hGAA antibodies against hTfR 3N (IgG4) and hGAA in the cerebellum. In the cerebellum of monkeys injected with hGAA antibodies against hTfR 3N (IgG4), specific staining of blood vessels and Purkinje cells was observed (Fig. 9a). On the other hand, no such staining was observed in the cerebellum of monkeys injected with hGAA (Fig. 9b). These results indicate that by fusing hGAA, which normally does not cross the blood-brain barrier, with an anti-hTfR 3N (IgG4) antibody, hGGA can be allowed to cross the blood-brain barrier and be taken up by Purkinje cells in the cerebellum.

Example 29 Evaluation of the pharmacological action of hGAA antibodies against hTfR 3N (IgG4) in mice: determination of glycogen concentration (1).

Based on knockout mice as an animal model of Pompe disease in which the acid α-glucosidase (GAA) gene is deficient, mice were generated that lack the GAA gene when homologous and have a chimeric TfR gene when heterologous (GAA-KO/hTfR-KI mice) by replacing the gene encoding the extracellular region of the mouse TfR with the gene encoding the extracellular region of the human TfR receptor gene by the method described in Example 7-2. Mice were divided into seven groups and injected intravenously with hGAA antibodies against hTfR 3N (IgG4) or hGAA according to the use and dosage shown in Table 1. 19. Normal mice not treated with pharmaceutical agent were classified as normal controls (first group) and GAA-KO/hTfR-KI mice not treated with pharmaceutical agent were classified as disease controls (second group). Each group contained five mice. In addition, male and female mice at 914 weeks of age (at the start of administration) were used.

Table 19 Grouping mice

Group Test Substance Mouse Dosage (mg/kg) Injection interval Number of injections 1 - Normal - - - mouse 2 - Mouse GAAKO/hTfR-KI - - - 3 hGAA Mouse GAAKO/hTfR-KI 20 2 weeks 4 weeks 4 hGAAantibody against hTfR 3N (IgG4) Mouse GAAKO/hTfR-KI 2.5 5 5.0 6 10 7 20

For the second and third administrations of the test substance, diphenhydramine was administered at a dosage of 10 ml/kg to mice 10-20 minutes before administration. This treatment aims to prevent the experimental animals from developing an immunological reaction, such as anaphylactic shock, due to the administration of the test substance. 13 days after the last injection, the mouse was decapitated under isoflurane anesthesia, and the right cerebral hemisphere and cervical spinal cord were quickly dissected, and then quickly immersed in liquid nitrogen for quick freezing. After freezing, the wet weight of the tissues of each organ was measured. For the heart, diaphragm, liver, spleen, quadriceps femoris, soleus, rectus femoris, extensor digitorum longus, and gastrocnemius (collected skeletal muscles: right leg), sufficient blood was removed by bloodletting, then tissues were collected and washed physiological saline solution

- 91 042007 and measured the wet weight of the tissues of each of the organs. After measurement, each organ was sealed in a 1.5 ml tube and flash-frozen in liquid nitrogen.

Water for injection was added to the tissues of each organ in an amount of 20 times the wet weight of the tissues, and the tissues were homogenized using a ball mill. Homogenized tissues were transferred to a centrifuge tube, heated at 100°C for 5 minutes and left to stand on ice. Thereafter, the tissues were centrifuged at 13,000 rpm at 4° C. for 5 minutes, and the supernatant was collected. The supernatant was frozen and kept at -70°C or below.

The concentration of glycogen contained in the supernatant was measured using a glycogen assay kit (Viovision Inc.). The measurement results are shown in Figures 10 and 11. In FIG. 10 shows glycogen concentrations in the right cerebral hemisphere, cervical spinal cord, heart, diaphragm, liver, and spleen (FIGS. 10a-10f, respectively). In addition, in FIG. 11 shows glycogen concentrations in the quadriceps femoris, soleus, rectus femoris, extensor digitorum longus, and gastrocnemius muscles (FIGS. 11a-11e, respectively). However, the group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg was excluded due to their death 5 days after the last injection.

The results in FIG. 10 and 11 will be discussed below.

Brain.

Glycogen concentrations in the brain tissues of the normal control group, the disease control group, the group of mice that were injected with hGAA at a dosage of 20 mg/kg, and the group of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5, 0, 10 and 20 mg/kg were 0.0815 mg/g, 3.08 mg/g, 2.36 mg/g, 2.07 mg/g, 1.00 mg/g, 0.891 mg/g and 0.592 mg/g, respectively, in terms of wet weight (Fig. 10(a)). Groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of not less than 5.0 mg/kg showed a significant decrease in tissue glycogen concentration compared with the control group of the disease and the group of mice that were injected with hGAA at a dosage of 20 mg /kg. In addition, the decrease in glycogen concentration due to hGAA antibodies against hTfR 3N (IgG4) depended on the dosage.

The cervical region of the spinal cord.

Glycogen concentrations in the tissues of the cervical spinal cord of the normal control group, the disease control group, the group of mice that were injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5.0, 10 and 20 mg/kg were 0.0459 mg/g, 3.10 mg/g, 2.68 mg/g, 2.26 mg/g, 1.77 mg/g, 1.06 mg/g and 0.795 mg/g, respectively, in terms of wet weight (FIG. 10(b)). All groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) showed a significant decrease in tissue glycogen concentration compared to the disease control group. In addition, groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of not less than 5.0 mg/kg showed a significant decrease in tissue glycogen concentration compared to a group of mice that were injected with hGAA at a dosage of 20 mg/kg. In addition, the decrease in glycogen concentration due to hGAA antibodies against hTfR 3N (IgG4) depended on the dosage.

Heart.

The concentrations of glycogen in the heart of the normal control group, the disease control group, the group of mice that were injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5.0, 10 and 20 mg/kg were 0.0816 mg/g, 32.4 mg/g, 4.54 mg/g, 12.3 mg/g, 3.45 mg/g, 1.10 mg/g and 0.205 mg/g, respectively, in terms of wet weight (Fig. 10(c)). All groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) showed a significant decrease in tissue glycogen concentration compared to the disease control group. A group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg showed a significant decrease in glycogen concentration compared to a group of mice that were injected with hGAA at a dosage of 20 mg/kg.

Diaphragm.

Diaphragm glycogen concentrations in the normal control group, the disease control group, the group of mice injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5.0 , 10 and 20 mg/kg, were 0.0824 mg/g, 14.1 mg/g, 6.96 mg/g, 12.4 mg/g, 5.57 mg/g, 2.68 mg/g and 0.564 mg/g, respectively, in terms of wet weight (Fig. 10(d)). Groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of not less than 5.0 mg/kg showed a significant decrease in the concentration of glycogen in tissues compared with the control group of the disease. A group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 10 mg/kg showed a significant decrease in glycogen concentration compared to a group of mice that were injected with hGAA at a dosage of 20 mg/kg.

Liver.

Liver glycogen concentrations of the normal control group, the disease control group, the group of mice injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5.0, 10 and 20 mg/kg were 1.16 mg/g, 22.5

- 92 042007 mg/g, 2.65 mg/g, 8.88 mg/g, 2.95 mg/g, 3.01 mg/g and 1.46 mg/g, respectively, in terms of wet weight (Fig. .10(e)). All groups of mice injected with hGAA antibodies against hTfR 3N (IgG4), as well as the group of mice that were injected with hGAA at a dosage of 20 mg/kg, showed a significant decrease in the concentration of glycogen in tissues compared with the control group of the disease.

Spleen.

Glycogen concentrations in the spleen of the normal control group, the disease control group, the group of mice that were injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5.0, 10 and 20 mg/kg were 0.0295 mg/g, 3.15 mg/g, 0.182 mg/g, 0.368 mg/g, 0.165 mg/g, 0.122 mg/g and 0.0990 mg/g, respectively , in terms of fresh weight (Fig. 10(f)). All groups of mice injected with hGAA antibodies against hTfR 3N (IgG4), as well as the group of mice that were injected with hGAA at a dosage of 20 mg/kg, showed a significant decrease in the concentration of glycogen in tissues compared with the control group of the disease.

Quadriceps femoris.

Glycogen concentrations in the tissues of the quadriceps femoris muscle of the normal control group, the control group of the disease, the group of mice that were injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5 0, 10 and 20 mg/kg were 0.0529 mg/g, 8.66 mg/g, 7.41 mg/g, 6.40 mg/g, 2.68 mg/g, 1.71 mg /g and 0.282 mg/g, respectively, in terms of fresh weight (Fig. 11(a)). Groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of not less than 5.0 mg/kg showed a significant decrease in tissue glycogen concentration compared with the control group of the disease and the group of mice that were injected with hGAA at a dosage of 20 mg /kg. The group of mice treated with hGAA at 20mg/kg did not show a significant decrease in tissue glycogen concentration compared to the disease control group.

Calf muscle.

Glycogen concentrations in the tissues of the gastrocnemius muscle of the normal control group, the disease control group, the group of mice that were injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5, 0, 10 and 20 mg/kg were 0.173 mg/g, 7.68 mg/g, 5.09 mg/g, 5.23 mg/g, 2.40 mg/g, 1.58 mg/g and 0.221 mg/g, respectively, in terms of wet weight (Fig. 11(b)). Groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of not less than 5.0 mg/kg showed a significant decrease in the concentration of glycogen in tissues compared with the control group of the disease. The group of mice treated with hGAA at 20mg/kg did not show a significant decrease in tissue glycogen concentration compared to the disease control group.

soleus muscle.

Glycogen concentrations in the tissues of the soleus muscle of the normal control group, the control group of the disease, the group of mice that were injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5, 0, 10 and 20 mg/kg were 0.116 mg/g, 12.08 mg/g, 2.233 mg/g, 7.20 mg/g, 2.74 mg/g, 1.07 mg/g and 0.378 mg /g, respectively, in terms of wet weight (Fig. 11(c)). All groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) showed a significant decrease in tissue glycogen concentration compared to the disease control group. In addition, groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of not less than 5.0 mg/kg, as well as a group of mice that were injected with hGAA at a dosage of 20 mg/kg, showed a significant decrease in glycogen concentration in tissues.

Rectus femoris.

Glycogen concentrations in the tissues of the rectus femoris of the normal control group, the control group of the disease, the group of mice that were injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5 .0, 10 and 20 mg/kg were 0.0419 mg/g, 9.68 mg/g, 8.23 mg/g, 4.13 mg/g, 2.56 mg/g, 0.895 mg/g and 0.277 mg/g, respectively, in terms of wet weight (Fig. 11(d)). All groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) showed a significant decrease in tissue glycogen concentration compared to the control group of the disease and the group of mice that were injected with hGAA at a dosage of 20 mg/kg. The group of mice treated with hGAA at 20mg/kg did not show a significant decrease in tissue glycogen concentration compared to the disease control group.

The muscle of the long extensor of the fingers.

