Peptide having affinity for human transferrin receptor

Abstract

In one embodiment, the present invention pertains to a peptide having affinity for the human transferrin receptor, and to applications of the same, wherein in order to pass through the blood-brain barrier, the peptide can be used by binding to any compound (such as proteins, nucleic acids, and low-molecular weight compounds) that should function in the central nervous system (CNS). The peptide includes, for example, an amino acid sequence selected from the group consisting of amino acid sequences expressed by SEQ ID NOs: 3-7 and SEQ ID NO: 55, or a mutant of the same.

Description

Human transferrin receptor affinity peptide

[0001] In one embodiment, the present invention relates to a peptide having affinity for the human transferrin receptor, which is used in combination with a compound (such as a protein, nucleic acid, or low-molecular-weight compound) that is to function in the central nervous system (CNS) in order to allow the compound to pass through the blood-brain barrier, and a method for using the peptide.

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

[0003] The existence of the blood-brain barrier (BBB) ​​allows most cells in the central nervous system to maintain biochemical homeostasis without being affected by fluctuations in the concentrations of hormones, cytokines, and other substances in the blood. However, the existence of the BBB poses challenges for drug development. For example, enzyme replacement therapy using intravenous administration of recombinant iduronate 2-sulfatase is currently being used to treat mucopolysaccharidosis type II (Hunter syndrome), a hereditary metabolic disorder caused by a deficiency of iduronate 2-sulfatase. However, this therapy is less effective for treating the significant central nervous system (CNS) abnormalities observed in Hunter syndrome because iduronate 2-sulfatase cannot penetrate the BBB. For other CNS disorders, such as Alzheimer's disease and brain tumors, drugs must reach the CNS, but drugs that cannot penetrate the BBB cannot be effective.

[0004] Various methods have been developed to allow macromolecular substances, such as proteins, to act in the central nervous system to pass through the blood-brain barrier. As such methods, various methods have been reported in which macromolecular substances are modified to have affinity for membrane proteins present on endothelial cells of intracerebral capillaries. Examples of membrane proteins present on endothelial cells of intracerebral capillaries include receptors for compounds such as insulin, transferrin, insulin-like growth factors (IGF-I, IGF-II), LDL, and leptin. Among these, antibodies against transferrin receptors have been reported to be able to pass through the blood-brain barrier and migrate into the brain parenchyma, and to deliver drugs to the brain by binding to the drugs to act in the central nervous system (Patent Documents 1 to 8).

[0005] Furthermore, a Hunter syndrome treatment drug (Isucargo (registered trademark)) was launched in 2021, which combines iduronate-2-sulfatase with an anti-transferrin receptor antibody and can pass through the blood-brain barrier to exert its therapeutic effect in the central nervous system (Non-patent document 1).

[0006] US2007 / 0082380US2008 / 0152645US2009 / 0053219US2011 / 0110935US2018 / 0171012US2018 / 0179291US2020 / 0317798US2020 / 0384061

[0007] Package insert "Isucargo 10mg for Intravenous Infusion" March 2021 (1st edition)

[0008] Under the above circumstances, one of the objects of the present invention is to provide a peptide having affinity for the human transferrin receptor, which can be used in combination with compounds (proteins, nucleic acids, low-molecular-weight compounds, etc.) that are to function in the central nervous system (CNS) in order to enable the compounds to pass through the blood-brain barrier, and a method for using the peptide.