Glycogen concentrations in the tissues of the extensor digitorum longus muscle of the normal control group, the disease control group, the group of mice that were injected with hGAA at a dosage of 20 mg/kg, and the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at dosages of 2.5, 5.0, 10 and 20 mg/kg were 0.0950 mg/g, 10.4 mg/g, 9.11 mg/g, 4.79 mg/g, 2.59 mg/g, 1.34 mg/g and 0.188 mg/g, respectively, in terms of fresh weight (Fig. 11(e)). All groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) showed a significant decrease in tissue glycogen concentration compared to the disease control group. In addition, groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of not less than 5.0 mg/kg showed a significant decrease in

- 93 042007 decrease in the concentration of glycogen in tissues compared with a group of mice that were injected with hGAA at a dosage of 20 mg/kg. The group of mice treated with hGAA at 20mg/kg did not show a significant decrease in tissue glycogen concentration compared to the disease control group.

Example 30 Evaluation of the pharmacological action of hGAA antibodies against hTfR 3N (IgG4) in mice: histopathological evaluation (2).

The left cerebral hemisphere, cervical spinal cord, quadriceps femoris, soleus, and extensor digitorum longus muscles, which were collected in Example 29, were immersed in a sufficient amount of 10% neutral formalin buffer, approximately 24 hours later, the 10% neutral formalin buffer was replaced . 3 days after tissue collection, the tissues were immersed in 80% ethanol and a timer was programmed for a 48 hour delay so as to replace the ethanol with toluene using an automatic fixation and immersion device (Sakura Rotary), finally the tissues were immersed in paraffin, and the obtained thus the samples were subjected to PAS staining and HE staining.

The results of PAS staining will be discussed below.

Cortex.

Regarding the staining of PAS in cells, all of the disease control group and the group of mice injected with hGAA at a dosage of 20 mg/kg had moderate staining, while the groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 2, 5-20 mg/kg showed a reduction in staining. Regarding the staining of PAS in blood vessels, a group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg showed a decrease in staining.

Hippocampus/dentate gyrus and basal ganglion.

A group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg showed a decrease in PAS staining in cells and blood vessels compared to the disease control group. In the basal ganglion, a group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 2.5 mg/kg, and a group of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 5.0 mg/kg, also showed a decrease in staining.

Intermediate brain (thalamus/hypothalamus).

Groups of mice treated with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 2.5-20 mg/kg showed a decrease in PAS staining compared to the disease control group.

The midbrain is the bridge of the brain.

Groups of mice treated with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 5.0-20 mg/kg showed a decrease in PAS staining.

Medulla.

A group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg showed a decrease in PAS staining compared to the disease control group.

Cerebellum.

In the Purkinje cell layer/granular layer, groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 2.5-20 mg/kg tended to show a decrease in PAS staining in cells at the periphery of Purkinje cells compared to the disease control group. . In white matter groups of mice treated with hGAA antibodies against hTfR 3N (IgG4) at doses of 2.5 and 20 mg/kg tended to show a decrease in PAS staining.

Quadriceps femoris.

Groups of mice injected with hGAA anti-hTfR 3N (IgG4) at doses of 5.0 and 10 mg/kg tended to show a decrease in PAS staining compared to disease controls, and a group of mice injected with hGAA anti-hTfR 3N (IgG4) at 20 mg/kg showed a marked reduction in staining.

soleus muscle.

The group of mice injected with hGAA at 20 mg/kg and the group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at 10 mg/kg showed a decrease in PAS staining compared to the disease control group, and the group of mice which were injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg, showed a marked decrease in staining.

Further staining results will NOT be discussed below.

Cortex.

Cortex: Groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 5.0-20 mg/kg tended to show a reduction in tumor cell growth compared to the disease control group. Cerebral white matter (corpus callosum, hippocampal fimbria, and anterior commissure) and cerebral ventricle: a group of mice injected with hGAA at a dose of 20 mg/kg and a group of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at a dose of 5, 0-20 mg/kg, showed a decrease in the content of white matter vacuoles compared with the control group of the disease.

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Only the group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg tended to show a reduction in cell tumor growth compared to the control group for medulla oblongata disease.

Cerebellum.

A group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg tended to show a reduction in tumor cell growth in the white matter.

Quadriceps femoris.

The group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 10 mg/kg tended to show a decrease in the formation of vacuoles / lacunae in muscle fibers compared to the disease control group, and the group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at 20 mg/kg showed an additional reduction in the formation of vacuoles/lacunae in muscle fibers.

soleus muscle.

A group of mice injected with hGAA at 20 mg/kg and groups of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dose of 5.0-10 mg/kg showed a reduction in the formation of vacuoles/lacunae in muscle fibers compared to with the control group of the disease, and the group of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 20 mg/kg showed a slight decrease in the formation of vacuoles/lacunae in muscle fibers.

The muscle of the long extensor of the fingers.

The group of mice injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 5.0 mg/kg showed a decrease in the formation of vacuoles / lacunae in muscle fibers compared with the control group of the disease, and groups of mice that were injected with hGAA antibodies against hTfR 3N (IgG4) at a dosage of 10-20 mg/kg, showed an additional reduction in the formation of vacuoles/lacunae in muscle fibers.

The above results of the pharmacological action of hGAA antibodies against hTfR 3N (IgG4) in mice show that the administration of hGAA antibodies against hTfR 3N (IgG4) at a dosage of not less than 5 mg/kg is more effective as a therapeutic agent for Pompe disease with histopathological point of view than administration of the commercially available hGAA at 20mg/kg and exhibits extremely marked effects in the central nervous system and skeletal muscle tissues.

Example 31 Preparation of fusion protein between anti-hTfR 3N Fab and hGAA (Fab GSGAA).

To obtain the expression vector pE-neo (Fab HC-GS-GAA), a DNA fragment having the nucleotide sequence of SEQ ID NO: 89 and containing a gene encoding a fusion protein between hGAA and the Fab heavy chain of a humanized anti-hTfR antibody was artificially synthesized and inserted into pE-neo vector between its MluI and NotI. In this case, a nucleotide sequence encoding a leader peptide serving as a secretion signal was introduced into the gene encoding the fusion protein at the 5'-terminal side of the gene. CHO cells (CHO-Ki: purchased from American Type Culture Collection) were transformed with pE-neo (Fab HC-GS-GAA) and pE-hygr (LC3) obtained in Example 11 to generate a cell line expressing an inter-hGAA fusion protein and an anti-hTfR 3N Fab (Fab GS-GAA). The culture supernatant was obtained by culturing the expression cell line as described in Example 22. The culture supernatant was loaded onto a CaptureSelect IgG-CH1 column, which is a CH1 affinity column (column volume: approximately 1.0 ml, Thermo Fisher Scientific Inc.), which was balanced with five column volumes of 20 mM HEPES-NaOH buffer (pH: 7. 0) containing 100 mM NaCl to adsorb the fusion protein on the CH1 affinity column. The column was then washed by feeding five column volumes of 20 mM HEPES-NaOH buffer (pH: 7.0) containing 100 mM NaCl. The fusion protein adsorbed on the CH1 affinity column was then eluted with five column volumes of 100 mM glycine buffer (pH: 3.0) containing 50 mM NaCl. The eluate was immediately neutralized by adding the eluate to a container that had previously been supplied with 1 M HEPES-NaOH buffer (pH: 7.5). the product thus obtained was used as the purification product of Fab GS-GAA in the following experiments.

Example 32 Evaluation of the pharmacological action of Fab GS-GAA in mice.

Mice (GAA-KO/hTfR-KI mice) described in Example 28 were intravenously injected with Fab GS-GAA prepared in Example 31 or hGAA according to the use and dosage described in Table 2. 19-2. Normal mice that were not injected with the pharmaceutical agent were classified as normal controls (first group), and GAA-KO/hTfR-KI mice that were not administered with the pharmaceutical agent were classified as disease controls (second group). Each group included three or four mice. In addition, male and female mice were used, which were 9-14 weeks old (at the start of administration). One week after administration, the right hemisphere of the brain, heart, diaphragm, quadriceps femoris, soleus, and rectus femoris were collected by the method described in Example 29, and the concentration of glycogen contained in the tissues of each of the organs was measured. The measurement results are shown in FIG. 12. Compared with a group of mice,

- 95 042007 injected with hGAA, the Fab GS-GAA group of mice showed a marked decrease in glycogen concentration in all organs where glycogen concentration was measured. The group of mice injected with Fab GS-GAA showed a particularly marked decrease in glycogen concentration in the right hemisphere of the brain and heart. In addition, the group of mice injected with Fab GS-GAA at 5.0 mg/kg showed a marked decrease in glycogen concentration in the right hemisphere of the brain and heart compared to the group of mice administered with hGAA at 20 mg/kg. These results indicate that GS-GAA Fab can be used as a therapeutic agent for Pompe disease, especially as a therapeutic agent for Pompe disease accompanied by impaired brain and/or heart function.

Table 19-2. Grouping mice

Group Test Substance Mouse Dosage (mg/kg) 1 - normal mouse - 2 - Mouse GAA-KO/hTfR-KI - 3 hGAA Mouse GAA-KO/hTfR-KI 20 4 Fab GS-GAA Mouse GAA-KO/hTfR-KI 50 5 20

Example 33 Preparation of fusion proteins between various human lysosomal enzymes and humanized anti-hTfR antibodies.

A DNA fragment was artificially synthesized that had a nucleotide sequence containing a gene encoding fusion proteins between various human lysosomal enzymes and the humanized anti-hTfR antibody heavy chain (Note: when the fusion protein was scFab-IDUA, the expression vector was a single-chain humanized anti-hTfR antibody). These fusion proteins have amino acid sequences shown as SEQ ID NONS shown in Tables 20-A to 20-C and are designated in the fusion protein designation column in Table 1. 20-A 20-C. To obtain an expression vector, the synthesized DNA fragment was inserted into the pEpeo vector between its MluI and NotI. In this case, a nucleotide sequence encoding a leader peptide serving as a secretion signal was introduced into the gene encoding the fusion protein at the 5'-terminal side of the gene. Expression vector designations are shown in Table 1. 20-A - 20-C. To obtain a cell line expressing fusion proteins between various lysosomal enzymes and humanized anti-hTfR antibodies, CHO cells (CHO-Ki: purchased from the American Type Culture Collection) were transformed with each expression vector obtained and pE-hygr (LC3) obtained in Example 11 The culture supernatant was obtained by culturing the expression cell line as described in Example 22.

The fusion proteins between the lysosomal enzyme and the humanized anti-hTfR antibody that were contained in the culture supernatant were obtained by the following method 1 for purifying proteins having a Fab antibody and by the following method 2 for purifying proteins with an antibody containing a full-length heavy chain.