[0009] In research aimed at the above-mentioned object, the present inventors conducted extensive research and found that a peptide having affinity for human transferrin receptor (hTfR), which contains the variable region of an anti-hTfR heavy chain antibody that specifically recognizes the extracellular region of hTfR and is obtained by the production method described in detail herein (anti-hTfR heavy chain antibody variable region peptide), efficiently crosses the blood-brain barrier, and thus completed the present invention. That is, the present invention includes the following. Note that in the inventions described in 1. to 104. below, the human transferrin receptor affinity peptide can be replaced with a peptide containing the variable region of a heavy chain antibody, and the peptide can also be replaced with a protein. 1. A peptide having affinity for the human transferrin receptor (human transferrin receptor affinity peptide), which comprises a variable region of a heavy chain antibody having three complementarity determining regions, CDR1, CDR2, and CDR3, and is selected from the group consisting of (1) to (5) below: (1) a peptide in which the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:13, (2) a peptide in which the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:17, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:19, (3) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:23, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:25; (4) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO:26 or SEQ ID NO:27, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:31; (5) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21, and the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO:56 or SEQ ID NO:57;and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25. 2. A human transferrin receptor affinity peptide selected from the group consisting of (1) to (6) below: (1) a peptide having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 3 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (1) of claim 1, (2) a peptide having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 4 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (2) of claim 1, (3) a peptide having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 5 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (3) of claim 1, (4) A peptide having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 6 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (4) of claim 1, (5) A peptide having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 7 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (3) of claim 1, and (6) A peptide having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 55 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (5) of claim 1. 3. A peptide with affinity for human transferrin receptor according to 2 above, wherein the amino acid sequence identity is 85% or more. 4. A peptide with affinity for human transferrin receptor according to 2 above, wherein the amino acid sequence identity is 90% or more. 5. 6. The peptide with affinity for the human transferrin receptor according to 2 above, which has an amino acid sequence identity of 95% or more. 6. The peptide with affinity for the human transferrin receptor according to 1 above, which comprises an amino acid sequence selected from the group consisting of the following (1) to (6): (1) the amino acid sequence of SEQ ID NO: 3 in which 1 to 10 amino acids have been substituted, deleted or added, and the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9,(1) an amino acid sequence in which the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; (2) an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted, or added in the amino acid sequence of SEQ ID NO: 4, and in which the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19; (3) an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted, or added in the amino acid sequence of SEQ ID NO: 5, and in which the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25; (4) An amino acid sequence in which 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 6 have been substituted, deleted, or added, and the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31; (5) An amino acid sequence in which 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 7 have been substituted, deleted, or added, and the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25; (6) An amino acid sequence in which 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 55 have been substituted, deleted, or added, and the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 56, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25. 7. Substitution,7. A peptide with affinity for the human transferrin receptor according to 5 above, wherein the number of deleted or added amino acids is 1 to 5. 8. A peptide with affinity for the human transferrin receptor according to 5 above, wherein the number of substituted, deleted or added amino acids is 1 to 3. 9. A peptide with affinity for the human transferrin receptor according to any of 1 to 8 above, which has affinity for both the extracellular domain of the human transferrin receptor and the extracellular domain of the simian transferrin receptor. 10. A peptide with affinity for the human transferrin receptor according to any of 1 to 8 above, wherein the dissociation constants with the extracellular domain of the human transferrin receptor are both 5 x 10 －９ ~1 x 10 －７ 11. The peptide with affinity to the human transferrin receptor according to 9 above, wherein the dissociation constant with the extracellular domain of the human transferrin receptor is 8 x 10 －９ ~2 x 10 －８11. The peptide with affinity for the human transferrin receptor according to claim 9, wherein the dissociation constant with the monkey transferrin receptor is 0.5 to 2.5 when the dissociation constant with the extracellular domain of the human transferrin receptor is set to 1. 12. The peptide with affinity for the human transferrin receptor according to claim 9, wherein the dissociation constant with the monkey transferrin receptor is 0.5 to 2.5 when the dissociation constant with the extracellular domain of the human transferrin receptor is set to 1. 13. The peptide with affinity for the human transferrin receptor according to claim 9, wherein the dissociation constant with the monkey transferrin receptor is 0.7 to 1.2 when the dissociation constant with the extracellular domain of the human transferrin receptor is set to 1. 14. A heavy chain antibody variable region peptide selected from the group consisting of (1) to (3) below, which is a peptide in which a plurality of the human transferrin receptor affinity peptides according to any one of 1 to 13 above are bound: (1) a peptide in which 2 to 10 of the human transferrin receptor affinity peptides according to 1 to 12 above are bound directly or via a linker, (2) a peptide in which 2 or 3 of the human transferrin receptor affinity peptides according to 1 to 12 above are bound directly or via a linker, (3) a peptide in which two of the human transferrin receptor affinity peptides according to 1 to 12 above are bound directly or via a linker. 15. The human transferrin receptor affinity peptide according to 14 above, wherein the linker linking the plurality of human transferrin receptor affinity peptides is a peptide linker consisting of 1 to 50 amino acids. 16. 16. The human transferrin receptor affinity peptide according to 14 above, wherein the amino acid sequence of the peptide linker linking a plurality of the human transferrin receptor affinity peptides is selected from the group consisting of a single amino acid, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 58, and SEQ ID NO: 69. 17. The human transferrin receptor affinity peptide according to 14 above, wherein the amino acid sequence of the peptide linker linking the heavy chain antibody variable region peptides is selected from the group consisting of a single amino acid, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 58, and SEQ ID NO: 69.The human transferrin receptor affinity peptide according to claim 14, which consists of a sequence of 2 to 10 amino acids connected in tandem. 18. A human transferrin receptor affinity peptide-drug complex, in which a drug is bound to the human transferrin receptor affinity peptide according to any one of claims 1 to 17 above. 19. The complex according to claim 18, in which the drug is any one of another protein (A), a nucleic acid, or a low molecular weight compound. 20. A fusion protein of the human transferrin receptor affinity peptide according to any one of claims 1 to 17 above and another protein (A). 21. The fusion protein according to claim 20, in which the human transferrin receptor affinity peptide is bound to the C-terminus of the other protein (A), either directly or via a linker. 22. The fusion protein according to claim 20, in which the human transferrin receptor affinity peptide is bound to the N-terminus of the other protein (A), either directly or via a linker. 23. The fusion protein according to claim 21 or 22, in which the linker is a peptide linker consisting of 1 to 50 amino acids. 24. 25. The fusion protein according to claim 23, wherein the amino acid sequence of the peptide linker is selected from the group consisting of a single amino acid, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 58, and SEQ ID NO: 69. 26. The fusion protein according to any of claims 20 to 25, wherein the other protein (A) is of human origin. 27. 27. The fusion protein according to any one of 20 to 26 above, wherein the other protein (A) is a cytokine, a growth factor, or an antibody drug. 28. The fusion protein according to any one of 20 to 26 above, wherein the other protein (A) is a brain-derived nerve growth factor (BDNF), nerve growth factor (NGF), a lysosomal enzyme,Ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, granulocyte-macrophage colony-stimulating factor (GM-C) 28. The fusion protein according to any one of items 20 to 26 above, wherein the antibody is selected from the group consisting of PD-1 ligand, PD-L1, PD-L2, an enzyme having an activity of degrading beta-amyloid, an anti-beta-amyloid antibody, an anti-BACE antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-HER2 antibody, an anti-TNF-α antibody, and an anti-CTLA-4 antibody. The other protein (A) is a lysosomal enzyme, and the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, acid α-glucosidase, glucocerebrosidase, β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. 27. The fusion protein according to any one of claims 20 to 26, wherein the fusion protein is selected from the group consisting of enzymes selected from the group consisting of acetyl-CoA, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2. 28. The fusion protein according to any one of claims 20 to 26, wherein the fusion protein is bound to serum albumin.The fusion protein according to any one of claims 20 to 29. 31. The fusion protein according to any one of claims 20 to 29, to which a human IgG Fc region or a part thereof is bound. 