Purification Method 1: The culture supernatant was loaded onto a CaptureSelect IgG-CH1 column, which is a CH1 affinity column (column volume: approximately 1.0 ml, Thermo Fisher Scientific Inc.), which was equilibrated with five column volumes of 20 mM HEPES-NaOH buffer ( pH: 7.0) containing 100 mM NaCl to adsorb the fusion protein on the CH1 affinity column. The column was then washed by feeding five column volumes of 20 mM HEPES-NaOH buffer (pH: 7.0) containing 100 mM NaCl. The fusion protein adsorbed on the CH1 affinity column was then eluted with five column volumes of 100 mM glycine buffer (pH: 3.0) containing 50 mM NaCl. The eluate was immediately neutralized by adding the eluate to a container that had previously been supplied with 1 M HEPES-NaOH buffer (pH: 7.5).

Purification Method 2: The culture supernatant was loaded onto a MabSelect SuRe LX protein affinity column (column volume: approximately 1.0 ml, GE Healthcare Inc.) which was equilibrated with five column volumes of 20 mM HEPES-NaOH buffer (pH: 7.0) containing 100 mM NaCl to adsorb the fusion protein to Protein A. The column was then washed by feeding five column volumes of 20 mM HEPES-NaOH buffer (pH: 7.0) containing 100 mM NaCl. The protein adsorbed fusion protein was then eluted with five column volumes of 100 mM glycine buffer (pH: 3.0) containing 50 mM NaCl. The eluate was immediately neutralized by adding the eluate to a container that had previously been supplied with 1 M HEPES-NaOH buffer (pH: 7.5).

Using the fusion protein purification products thus obtained, the following experiments were carried out. In addition, the thus obtained fusion proteins between lysosomal enzymes and humanized antibodies against hTfR were designated as shown in the rightmost column in Table. 20-A - 20-C.

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Table 20(A). List of fusion proteins between various human lysosomal enzymes and humanized anti-hTfR antibodies

Lysosome designation SEQ ID NO Fusion protein notation Expression vector designation Light chain expression vector Fusion protein designation lysosomal enzyme humanized anti-hTfR antibody hGAA 89 Fab HC-GS3- GAA pE-neo (Fab HCGS3-GAA) pE-hygr (LC3) Fab GS3-GAA hIDUA 90 IgG4 HCIDUA pE-neo (IgG4 HCIDUA) pE-hygr (LC3) IgG4IDUA 91 IgG4 HC-GS8IDUA pE-neo (IgG4 HCGS8-IDUA) pE-hygr (LC3) IgG4 GS8-IDUA 92 IgG4 HCGS20-IDUA pE-neo (IgG4 HCGS20-IDUA) pE-hygr (LC3) IgG4 GS20-IDUA 93 Fab HC-GS3- IDUA pE-neo (Fab HCGS3-IDUA) pE-hygr (LC3) Fab GS3-IDUA 94 Fab HC-GS5- IDUA pE-neo (Fab HCGS5-IDUA) pE-hygr (LC3) Fab GS5-IDUA 95 FabHC-GSlO- IDUA pE-neo (Fab HCGS10-IDUA) pE-hygr (LC3) Fab GS10-IDUA 96 Fab HC-GS20- IDUA pE-neo (Fab HCGS20-IDUA) pE-hygr (LC3) Fab GS20-IDUA 97 Tandem Fab HC-IDUA pE-neo (Tandem Fab HC-IDUA) pE-hygr (LC3) Tandem Fab IDUA 99 scFab-IDUA pE-neo (scFabIDUA) - scFab-IDUA hPPT-1 100 IgG4 HC-PPT1 pE-neo (IgG4 HC- pE-hygr (LC3) IgG4 PPT1

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PPT1) 101 IgG4 HC-GS5- PPT1 pE-neo (IgG4 HCGS5-PPT1) pE-hygr (LC3) IgG4 GS5-PPT1 102 IgG4 HCGS10-PPT1 pE-neo (IgG4 HCGS10-PPT1) pE-hygr (LC3) IgG4 GS10-PPT1 103 IgG4 HCGS20-PPT1 pE-neo (IgG4 HCGS20-PPT1) pE-hygr (LC3) IgG4 GS20-PPT1 104 FabHC-GS3- PPT1 pE-neo (Fab HCGS3-PPT1) pE-hygr (LC3) Fab GS3-PPT1 105 Tandem Fab HC-PPT1 pE-neo (Tandem Fab HC-PPT1) pE-hygr (LC3) Tandem Fab PPT1 haSM 106 IgG4 HC-ASM pE-neo (IgG4 HCASM) pE-hygr (LC3) IgG4ASM 107 IgG4 HC-GS5ASM pE-neo (IgG4 HCGS5-ASM) pE-hygr (LC3) IgG4 GS5-ASM 108 IgG4 HC- GS10-ASM pE-neo (IgG4 HCGS10-ASM) pE-hygr (LC3) IgG4 GS10-ASM 109 IgG4 HC- GS20-ASM pE-neo (IgG4 HCGS20-ASM) pE-hygr (LC3) IgG4 GS20-ASM 110 FabHC-GS3- ASM pE-neo (Fab HCGS3-ASM) pE-hygr (LC3) Fab GS3-ASM 111 Fab HC-GS5- ASM pE-neo (Fab HCGS5-ASM) pE-hygr (LC3) Fab GS5-ASM 112 FabHC-GSlO- ASM pE-neo (Fab HCGS10-ASM) pE-hygr (LC3) Fab GS10-ASM 113 Fab HC-GS20- ASM pE-neo (Fab HCGS20-ASM) pE-hygr (LC3) Fab GS20-ASM 114 Tandem Fab HC-ASM pE-neo (Tandem Fab HC-ASM) pE-hygr (LC3) Tandem Fab ASM hARSA 115 IgG4 HCARSA pE-neo (IgG4 HCARSA) pE-hygr (LC3) IgG4 ARSA 116 IgG4 HC-GS5ARSA pE-neo (IgG4 HCGS5-ARSA) pE-hygr (LC3) IgG4 GS5-ARSA 117 IgG4 HC- GS10-ARSA pE-neo (IgG4 HCGS10-ARSA) pE-hygr (LC3) IgG4 GS10-ARSA 118 IgG4 HC- GS20-ARSA pE-neo (IgG4 HCGS20-ARSA) pE-hygr (LC3) IgG4 GS20-ARSA 119 FabHC-GS3- ARSA pE-neo (Fab HCGS3-ARSA) pE-hygr (LC3) Fab GS3-ARSA 120 Fab HC-GS5- ARSA pE-neo (Fab HCGS5-ARSA) pE-hygr (LC3) Fab GS5-ARSA 121 FabHC-GSlO- ARSA pE-neo (Fab HCGS10-ARSA) pE-hygr (LC3) Fab GS10-ARSA 122 Fab HC-GS20- ARSA pE-neo (Fab HCGS20-ARSA) pE-hygr (LC3) Fab GS20-ARSA 123 Tandem Fab HC-ARSA pE-neo (Tandem Fab HC-ARSA) pE-hygr (LC3) Tandem Fab ARSA

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Table 20(B). List of fusion proteins between various human lysosomal enzymes and humanized anti-hTfR antibodies

Lysosome designation SEQ ID NO Fusion protein notation Expression vector designation Light chain expression vector Fusion protein designation lysosomal enzyme humanized anti-hTfR antibody hSGSH 124 IgG4 HC-SGSH pE-neo (IgG4 HCSGSH) pE-hygr (LC3) IgG4SGSH 125 IgG4 HC-GS5SGSH pE-neo (IgG4 HCGS5-SGSH) pE-hygr (LC3) IgG4 GS5-SGSH 126 IgG4 HC-GS10SGSH pE-neo (IgG4 HCGS10-SGSH) pE-hygr (LC3) IgG4 GS10-SGSH 127 IgG4 HC-GS20SGSH pE-neo (IgG4 HCGS20-SGSH) pE-hygr (LC3) IgG4 GS20-SGSH 128 Fab HC-GS3- SGSH pE-neo (Fab HC-GS3SGSH) pE-hygr (LC3) Fab GS3-SGSH 129 Tandem Fab HC-SGSH pE-neo (Tandem Fab HC-SGSH) pE-hygr (LC3) Tandem Fab SGSH hGBA 130 IgG4 HC-GBA pE-neo (IgG4 HCGBA) pE-hygr (LC3) IgG4GBA 131 IgG4 HC-GS5GBA pE-neo (IgG4 HCGS5-GBA) pE-hygr (LC3) IgG4 GS5-GBA 132 IgG4 HC-GS10GBA pE-neo (IgG4 HCGS10-GBA) pE-hygr (LC3) IgG4 GS10-GBA 133 IgG4HC-GS20GBA pE-neo (IgG4 HCGS20-GBA) pE-hygr (LC3) IgG4 GS20-GBA 134 Fab HC-GS3- GBA pE-neo (Fab HC-GS3GBA) pE-hygr (LC3) Fab GS3-GBA 135 Tandem Fab HC-GBA pE-neo (Tandem Fab HC-GBA) pE-hygr (LC3) Tandem Fab GBA

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hTPP-1 136 IgG4 HC-TPP1 pE-neo (IgG4 HCTPP1) pE-hygr (LC3) IgG4 TPP1 137 IgG4 HC-GS5- TPP1 pE-neo (IgG4 HCGS5-TPP1) pE-hygr (LC3) IgG4 GS5-TPP1 138 IgG4 HC-GS10- TPP1 pE-neo (IgG4 HCGS10-TPP1) pE-hygr (LC3) IgG4 GS10-TPP1 139 IgG4 HC-GS20- TPP1 pE-neo (IgG4 HCGS20-TPP1) pE-hygr (LC3) IgG4 GS20-TPP1 140 Fab HC-GS3- TPP1 pE-neo (Fab HC-GS3TPP1) pE-hygr (LC3) Fab GS3-TPP1 141 Tandem Fab HC-TPP1 pE-neo (Tandem Fab HC-TPP1) pE-hygr (LC3) Tandem Fab TPP1 hNAGLU 142 IgG4 HCNAGLU pE-neo (IgG4 HCNAGLU) pE-hygr (LC3) IgG4 NAGLU 143 IgG4 HC-GS5NAGLU pE-neo (IgG4 HCGS5-NAGLU) pE-hygr (LC3) IgG4 GS5-NAGLU 144 IgG4 HC-GS10- NAGLU pE-neo (IgG4 HCGS10-NAGLU) pE-hygr (LC3) IgG4 GS10NAGLU 145 IgG4 HC-GS20NAGLU pE-neo (IgG4 HCGS20-NAGLU) pE-hygr (LC3) IgG4 GS20NAGLU 146 Fab HC-GS3- NAGLU pE-neo (Fab HC-GS3NAGLU) pE-hygr (LC3) Fab GS3-NAGLU 147 Fab HC-GS5- NAGLU pE-neo (Fab HC-GS5NAGLU) pE-hygr (LC3) Fab GS5-NAGLU 148 FabHC-GSlO- NAGLU pE-neo (Fab HCGS10-NAGLU) pE-hygr (LC3) Fab GS10-NAGLU 149 Fab HC-GS20- NAGLU pE-neo (Fab HCGS20-NAGLU) pE-hygr (LC3) Fab GS20-NAGLU hGUSB 150 IgG4 HCG USB pE-neo (IgG4 HCGUSB) pE-hygr (LC3) IgG4 GUSB 151 IgG4 HC-GS5GUSB pE-neo (IgG4 HCGS5-GUSB) pE-hygr (LC3) IgG4 GS5-GUSB 152 IgG4 HC-GS10GUSB pE-neo (IgG4 HCGS10-GUSB) pE-hygr (LC3) IgG4 GS10-GUSB 153 IgG4 HC-GS20GUSB pE-neo (IgG4 HCGS20-GUSB) pE-hygr (LC3) IgG4 GS20-GUSB 154 Fab HC-GS3- GUSB pE-neo (Fab HC-GS3GUSB) pE-hygr (LC3) Fab GS3-GUSB 155 Fab HC-GS5- GUSB pE-neo (Fab HC-GS5GUSB) pE-hygr (LC3) Fab GS5-GUSB 156 FabHC-GSlO- pE-neo (Fab HC- pE-hygr (LC3) Fab GS10-GUSB GUSB GS10-GUSB) 157 Fab HC-GS20- GUSB pE-neo (Fab HCGS20-GUSB) pE-hygr (LC3) Fab GS20-GUSB