32. A nucleic acid encoding the peptide with affinity for the human transferrin receptor according to any one of claims 1 to 17. 33. A nucleic acid encoding the fusion protein according to any one of claims 20 to 31. 34. An expression vector incorporating the nucleic acid according to claim 32 or 33. 35. A cell transformed with the expression vector according to claim 34. 36. The cell according to claim 35, wherein the cell is derived from a mammal. 37. A peptide with affinity for the human transferrin receptor, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:55, in which 1 to 10 amino acids have been substituted, deleted, or added. 38. The peptide with affinity for the human transferrin receptor according to claim 37, in which the number of substituted, deleted, or added amino acids is 1 to 5. 39. 40. The peptide having affinity for the human transferrin receptor according to 37 above, wherein the number of substituted, deleted or added amino acids is 1 to 3. 41. The peptide having affinity for the human transferrin receptor according to 1 above, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 55. The human transferrin receptor affinity peptide according to 1 above, which is selected from the group consisting of the following (1) to (6): (1) a peptide having 80% or more identity with the amino acid sequence of SEQ ID NO:3, and having an amino acid sequence of CDR1 set forth in SEQ ID NO:8 or SEQ ID NO:9, an amino acid sequence of CDR2 set forth in SEQ ID NO:10 or SEQ ID NO:11, and an amino acid sequence of CDR3 set forth in SEQ ID NO:12 or SEQ ID NO:13, (2) a peptide having 80% or more identity with the amino acid sequence of SEQ ID NO:4, and having an amino acid sequence of CDR1 set forth in SEQ ID NO:14 or SEQ ID NO:15, an amino acid sequence of CDR2 set forth in SEQ ID NO:16 or SEQ ID NO:17, and an amino acid sequence of CDR3 set forth in SEQ ID NO:18 or SEQ ID NO:19, (3) a peptide having 80% or more identity with the amino acid sequence of SEQ ID NO:5, and having an amino acid sequence of CDR1 set forth in SEQ ID NO:20 or SEQ ID NO:21,(4) A CDR2 having an amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and a CDR3 having an amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, (5) A CDR1 having an amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27, a CDR2 having an amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29, and a CDR3 having an amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31, (6) A CDR1 having an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, a CDR2 having an amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and a CDR3 having an amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, (6) A peptide having 80% or more identity with the amino acid sequence of SEQ ID NO: 55, and wherein the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25. 42. The peptide with affinity for human transferrin receptor of above 41, wherein the amino acid sequence identity is 85% or more. 43. The peptide with affinity for human transferrin receptor of above 41, wherein the amino acid sequence identity is 90% or more. 44. The peptide with affinity for human transferrin receptor of above 41, wherein the amino acid sequence identity is 95% or more. 45. A heavy chain antibody-drug conjugate in which a drug is bound to a heavy chain antibody whose antigen is a molecule present on the surface of vascular endothelial cells. 46. The conjugate of above 45, wherein the drug is any one of other protein (A), nucleic acid, or low molecular weight compound. 47. 48. A fusion protein of a heavy chain antibody whose antigen is a molecule present on the surface of a vascular endothelial cell and another protein (A), wherein the heavy chain antibody is bound to the C-terminus of the other protein (A) directly or via a linker, or the heavy chain antibody is bound to the N-terminus of the other protein (A) directly or via a linker. 49. The fusion protein of 48 above, wherein the linker is a peptide linker consisting of 1 to 50 amino acids. 49. The amino acid sequence of the peptide linker is one amino acid,50. The fusion protein of claim 47, wherein the amino acid sequence is selected from the group consisting of the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, each of the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO: 11. 51. The complex or fusion protein of any of claims 46 to 50, wherein the other protein (A) is of human origin. 52. 52. The complex or fusion protein of any one of 46 to 51 above, wherein the other protein (A) is a cytokine, a growth factor, or an antibody drug. 53. The other protein (A) is brain-derived nerve growth factor (BDNF), nerve growth factor (NGF), lysosomal enzyme, ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, or interferon γ. , interleukin 6, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor α receptor (TNF-α receptor), PD-1 ligand, PD-L1, PD-L2, an enzyme having the activity of degrading beta-amyloid, anti-beta-amyloid antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-HER2 antibody, anti-TNF-α antibody, and anti-CTLA-4 antibody,A complex or fusion protein according to any one of 46 to 51 above. 54. The other protein (A) is a lysosomal enzyme, and the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, acid α-glucosidase, glucocerebrosidase, β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. 53. The complex or fusion protein of any of 46 to 51 above, wherein the enzyme is selected from the group consisting of A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1 and CLN2. 