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Table 20(C). List of fusion proteins between various human lysosomal enzymes and humanized anti-hTfR antibodies

Designation lysosomes SEQ ID NO Fusion protein notation Designation expression vector Light chain expression vector Fusion protein designation lysosomal enzyme humanized anti-hTfR antibody hGALC 158 IgG4 HCGALC pE-neo (IgG4 HCGALC) pE-hygr (LC3) IgG4 GALC 159 IgG4 HC-GS5GALC pE-neo (IgG4 HCGS5-GALC) pE-hygr (LC3) IgG4 GS5-GALC 160 IgG4HO- GS 10-GALC pE-neo (IgG4 HCGS 10-GALC) pE-hygr (LC3) IgG4GS 10-GALC 161 IgG4 HCGS20-GALC pE-neo (IgG4 HCGS20-GALC) pE-hygr (LC3) IgG4 GS20-GALC 162 Fab HC-GS3- GALC pE-neo (Fab HC-GS3GALC) pE-hygr (LC3) Fab GS3-GALC 163 Fab HC-GS5- GALC pE-neo (Fab HC-GS5GALC) pE-hygr (LC3) Fab GS5-GALC 164 Fab HC-GS 10- GALC pE-neo (Fab HCGS 10-GALC) pE-hygr (LC3) Fab GS 10-GALC 165 Fab HC-GS20- GALC pE-neo (Fab HCGS20-GALC) pE-hygr (LC3) Fab GS20-GALC hAC 166 IgG4 HC-AC pE-neo (IgG4 HC-AC) pE-hygr (LC3) IgG4 AC 167 IgG4 HC-GS5AC pE-neo (IgG4 HCGS5-AC) pE-hygr (LC3) IgG4 GS5-AC 168 IgG4 HC- GS 10-AC pE-neo (IgG4 HCGS 10-AC) pE-hygr (LC3) IgG4 GS 10-AC 169 IgG4 HC- GS20-AC pE-neo (IgG4 HCGS20-AC) pE-hygr (LC3) IgG4 GS20-AC 170 Fab HC-GS3- AC pE-neo (Fab HC-GS3AC) pE-hygr (LC3) Fab GS3-AC 171 Fab HC-GS5- AC pE-neo (Fab HC-GS5AC) pE-hygr (LC3) Fab GS5-AC

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172 Fab HC-GS 10- AC pE-neo (Fab HCGS10-AC) pE-hygr (LC3) Fab GS10-AC 173 Fab HC-GS20- AC pE-neo (Fab HCGS20-AC) pE-hygr (LC3) Fab GS20-AC hFUCAl 174 IgG4 HCFUCA1 pE-neo (IgG4 HC- FUCA1) pE-hygr (LC3) IgG4FUCA1 175 IgG4 HC-GS5- FUCA1 pE-neo (IgG4 HCGS5-FUCA1) pE-hygr (LC3) IgG4 GS5-FUCA1 176 IgG4 HCGS10-FUCA1 pE-neo (IgG4 HCGS10-FUCA1) pE-hygr (LC3) IgG4GS10- FUCA1 177 IgG4 HCGS20-FUCA1 pE-neo (IgG4 HCGS20-FUCA1) pE-hygr (LC3) IgG4 GS20- FUCA1 178 Fab HC-GS3- FUCA1 pE-neo (Fab HC-GS3FUCA1) pE-hygr (LC3) Fab GS3-FUCA1 179 Fab HC-GS5- FUCA1 pE-neo (Fab HC-GS5FUCA1) pE-hygr (LC3) Fab GS5-FUCA1 180 FabHC-GSlO- FUCA1 pE-neo (Fab HCGS10-FUCA1) pE-hygr (LC3) Fab GS10-FUCA1 181 Fab HC-GS20- FUCA1 pE-neo (Fab HCGS20-FUCA1) pE-hygr (LC3) Fab GS20-FUCA1 hLAMAN 182 IgG4 HCLAMAN pE-neo (IgG4 HCLAMAN) pE-hygr (LC3) IgG4 LAMAN 183 IgG4 HC-GS5LAMAN pE-neo (IgG4 HCGS5-LAMAN) pE-hygr (LC3) IgG4 GS5LAMAN 184 IgG4 HC- GS10- LAMAN pE-neo (IgG4 HCGS10-LAMAN) pE-hygr (LC3) IgG4GS10LAMAN 185 IgG4 HC- GS20- LAMAN pE-neo (IgG4 HCGS20-LAMAN) pE-hygr (LC3) IgG4 GS20LAMAN 186 Fab HC-GS3- LAMAN pE-neo (Fab HC-GS3LAMAN) pE-hygr (LC3) Fab GS3-LAMAN 187 Fab HC-GS5- LAMAN pE-neo (Fab HC-GS5LAMAN) pE-hygr (LC3) Fab GS5-LAMAN 188 FabHC-GSlO- LAMAN pE-neo (Fab HCGS10-LAMAN) pE-hygr (LC3) Fab GS10-LAMAN 189 Fab HC-GS20LAMAN pE-neo (Fab HCGS20-LAMAN) pE-hygr (LC3) Fab GS20-LAMAN

Example 34 Measurement of the affinity of fusion proteins between various human lysosomal enzymes and humanized anti-human and monkey TfR hTfR antibodies and the enzymatic activity of the fusion proteins.

Affinity for human TfR and monkey TfR was measured using the method described in Example 7. Human lysosomal enzyme activity was measured by the method described below. However, the enzymatic activity of hAC and hLAMAN was not measured.

Measurement of hIDUA activity.

The purified anti-TfR hIDUA antibody fusion protein was diluted to the desired concentration with 50 mM citrate buffer solution (pH: 3.5) containing 0.1% BSA and 20 µl of the resulting FluoroNunc F96 plate dilution (Nunc Inc.) was added to each well. .). 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next, as a substrate, 4methylumbelliferyl a-L-iduronide (glycosynth Inc.) was diluted to 1 mM with the buffer solution described above, and 60 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 200 μl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 365 nm and a detection wavelength of 460 nm by

- 102 042007 tablet reader. Enzymatic activity was determined in terms of specific activity per unit weight of protein (mg), with one unit being defined as 4 MU µmol/minute at 37°C. For the following fusion proteins, specific activity was also determined.

Measurement of hPPT-1 activity.

β-glucosidase (Oriental Yeast Co., Ltd.) was diluted to 0.25 mg/ml with 0.4 M Na ₂ HPO4/0.2 M citrate buffer solution (pH: 4.0) containing 0.2% BSA and 0.00625% Triton X-100, and 20 μl of the resulting dilution was added to each well of a FluoroNunc F96 plate (Nunc Inc.). Next, the purified anti-TfR hPPT-1 fusion protein was diluted to the desired concentration with the buffer solution described above, and 20 μl of the resulting dilution was added to each well. 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Further, as a substrate, 4-methylumbelliferyl 6-thio-palmitate-β-D-glucopyranoside (Santa Cruz Biotechnology Inc.) was diluted to 0.25 mM with the buffer solution described above, and 20 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 150 µl of 0.05 M glycine/NaOH buffer (pH: 10.6) was added to each well to stop the reaction, and measured at an excitation wavelength of 365 nm and a detection wavelength of 460 nm by means of a plate reader. Enzymatic activity was determined, with one unit being defined as 4 MU µmol/minute at 37°C. haSM activity measurement:

The purified anti-TfR haSM fusion protein was diluted to the desired concentration in 250 mM acetate buffer (pH: 5.0) containing 0.1 mM zinc acetate, 0.25 mg/ml BSA and 0.15% Tween 20, and into each 20 μl of the resulting dilution was added to a well of a FluoroNunc F96 plate (Nunc Inc.). 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next, as a substrate, β-hexadecanoylamino-4-methylumbelliferyl phosphorylcholine (Carbosynth Inc.) was diluted to 1 mM with the buffer solution described above, and 20 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 200 µl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 365 nm and a detection wavelength of 460 nm with a plate reader. Enzymatic activity was determined in terms of specific activity per unit weight of protein (mg), with one unit being defined as 4 MU µmol/minute at 37°C.

Measurement of hARSA activity.

Purified anti-TfR hARSA fusion protein was diluted to the desired concentration in 50 mM acetate buffer (pH: 5.0) containing 0.5% BSA and 1.0% Triton-X 100 and into each well of a FluoroNunc F96 plate (Nunc Inc. .) was added 20 μl of the resulting dilution. 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next, as a substrate, 4-methylumbelliferyl potassium sulfate salt (Sigma Inc.) was diluted to 5 mM with the buffer solution described above, and 100 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 150 µl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 365 nm and a detection wavelength of 460 nm with a plate reader. Enzymatic activity was determined, with one unit being defined as 4 MU µmol/minute at 37°C.

Measurement of hSGSH activity.

The purified anti-TfR hSGSH fusion protein was diluted to the desired concentration in 50 mM acetate buffer (pH: 5.0) containing 0.2% BSA, and 20 µl of the resulting dilution was added to each well of a FluoroNunc F96 plate (Nunc Inc.). . 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Further, as a substrate, 4-methylumbelliferyl 2-deoxy-2-sulfamino-a-O-glucopyranoside (Carbosynth Inc.) was diluted to 5 mM with the buffer solution described above, and 20 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 2 hours. After the reaction, 0.5 mg/ml of α-glucosidase (Oriental Yeast Co., Ltd.) was diluted with the above buffer solution, 20 μl of the resulting dilution was added to each well, and the mixture was reacted at 37° C. for 17 hours. After the reaction, 150 µl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 3-65 nm and a detection wavelength of 460 nm by means of a plate reader. Enzymatic activity was determined, with one unit being defined as 4 MU µmol/minute at 37°C.

Measurement of hGBA activity:

The purified anti-TfR hGBA fusion protein was diluted to the desired concentration in 100 mM phosphate buffer solution (pH: 6.0) containing 0.1% BSA, 0.15% Triton-X 100 and 0.125% sodium taurocholate, and into each well of the plate FluoroNunc F96 (Nunc Inc.) was added 20 μl of the resulting dilution. 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard.

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Next, as a substrate, 4-methylumbelliferyl p-D-glucopyranoside (Sigma Inc.) was diluted to 3.5 mM with the buffer solution described above, and 70 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 150 µl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 365 nm and a detection wavelength of 460 nm with a plate reader. Enzymatic activity was determined, with one unit being defined as 4 MU µmol/minute at 37°C.

Measurement of hTPP-1 activity.

The purified anti-TfR hTPP-1 fusion protein was diluted to the desired concentration in 50 mM sodium formate buffer (pH: 3.5) containing 150 mM NaCl and 0.1% Triton-X 100, and the resulting dilution was preincubated at 37 °C for 1 hour, 7-amino-4-methicoumarin (AMC) (Sigma Inc.) was used as a standard, and processed in a similar manner. After 1 hour, 20 µl of the dilution was added to each well of a FluoroNunc F96 plate (Nunc Inc.). Further, as a substrate, Ala-Ala-Phe-7-amido-4-methicoumarin (Sigma Inc.) was diluted to 25 mM with 100 mM acetate buffer solution (pH: 4.0) containing 150 mM NaCl and 0.1% Triton-X 100 and 40 µl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 150 µl of 100 mM monochloroacetate/130 mM NaOH/100 mM acetate buffer solution (pH: 4.3) was added to each well to stop the reaction, and measured at an excitation wavelength of 365 nm and a detection wavelength of 4-60 nm by means of a plate -reader. Enzymatic activity was determined, with one unit being defined as AMS µmol/minute at 37°C.

Measurement of hGAA activity.

The purified anti-TfR hGAA fusion protein was diluted to the desired concentration with PBS (-) (pH: 7.2) containing 0.5% BSA, and 20 μl of the resulting dilution was added to each well of a FluoroNunc F96 plate (Nunc Inc.). 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next, as a substrate, 4-methylumbelliferyl α-D-glucopyranoside (Sigma Inc.) was diluted to 2.2 mM with 200 mM acetate buffer (pH: 4.3), and 20 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 200 µl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 365 nm and a detection wavelength of 460 nm with a plate reader. Enzymatic activity was determined, with one unit being defined as 4 MU µmol/minute at 37°C. Measurement of hNAGLU activity:

The purified anti-TfR hNAGLU fusion protein was diluted to the desired concentration in 50 mM acetate buffer solution (pH: 4.3) containing 0.1% BSA, and 20 μl of the resulting dilution was added to each well of a FluoroNunc F96 plate (Nunc Inc.). . 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next, as a substrate, 4-methylumbelliferyl N-acetyl-α-D-glucosaminide (Calbiochem) was diluted to 1 mM with the buffer solution described above, and 20 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 150 µl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 3-65 nm and a detection wavelength of 460 nm by means of a plate reader. Enzymatic activity was determined, with one unit being defined as 4 MU µmol/minute at 37°C.

Measurement of hGUSB activity.

The purified anti-TfR hGUSB fusion protein was diluted to the desired concentration in 25 mM acetate buffer (pH: 4.5) containing 50 mM NaCl, and 20 μl of the resulting dilution was added to each well of a FluoroNunc F96 plate (Nunc Inc.). 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next, as a substrate, 4-methylumbelliferyl-O-glucuronide hydrate (Sigma Inc.) was diluted to 2 mM with the buffer solution described above, and 20 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 150 µl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 365 nm and a detection wavelength of 460 nm with a plate reader. Enzymatic activity was determined, with one unit being defined as 4 MU µmol/minute at 37°C.

Measurement of hGALC activity.

Purified anti-TfR GALC fusion protein was diluted to the desired concentration in 50 mM acetate buffer (pH: 4.5) containing 125 mM NaCl and 0.5% Triton X-100 and into each well of a FluoroNunc F96 plate (Nunc Inc.) 20 μl of the resulting dilution was added. 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Further as a substrate

- 104 042007

4-methylumbelliferyl-β-D-galactopyranoside (Sigma Inc.) was diluted to 1 mM with the buffer solution described above, and 20 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 150 µl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 365 nm and a detection wavelength of 460 nm with a plate reader. Enzymatic activity was determined, with one unit being defined as 4 MU µmol/minute at 37°C.

Measurement of hFUCA1 activity.

The purified anti-TfR FUCA1 fusion protein was diluted to the desired concentration in 100 mM citrate buffer (pH: 5.5) containing 0.2% BSAI, and 20 µl of the resulting dilution was added to each well of a FluoroNunc F96 plate (Nunc Inc.). . 4-methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next, as a substrate, 4-MU-a-L-fucopyranoside (Sigma Inc.) was diluted to 2 mM with the buffer solution described above, and 20 μl of the resulting dilution was added to each well. The mixture was shaken with a plate mixer, and then reacted at 37° C. for 1 hour. After the reaction, 150 µl of 0.05 M glycine/NaOH buffer solution (pH: 10.6) was added to each well to stop the reaction, and measurement was performed at an excitation wavelength of 365 nm and a detection wavelength of 460 nm with a plate reader. Enzymatic activity was determined, with one unit being defined as 4 MU µmol/minute at 37°C.

Tab. 21 shows measurements of the affinity of fusion proteins between various human lysosomal enzymes and humanized anti-human and monkey TfR hTfR antibodies, and the enzymatic activity of the fusion proteins. Affinity and enzymatic activity were measured only for some of the fusion proteins between lysosomal enzymes and the humanized anti-hTfR antibody that were prepared in Example 33. Specifically, affinity for human TfR and monkey TfR was measured for 24 types of fusion proteins, and enzymatic activity was measured for 20 types of fusion proteins .

Each of the fusion proteins whose enzymatic activity was measured had enzymatic activity as a lysosomal enzyme. In addition, each of the fusion proteins whose affinity for human TfR was measured had a high affinity for human TfR, even those with the lowest affinity showed a KD value of 2.09x10'1, and 19 types of fusion proteins showed a _KD value below than 1.00x10 ^-1 . In addition, each of the fusion proteins whose affinity for monkey TfR was measured had a high affinity for monkey TfR, even those with the lowest affinity showed a KD value of 3.05x10 ^-8 , and 12 types of fusion proteins showed a KD value lower, than 1.00x10 ^-12 .

The anti-hTfR antibody used herein is an antibody whose heavy chain is a heavy chain (IgG) having the amino acid sequence of SEQ ID NO: 65 as its variable region, or a Fab heavy chain, and whose light chain has the amino acid sequence as its variable region SEQ ID NO: 18. These results show that by converting the lysosomal enzyme into the form of a fusion protein between the lysosomal enzyme and an antibody containing these amino acid sequences as variable regions, the lysosomal enzyme can be passed through the BBB by the human TfR and exhibit its enzymatic activity. in the brain.

Table 21. Fusion protein affinity between various human lysosomal enzymes and humanized anti-human and monkey TfR hTfR antibodies and enzymatic activity of the fusion proteins

Fusion protein designation lysosomal enzyme humanized anti-hTfR antibody Specific Enzymatic Activity (Unit/mg) Affinity for human TfR and monkey TfR Type kon (1/Ms) koff (1/s) K _D (M) Fab-GS3-GAA 4.81 Human TfR 5.95x10 ^{5 3.39x10-4 2.09x10-10} TfR Monkeys 4.73x10 ^{5 1.45x10-2 3.05x10-5} IgG4-IDUA 1.55 Human TfR 9.80x10 ⁵ < ^1.00х10-7 < ^1.00х10-12 TfR Monkeys 5.61x10 ⁵ < ^1.00х10-7 < ^1.00х10-12 IgG4-GS20-IDUA 1.37 Human TfR 9.24x10 ⁵ < ^1.00х10-7 < ^1.00х10-12

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TfR Monkeys 4.81xl0 ⁵ <l,00xl0- ⁷ <l,00xl0- ¹² Fab-GS3-IDUA 1.7 Human TfR 2.93xl0 ^{5 5.28xl0-5} l,08xl0- ⁿ TfR Monkeys 2.21xl0 ^{5 2.92xl0'4} l,32xl0- ² IgG4-PPTl 9.4 Human TfR 7.37xl0 ⁵ <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys 3.07xl0 ⁵ <l,00xl0- ⁷ <l,00xl0- ¹² Fab-GS3-PPTl 9.1 Human TfR 4.17xl0 ⁵ <l,00xl0- ⁷ <l,00xl0' ¹² TfR Monkeys 9.53xl0 ^{4 9.13xl0'4} l,32xl0- ² Fab-GS3-ASM 0.105 Human TfR ^{5.26x10s 3.63xl0-5 6.89X10-11} TfR Monkeys ^{2.74x10s 5.49xl0'4 2.00xl0-2} IgG4-GS10-ARSA 0.72 Human TfR ^4.60x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^1.69x10s <l,00xl0- ⁷ <l,00xl0- ¹² Fab-GS3-ARSA 0.55 Human TfR ^{6.70x10s 4.02xl0-5 6.00xl0-12} TfR Monkeys ^{2.58x10s 2.8lxIO'4} l,09xl0- ² Fab-GS3-SGSH 0.068 Human TfR ^7.21x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^3.88x10s <l,00xl0- ⁷ <l,00xl0- ¹² IgG4-GS10-GBA 7.0 Human TfR ^9.57x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^4.49x10s <l,00xl0- ⁷ <l,00xl0- ¹² Fab-GS3-GBA 9.2 Human TfR ^6.23x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^2.99x10s 1.42x10'5 ^4.75xl0-2 IgG4-TPPl 0.26 Human TfR ^7.58x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^3.41x10s <l,00xl0- ⁷ <l,00xl0- ¹² IgG4-GS20-TPPl 0.21 Human TfR ^6.82x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^3.02x10s <l,00xl0- ⁷ <l,00xl0- ¹² Fab-GS3-NAGLU He measured Human TfR ^2.91x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^{1.54x10s 4.02xl0-4 2.62xl0'2} Fab-GS3-GUSB 8.49 Human TfR ^4.49x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^2.50x10s <l,00xl0- ⁷ <l,00xl0- ¹² IgG4-GS 10-GALC 0.35 Human TfR 3.14xl0 ⁴ <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys 2.80xl0 ⁴ <l,00xl0- ⁷ <l,00xl0- ¹² Fab-GS3-GALC 1.1 Human TfR ^2.24x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys Ι,ΙΙχΙΟ ⁵ l,54xl0- ⁴ l,40xl0- ² IgG4-GS10-AC He measured Human TfR ^5.76x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys 3.01xl0 ⁵ <l,00xl0- ⁷ <l,00xl0- ¹² Fab-GS3-AC He measured Human TfR ^5.01x10s l,05xl0- ^{4 2.09xl0-10} TfR Monkeys ^2.77x10s 3.27x10'5 1.18x10'5 IgG4-GS10-FUCAl 24.2 Human TfR ^4.66x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^2.46x10s <l,00xl0- ⁷ <l,00xl0- ¹² Fab-GS3-FUCAl 27.6 Human TfR ^2.82x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^1.51x10s 4.88x10'5 ^3.23xl0-10 Fab-GS3-LAMAN He measured Human TfR ^7.29x10s <l,00xl0- ⁷ <l,00xl0- ¹² TfR Monkeys ^3.57x10s <l,00xl0- ⁷ <l,00xl0- ¹²

Industrial Applicability

The anti-hTfR antibodies of the present invention, when fused with physiologically active proteins, small molecule compounds, and the like of interest, can allow them to pass through the blood-brain barrier and are therefore very useful in providing a means of delivering physiologically active proteins, small molecule compounds, and the like to the brain. , whose action is necessary in the central nervous system.

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List of reference designations

Blood vessel.

Parenchyma of the brain.

Nerve-like cells.

Purkinje cells.

Arbitrary sequence list text

SEQ ID NO: 3: amino acid sequence of exemplary linker 1

SEQ ID NO: 4: amino acid sequence of exemplary linker 2

SEQ ID NO: 5: amino acid sequence of exemplary linker 3

SEQ ID NO: 6: Amino acid sequence 1 of CDR1 in light chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 7: Amino acid sequence 2 of CDR1 in light chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 8: Amino acid sequence 1 of CDR2 in light chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 9: Amino acid sequence 2 of CDR2 in light chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 10: Amino acid sequence of CDR3 in light chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 11: Amino acid sequence 1 of CDR1 in heavy chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 12: Amino acid sequence 2 of CDR1 in mouse anti-hTfR antibody heavy chain #3

SEQ ID NO: 13: Amino acid sequence 1 of CDR2 in heavy chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 14: Amino acid sequence 2 of CDR2 in the heavy chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 15: Amino acid sequence 1 of CDR3 in the heavy chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 16: Amino acid sequence 2 of CDR3 in the heavy chain of mouse anti-hTfR antibody No. 3

SEQ ID NO: 17: Humanized anti-hTfR antibody light chain amino acid sequence 1 #3

SEQ ID NO: 18: Humanized anti-hTfR antibody light chain amino acid sequence 2 #3

SEQ ID NO: 19: Humanized anti-hTfR antibody light chain amino acid sequence 3 #3

SEQ ID NO: 20: Humanized anti-hTfR antibody light chain amino acid sequence 4 #3

SEQ ID NO: 21: Humanized anti-hTfR antibody light chain amino acid sequence 5 #3

SEQ ID NO: 22: amino acid sequence b of humanized anti-hTfR antibody light chain variable region #3

SEQ ID NO: 23: Light chain amino acid sequence of humanized anti-hTfR antibody No. 3 containing amino acid sequence 2 as variable region

SEQ ID NO: 24: Nucleotide sequence encoding the light chain amino acid sequence of a humanized anti-hTfR antibody No. 3 containing amino acid sequence 2 as variable region, synthetic sequence

SEQ ID NO: 25: Light chain amino acid sequence of humanized anti-hTfR antibody No. 3 containing amino acid sequence 4 as variable region

SEQ ID NO: 26: Nucleotide sequence encoding the light chain amino acid sequence of a humanized anti-hTfR antibody No. 3 containing amino acid sequence 4 as variable region, synthetic sequence

SEQ ID NO: 27: Light chain amino acid sequence of humanized anti-hTfR antibody No. 3 containing amino acid sequence 5 as variable region

SEQ ID NO: 28: Nucleotide sequence encoding the light chain amino acid sequence of a humanized anti-hTfR antibody No. 3 containing amino acid sequence 5 as variable region, synthetic sequence

SEQ ID NO: 29: Light chain amino acid sequence of humanized anti-hTfR antibody No. 3 containing amino acid sequence 6 as variable region

SEQ ID NO: 30: Nucleotide sequence encoding the light chain amino acid sequence of a humanized anti-hTfR antibody No. 3 containing amino acid sequence 6 as variable region, synthetic sequence

SEQ ID NO: 31: Humanized anti-hTfR antibody heavy chain variable region amino acid sequence 1 #3

SEQ ID NO: 32: Humanized anti-hTfR antibody heavy chain variable region amino acid sequence 2 #3

SEQ ID NO: 33: Humanized anti-hTfR antibody heavy chain variable region amino acid sequence 3 #3

SEQ ID NO: 34: Humanized anti-hTfR antibody heavy chain variable region amino acid sequence 4 #3

SEQ ID NO: 35: Humanized anti-hTfR antibody heavy chain variable region amino acid sequence 5 #3

SEQ ID NO: 36: Humanized anti-hTfR antibody heavy chain variable region amino acid sequence 6 #3

SEQ ID NO: 37: Heavy chain amino acid sequence of humanized anti-hTfR antibody No. 3 containing amino acid sequence 2 as variable region

SEQ ID NO: 38: Nucleotide sequence encoding the heavy chain amino acid sequence of humanized anti-hTfR antibody No. 3 containing amino acid sequence 2 as variable region, synthetic sequence of SEQ ID NO: 39: Amino acid sequence of heavy chain (IgG4) of humanized anti-hTfR antibody No. 3 containing the amino acid sequence 2 as the variable region

SEQ ID NO: 40: Nucleotide sequence encoding the heavy chain amino acid sequence (IgG4) of a humanized anti-hTfR antibody No. 3 containing amino acid sequence 2 as variable region, synthetic sequence

SEQ ID NO: 41: primer hTfR5', synthetic sequence

SEQ ID NO: 42: primer hTfR3', synthetic sequence

SEQ ID NO: 43: Hyg-Sfi5' primer, synthetic sequence

SEQ ID NO: 44: primer Hyg-BstX3', synthetic sequence

SEQ ID NO: 45: DNA nucleotide sequence in which a neomycin resistance gene flanked by 1oxP sequences is placed on the 3' side of a cDNA encoding a chimeric hTfR, synthetic sequence

SEQ ID NO: 46: 5' arm targeting vector sequence, synthetic sequence

SEQ ID NO: 47: 3' arm targeting vector sequence, synthetic sequence

SEQ ID NO: 48: Amino acid sequence of light chain variable region of mouse anti-hTfR antibody No. 3

SEQ ID NO: 49: Amino acid sequence of heavy chain variable region of mouse anti-hTfR antibody #3

SEQ ID NO: 51: Anti-hTfR antibody heavy chain fusion protein amino acid #3 (humanized 2) and hI2S, synthetic sequence

SEQ ID NO: 52: Nucleotide sequence encoding the amino acid sequence of anti-hTfR antibody heavy chain fusion protein No. 3 (humanized 2) and hI2S, synthetic sequence

SEQ ID NO: 53: Amino acid sequence of humanized anti-hTfR antibody heavy chain fusion protein #3N and hI2S

SEQ ID NO: 54: Nucleotide sequence encoding the amino acid sequence of the heavy chain fusion protein of the humanized anti-hTfR antibody No. 3N and hI2S, synthetic sequence

SEQ ID NO: 57: Amino acid sequence of humanized anti-hTfR antibody heavy chain fusion protein #3N and hGAA

SEQ ID NO: 58: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody No. 3N and hGAA

SEQ ID NO: 59: Nucleotide sequence encoding the amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody No. 3N and hGAA, synthetic sequence

SEQ ID NO: 60: Amino acid sequence of single chain humanized anti-hTfR antibody No. 3N

SEQ ID NO: 61: Humanized anti-hTfR antibody Fab heavy chain amino acid sequence #3N

SEQ ID NO: 62: Amino acid sequence 1 of CDR1 in heavy chain of humanized anti-hTfR antibody No. 3N

SEQ ID NO: 63: Amino acid sequence 2 of CDR1 in the humanized heavy chain

- 108 042007 anti-hTfR antibodies no. 3N

SEQ ID NO: 64: Amino acid sequence of framework region 3 in heavy chain of humanized anti-hTfR antibody #3N

SEQ ID NO: 65: Humanized anti-hTfR antibody heavy chain amino acid sequence #3N

SEQ ID NO: 66: Humanized anti-hTfR antibody heavy chain amino acid sequence #3N

SEQ ID NO: 67: Nucleotide sequence encoding humanized anti-hTfR antibody heavy chain amino acid sequence #3N, synthetic sequence

SEQ ID NO: 68: Heavy chain amino acid sequence (IgG4) of humanized anti-hTfR antibody No. 3N

SEQ ID NO: 69: Nucleotide sequence encoding the heavy chain amino acid sequence (IgG4) of humanized anti-hTfR antibody No. 3N, synthetic sequence

SEQ ID NO: 70: One example amino acid sequence of a human IgG Fc region

SEQ ID NO: 71: Amino acid sequence of heavy chain Fab with humanized anti-hTfR 3N IgG Fc region added

SEQ ID NO: 72: Nucleotide sequence encoding the Fab heavy chain amino acid sequence of a humanized anti-hTfR 3N IgG Fc region added, synthetic sequence

SEQ ID NO: 73: Amino acid sequence of framework region 3 in heavy chain of humanized anti-hTfR antibody #3

SEQ ID NO: 74: amino acid sequence of the albumin-binding domain

SEQ ID NO: 75: human IDUA amino acid sequence 1

SEQ ID NO: 76: human IDUA amino acid sequence 2

SEQ ID NO: 77: amino acid sequence of human PPT-1

SEQ ID NO: 78: amino acid sequence of human ASM

SEQ ID NO: 79: amino acid sequence of human ARSA

SEQ ID NO: 80: amino acid sequence of human SGSH

SEQ ID NO: 81: amino acid sequence of human GBA

SEQ ID NO: 82: amino acid sequence of human TPP-1

SEQ ID NO: 83: amino acid sequence of human NAGLU

SEQ ID NO: 84: amino acid sequence of human GUSB

SEQ ID NO: 85: amino acid sequence of human GALC

SEQ ID NO: 86: amino acid sequence of human AC

SEQ ID NO: 87: amino acid sequence of human FUCA1

SEQ ID NO: 88: amino acid sequence of human LAMAN

SEQ ID NO: 89: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hGAA, Fab HC-GS3-GAA

SEQ ID NO: 90: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hIDUA, IgG4 HC-IDUA

SEQ ID NO: 91: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hIDUA, IgG4 HC-GS8-IDUA

SEQ ID NO: 92: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hIDUA, IgG4 HC-GS20-IDUA

SEQ ID NO: 93: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hIDUA, Fab HC-GS3-IDUA

SEQ ID NO: 94: Amino acid sequence of fusion protein between humanized anti-hTfR antibody Fab heavy chain #3N and hIDUA, Fab HC-GS5-IDUA

SEQ ID NO: 95: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hIDUA, Fab HC-GS10-IDUA

SEQ ID NO: 96: Amino acid sequence of fusion protein between humanized anti-hTfR antibody Fab #3N heavy chain and hIDUA, Fab HC-GS20-IDUA

SEQ ID NO: 97: Amino acid sequence of fusion protein between heavy chain Fab of humanized anti-hTfR antibody #3N and hIDUA, Tandem Fab HC-IDUA

SEQ ID NO: 98: Amino acid sequence of single chain humanized anti-hTfR antibody No. 3N(2)

SEQ ID NO: 99: Amino acid sequence of fusion protein between single chain humanized anti-hTfR antibody #3N and IDUA, scFab-IDUA

SEQ ID NO: 100: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody No. 3N and hPPT-1, IgG4 HC-PPT1

SEQ ID NO: 101: Amino acid sequence of inter-heavy chain fusion protein (IgG4)

- 109 042007 humanized antibody against hTfR No. 3N and hPPT-1, IgG4 HC-GS5-PPT1

SEQ ID NO: 102: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody No. 3N and hPPT-1, IgG4 HC-GS10-PPT1

SEQ ID NO: 103: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody No. 3N and hPPT-1, IgG4 HC-GS20-PPT1

SEQ ID NO: 104: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hPPT-1, Fab HC-GS3-PPT1

SEQ ID NO: 105: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hPPT-1, Tandem Fab HC-PPT1

SEQ ID NO: 106: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody No. 3N and haSM, IgG4 HC-ASM

SEQ ID NO: 107: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and haSM, IgG4 HC-GS5-ASM

SEQ ID NO: 108: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and haSM, IgG4 HC-GS10-ASM

SEQ ID NO: 109: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and haSM, IgG4 HC-GS20-ASM

SEQ ID NO: 110: Amino acid sequence of fusion protein between humanized anti-hTfR antibody heavy chain Fab #3N and haSM, Fab HC-GS3-ASM

SEQ ID NO: 111: Amino acid sequence of fusion protein between humanized anti-hTfR antibody heavy chain Fab #3N and haSM, Fab HC-GS5-ASM

SEQ ID NO: 112: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and haSM, Fab HC-GS10-ASM

SEQ ID NO: 113: Amino acid sequence of fusion protein between humanized anti-hTfR antibody heavy chain Fab #3N and haSM, Fab HC-GS20-ASM

SEQ ID NO: 114: Amino acid sequence of fusion protein between humanized anti-hTfR antibody heavy chain Fab #3N and haSM, Tandem Fab HC-ASM

SEQ ID NO: 115: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hARSA, IgG4 HC-ARSA

SEQ ID NO: 116: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hARSA, IgG4 HC-GS5-ARSA

SEQ ID NO: 117: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hARSA, IgG4 HC-GS10-ARSA

SEQ ID NO: 118: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hARSA, IgG4 HC-GS20-ARSA

SEQ ID NO: 119: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hARSA, Fab HC-GS3-ARSA

SEQ ID NO: 120: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hARSA, Fab HC-GS5-ARSA

SEQ ID NO: 121: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hARSA, Fab HC-GS10-ARSA

SEQ ID NO: 122: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hARSA, Fab HC-GS20-ARSA

SEQ ID NO: 123: Amino acid sequence of fusion protein between humanized anti-hTfR antibody heavy chain Fab #3N and hARSA, Tandem Fab HC-ARSA

SEQ ID NO: 124: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hSGSH, IgG4 HC-SGSH

SEQ ID NO: 125: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hSGSH, IgG4 HC-GS5-SGSH

SEQ ID NO: 126: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hSGSH, IgG4 HC-GS10-SGSH

SEQ ID NO: 127: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hSGSH, IgG4 HC-GS20-SGSH

SEQ ID NO: 128: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hSGSH, Fab HC-GS3-SGSH

SEQ ID NO: 129: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hSGSH, Tandem Fab HC-SGSH

SEQ ID NO: 130: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGBA, IgG4 HC-GBA

SEQ ID NO: 131: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGBA, IgG4 HC-GS5-GBA

SEQ ID NO: 132: Amino acid sequence of inter-heavy chain fusion protein (IgG4)

- 110 042007 humanized antibody against hTfR No. 3N and hGBA, IgG4 HC-GS10-GBA

SEQ ID NO: 133: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGBA, IgG4 HC-GS20-GBA

SEQ ID NO: 134: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hGBA, Fab HC-GS3-GBA

SEQ ID NO: 135: Amino acid sequence of fusion protein between heavy chain Fab of humanized anti-hTfR antibody #3N and hGBA, Tandem Fab HC-GBA

SEQ ID NO: 136: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hTPP-1, IgG4 HC-TPP1

SEQ ID NO: 137: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hTPP-1, IgG4 HC-GS5-TPP1

SEQ ID NO: 138: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hTPP-1, IgG4 HC-GS10-TPP1

SEQ ID NO: 139: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody No. 3N and hTPP-1, IgG4 HC-GS20-TPP1

SEQ ID NO: 140: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hTPP-1, Fab HC-GS3-TPP1

SEQ ID NO: 141: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hTPP-1, Tandem Fab HC-TPP1

SEQ ID NO: 142: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hNAGLU, IgG4 HC-NAGLU

SEQ ID NO: 143: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hNAGLU, IgG4 HC-GS5-NAGLU

SEQ ID NO: 144: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hNAGLU, IgG4 HC-GS10-NAGLU

SEQ ID NO: 145: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hNAGLU, IgG4 HC-GS20-NAGLU

SEQ ID NO: 146: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hNAGLU, Fab HC-GS3-NAGLU

SEQ ID NO: 147: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hNAGLU, Fab HC-GS5-NAGLU

SEQ ID NO: 148: Amino acid sequence of fusion protein between humanized anti-hTfR antibody Fab #3N heavy chain and hNAGLU, Fab HC-GS10-NAGLU

SEQ ID NO: 149: Amino acid sequence of fusion protein between humanized anti-hTfR antibody Fab #3N heavy chain and hNAGLU, Fab HC-GS20-NAGLU

SEQ ID NO: 150: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGUSB, IgG4 HC-GUSB

SEQ ID NO: 151: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGUSB, IgG4 HC-GS5-GUSB

SEQ ID NO: 152: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGUSB, IgG4 HC-GS10-GUSB

SEQ ID NO: 153: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGUSB, IgG4 HC-GS20-GUSB

SEQ ID NO: 154: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hGUSB, Fab HC-GS3-GUSB

SEQ ID NO: 155: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hGUSB, Fab HC-GS5-GUSB

SEQ ID NO: 156: Amino acid sequence of fusion protein between humanized anti-hTfR antibody heavy chain Fab #3N and hGUSB, Fab HC-GS10-GUSB

SEQ ID NO: 157: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hGUSB, Fab HC-GS20-GUSB

SEQ ID NO: 158: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGALC, IgG4 HC-GALC

SEQ ID NO: 159: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGALC, IgG4 HC-GS5-GALC

SEQ ID NO: 160: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGALC, IgG4 HC-GS10-GALC

SEQ ID NO: 161: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hGALC, IgG4 HC-GS20-GALC

SEQ ID NO: 162: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hGALC, Fab HC-GS3-GALC

SEQ ID NO: 163: Amino acid sequence of Fab zy heavy chain fusion protein

- 111 -

Claims (47)

Manized antibody against hTfR No. 3N and hGALC, Fab HC-GS5-GALC SEQ ID NO: 164: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hGALC, Fab HC-GS10-GALC SEQ ID NO: 165: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hGALC, Fab HC-GS20-GALC SEQ ID NO: 166: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody No. 3N and hAC, IgG4 HC-AC SEQ ID NO: 167: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hAC, IgG4 HC-GS5-AC SEQ ID NO: 168: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hAC, IgG4 HC-GS10-AC SEQ ID NO: 169: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hAC, IgG4 HC-GS20-AC SEQ ID NO: 170: Amino acid sequence of fusion protein between humanized anti-hTfR antibody heavy chain Fab #3N and hAC, Fab HC-GS3-AC SEQ ID NO: 171: Amino acid sequence of fusion protein between humanized anti-hTfR antibody heavy chain Fab #3N and hAC, Fab HC-GS5-AC SEQ ID NO: 172: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hAC, Fab HC-GS10-AC SEQ ID NO: 173: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hAC, Fab HC-GS20-AC SEQ ID NO: 174: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hFUCA1, IgG4 HC-FUCA1 SEQ ID NO: 175: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody No. 3N and hFUCA1, IgG4 HC-GS5-FUCA1 SEQ ID NO: 176: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hFUCA1, IgG4 HC-GS10-FUCA1 SEQ ID NO: 177: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hFUCA1, IgG4 HC-GS20-FUCA1 SEQ ID NO: 178: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hFUCA1, Fab HC-GS3-FUCA1 SEQ ID NO: 179: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hFUCA1, Fab HC-GS5-FUCA1 SEQ ID NO: 180: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hFUCA1, Fab HC-GS10-FUCA1 SEQ ID NO: 181: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hFUCA1, Fab HC-GS20-FUCA1 SEQ ID NO: 182: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hLAMAN, IgG4 HC-LAMAN SEQ ID NO: 183: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hLAMAN, IgG4 HC-GS5-LAMAN SEQ ID NO: 184: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hLAMAN, IgG4 HC-GS10-LAMAN SEQ ID NO: 185: Amino acid sequence of heavy chain fusion protein (IgG4) of humanized anti-hTfR antibody #3N and hLAMAN, IgG4 HC-GS20-LAMAN SEQ ID NO: 186: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hLAMAN, Fab HC-GS3-LAMAN SEQ ID NO: 187: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hLAMAN, Fab HC-GS5-LAMAN SEQ ID NO: 188: Amino acid sequence of fusion protein between heavy chain humanized anti-hTfR antibody Fab #3N and hLAMAN, Fab HC-GS10-LAMAN SEQ ID NO: 189: Amino acid sequence of fusion protein between humanized anti-hTfR antibody heavy chain Fab #3N and hLAMAN, Fab HC-GS20-LAMAN CLAIM 1. An antibody against human transferrin receptor, where in the variable region of the heavy chain of the antibody: (a) CDR1 contains the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, (b) CDR2 contains the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and (c) CDR3 contains the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, and where in the variable region of the antibody light chain: - 112 042007 (a) CDR1 contains the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, (b) CDR2 contains the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence of Lys-Val-Ser, and (c) CDR3 contains the amino acid sequence of SEQ ID NO: 10. 2. The antibody of claim 1, wherein the heavy chain framework region 3 comprises the amino acid sequence of SEQ ID NO: 64. 3. The antibody of claim 2, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 65. 4. An antibody according to claim 1 or 2, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68. 5. An antibody according to any one of claims 1 to 4, wherein the antibody light chain variable region comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22. 6. An antibody according to any one of claims 1 to 4, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29. 7. An antibody according to any one of claims 1 to 6, wherein the antibody has affinity for both the extracellular region of the human transferrin receptor and the extracellular region of the monkey transferrin receptor. 8. An antibody according to claim 7, wherein the dissociation constant of its complex with the extracellular region of the human transferrin receptor is no more than 1x10'1° M and the dissociation constant of its complex with the extracellular region of the monkey transferrin receptor is no more than 1x10'9 M. 9. An antibody according to any one of claims 1-8, wherein the antibody is Fab, F(ab') ₂ or F(ab'). 10. An anti-human transferrin receptor antibody according to any one of claims 1 to 8, wherein the antibody is a single chain antibody selected from the group consisting of scFab, scF(ab'), scF(ab') ₂ and scFv. 11. An antibody according to claim 10, wherein the light chain and heavy chain of the antibody are linked via a linker sequence. 12. The antibody of claim 11, wherein the heavy chain is linked via a linker sequence to the light chain at the C-terminus of the light chain. 13. The antibody of claim 10 wherein the light chain is linked via a linker sequence to a heavy chain at the C-terminus of the heavy chain. 14. An antibody according to any one of claims 11-13, wherein the linker sequence consists of 2-50 amino acid residues. 15. An antibody according to claim 14, wherein the linker sequence is selected from the group consisting of Gly-Ser amino acid sequence, Gly-Gly-Ser amino acid sequence, Gly-Gly-Gly amino acid sequence, SEQ ID NO: 3, SEQ ID NO : 4 and SEQ ID NO: 5, an amino acid sequence consisting of three sequentially linked amino acid sequences, each is SEQ ID NO: 3, and amino acid sequences consisting of 2-10 repeats of any of the above sequences that are sequentially linked. 16. A fusion protein comprising an anti-human transferrin receptor antibody and another protein (A), wherein the anti-human transferrin receptor antibody is an antibody according to any one of claims 1 to 15, wherein the other protein (A) is a protein that is capable of expressing its physiological activity in the body, and where the protein (A) is linked to the light chain of the antibody at the C-terminus or N-terminus of the light chain. 17. The fusion protein of claim 16 wherein the protein (A) is linked directly or via a linker to the light chain at the C-terminus or N-terminus of the light chain. 18. The fusion protein of claim 16 or 17, wherein the protein (A) is linked via a linker to the light chain at the C-terminus or N-terminus of the light chain. 19. A fusion protein according to claim 18, wherein the linker is a peptide consisting of 1-50 amino acid residues. 20. The fusion protein according to claim 19, wherein the linker is a peptide containing an amino acid sequence selected from the group consisting of one glycine, one serine, Gly-Ser amino acid sequence, Gly-Gly-Ser amino acid sequence, SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and amino acid sequences consisting of 2-10 repeats of any of the above sequences that are sequentially linked. 21. A fusion protein of an anti-human transferrin receptor antibody and another protein (A), wherein the anti-human transferrin receptor antibody is an antibody according to any one of claims 1 to 15, wherein the other protein (A) is a protein that is capable of exhibiting its physiological - 113 042007 activity in the body, and where the protein (A) is associated with the heavy chain of the antibody at the C-terminus or N-terminus of the heavy chain. 22. The fusion protein of claim 21 wherein protein (A) is linked directly or via a linker to a heavy chain at the C-terminus or N-terminus of the heavy chain. 23. The fusion protein of claim 21 or 22, wherein the protein (A) is linked via a linker to the heavy chain at the C-terminus or N-terminus of the heavy chain. 24. The fusion protein of claim 23, wherein the linker sequence is a peptide consisting of 1-50 amino acid residues. 25. The fusion protein of claim 24, wherein the linker is a peptide containing an amino acid sequence selected from the group consisting of one glycine, one serine, Gly-Ser amino acid sequence, Gly-Gly-Ser amino acid sequence, SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and amino acid sequences consisting of 2-10 repeats of any of the above sequences that are sequentially linked. 26. A fusion protein according to any one of claims 16-25, wherein protein (A) is a protein derived from a human. 27. The fusion protein according to any one of claims 16-26, wherein the protein (A) is selected from the group consisting of nerve growth factor (NGF), lysosomal enzymes, ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin-3, neurotrophin-4/5, neurotrophin-6, neuregulin-1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), endothelial growth factor vascular (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), cytokine, receptor factor tumor necrosis α (TNF-α receptor), PD-1 ligand, PD-Ll, PD-L2, an enzyme that destroys β-amyloid activity, antibodies against β-amyloid, antibodies against BACE, antibodies against EGFR, antibodies against PD- 1, antibodies anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-HER2 antibodies, anti-TNF-α antibodies and anti-CTLA-4 antibodies. 28. The fusion protein of claim 16, wherein the protein (A) is a lysosomal enzyme, wherein the lysosomal enzyme is selected from the group consisting of a-L-iduronidase, iduronate-2-sulfatase, human acid α-glucosidase, glucocerebrosidase, β-galactosidase, protein -activator GM2, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, a-L-fucosidase, aspartylglucosaminidase, α-Nacetylgalactosaminidase, acid sphingomyelinase , a-galactosidase A, β-glucuronidase, heparan-Nsulfatase, a-N-acetylglucosaminidase, acetyl-KoA:a-a-glucosaminide-N-acetyltransferase, Nacetylglucosamine-6-sulfate-sulfatase, acid ceramidase, amyl-1,6-glucosidase, sialidase , palmitoyl protein thioesterase 1, tripeptidyl peptidase 1, hyaluronidase 1, CLN1 and CLN2. 29. A fusion protein according to any one of claims 16-26, wherein protein (A) is human iduronate-2-sulfatase, human acid α-glucosidase, or human α-L-iduronidase. 30. The fusion protein of claim 26, wherein the protein (A) is human acid α-glucosidase, wherein (1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain is linked at its C-terminus via the Gly-Ser amino acid sequence with human acid α-glucosidase, thus forming the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58. 31. The fusion protein of claim 26, wherein protein (A) is human acid α-glucosidase, where: (1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68, and the heavy chain is linked at its C-terminus via the GlySer amino acid sequence with human acid α-glucosidase with the amino acid sequence of SEQ ID NO: 55 or 56. 32. The fusion protein of claim 26, wherein protein (A) is human acid α-glucosidase, where the antibody is a Fab, and where: (1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain is linked at its C-terminus by an amino acid sequence consisting of three consecutively linked amino acid sequences, each of which is SEQ ID NO: 3 , with human acid α-glucosidase, thus forming the amino acid sequence of SEQ ID NO: 89. 33. The fusion protein of claim 26, wherein protein (A) is human acid α-glucosidase, where the antibody is a Fab, and where: (1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 61, and the heavy chain is linked at its C-terminus through an amino acid sequence consisting of three - 114 042007 consecutively linked amino acid sequences, each of which is SEQ ID NO: 3, with human acid α-glucosidase with the amino acid sequence of SEQ ID NO: 55 or 56. 34. The fusion protein of claim 26, wherein protein (A) is human a-L-iduronidase, where the antibody is Fab, and where: (1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain is linked at its C-terminus by an amino acid sequence consisting of three consecutively linked amino acid sequences, each of which is SEQ ID NO: 3 , with human a-L-iduronidase, thus forming the amino acid sequence of SEQ ID NO: 93. 35. The fusion protein of claim 26, wherein protein (A) is human a-L-iduronidase, where the antibody is a Fab, and where: (1) the antibody light chain contains the amino acid sequence of SEQ ID NO: 23, and (2) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 61, and the heavy chain is linked at its C-terminus by an amino acid sequence consisting of three consecutive amino acids sequences, each of which is SEQ ID NO: 3, with human a-L-iduronidase with the amino acid sequence of SEQ ID NO: 75 or 76. 36. The fusion protein of claim 16, wherein a human IgG Fc region is inserted between the protein (A) and the antibody. 37. The fusion protein of claim 36, wherein the human IgG Fc region is linked directly or via a linker sequence to the protein (A) at its C-terminus, and the antibody heavy chain or light chain is linked directly or via a linker sequence to the IgG Fc region human at its C-terminus. 38. The fusion protein of claim 36 or 37, wherein the human IgG Fc region contains the amino acid sequence of SEQ ID NO: 70. 39. The fusion protein of claim 38, wherein the human IgG Fc region is linked via a linker sequence to a Fab heavy chain consisting of the amino acid sequence of SEQ ID NO: 61 and contains the amino acid sequence of SEQ ID NO: 71 thus formed. 40. A DNA fragment encoding the amino acid sequence of an anti-human transferrin receptor antibody according to any one of claims 1-15. 41. A DNA fragment encoding the amino acid sequence of a fusion protein according to any one of claims 16-39. 42. An expression vector containing a DNA fragment according to claim 40 or 41. 43. A mammalian cell transformed with the expression vector of claim 42. 44. A pharmaceutical complex comprising an anti-human transferrin receptor antibody according to any one of claims 1 to 15 and a low molecular weight pharmacologically active compound, wherein the light chain and/or heavy chain of the anti-human transferrin receptor antibody is covalently linked to the pharmacologically active small molecular weight compound that needs to be able to passing through the blood-brain barrier and manifestations of its function in the brain. 45. The complex of claim 44, wherein the pharmacologically active compound is any compound selected from the group consisting of an anticancer agent, a therapeutic agent for Alzheimer's disease, a therapeutic agent used to treat Parkinson's disease, a therapeutic agent used to treat Huntington's disease, a therapeutic agent an agent used to treat schizophrenia, an antidepressant, a therapeutic agent used to treat multiple sclerosis, a therapeutic agent used to treat amyotrophic lateral sclerosis, a therapeutic agent used to treat tumors of the central nervous system, including a tumor of the brain, a therapeutic agent used to treat lysosomal storage disease accompanied by encephalopathy, therapeutic agent used to treat glycogenosis, therapeutic agent used to treat muscular dystrophy, therapeutic media a drug used to treat cerebral ischemia, a therapeutic agent used to treat prion diseases, a therapeutic agent used to treat traumatic disorders of the central nervous system, a therapeutic agent used to treat viral and bacterial diseases of the central nervous system, a pharmaceutical agent used to recover from brain surgery, a pharmaceutical used for recovery from spinal cord surgery, siRNA, antisense DNA, and a peptide. 46. The use of an anti-human transferrin receptor antibody according to any one of claims 1 to 15 for the preparation of a parenteral pharmaceutical agent for the treatment of a disease of the central nervous system by binding to it a molecule of a physiologically active protein or a pharmacologically active small molecule compound used to treat these disorders. 47. A method for treating a disorder of the central nervous system, comprising parenteral administration to a patient with a disorder of a therapeutically effective amount of a physiologically active - 115 -