54. The complex or fusion protein of any of 46 to 54 above, further bound to serum albumin. 55. The fusion protein of any of 46 to 54 above, further bound to a human IgG Fc region or a portion thereof. 56. The fusion protein of any of 46 to 54 above, further bound to a human IgG Fc region or a portion thereof. 57. 57. The complex or fusion protein of any one of 45 to 56 above, wherein the vascular endothelial cells are cerebrovascular endothelial cells. 58. The complex or fusion protein of any one of 45 to 57 above, wherein the molecule present on the surface of the vascular endothelial cells is selected from the group consisting of transferrin receptor (TfR), insulin receptor, leptin receptor, lipoprotein receptor, IGF receptor, OATP-F, organic anion transporter, MCT-8, and monocarboxylic acid transporter, particularly one of human origin. 59. The complex or fusion protein of any one of 45 to 57 above, wherein the molecule present on the surface of the vascular endothelial cells is transferrin receptor (TfR), particularly one of human origin. 60. The drug is a combination of the heavy chain antibody and polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol,60. The conjugate of any one of claims 45 to 46, wherein the antibody and the human lysosomal enzyme are linked via a linker selected from the group consisting of polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid, biotin-streptavidin, and derivatives thereof. 61. The conjugate or fusion protein of any one of claims 45 to 60, wherein the heavy chain antibody is a heavy chain antibody derived from a camelid or all or a part of an immunoglobulin new antigen receptor derived from a cartilaginous fish. 62. The conjugate or fusion protein of claim 61, wherein the heavy chain antibody is a single heavy chain antibody. 63. The complex or fusion protein of 61 or 62 above, wherein the heavy chain antibody is selected from the group consisting of the following (1) to (4): (1) one consisting of only the whole or a part of the heavy chain variable region, (2) one consisting of the whole or a part of the heavy chain variable region and a part of the hinge region, (3) one consisting of the whole of the heavy chain variable region and hinge region, and (4) one comprising the heavy chain variable region, hinge region, and a part of the constant region. 64. A nucleic acid encoding any of the fusion proteins of 47 to 59 and 61 to 63 above. 65. An expression vector incorporating the nucleic acid of 64 above. 66. A cell transformed with the expression vector of 65 above. 67. The cell of 66 above, wherein the cell is derived from a mammal. 68. A pharmaceutical composition comprising the complex or fusion protein of any of 45 to 63 above as an active ingredient, particularly a pharmaceutical composition in which the active ingredient passes through the blood-brain barrier and exhibits a medicinal effect in the central nervous system. 69. A heavy chain antibody variable region peptide having affinity for the human transferrin receptor, which has three complementarity determining regions, CDR1, CDR2, and CDR3, selected from the group consisting of (1) to (6) below: (1) the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9,a CDR2 having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13, wherein the amino acid sequences of CDR1, CDR2, and CDR3 may be any, but preferably, a CDR1 having the amino acid sequence of SEQ ID NO: 8, a CDR2 having the amino acid sequence of SEQ ID NO: 10, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, or a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 13; (2) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, wherein each of the amino acid sequences of CDR1, CDR2, and CDR3 may be any, but preferably, the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 14, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 16, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 18, or the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 15, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 17, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 19; (3) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, wherein each of the amino acid sequences of CDR1, CDR2, and CDR3 may be any, but preferably, the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 22, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24, or the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 23,and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 25; (4) the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31, wherein each of the amino acid sequences of CDR1, CDR2, and CDR3 may be any, but preferably the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 26, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 30, or the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 27, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 29, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 31; (5) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, wherein each of the amino acid sequences of CDR1, CDR2, and CDR3 may be any, but preferably, the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 22, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24, or the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 23, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 25; (6) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25. Here, the amino acid sequences of CDR1, CDR2, and CDR3 may be any, but preferably, the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20, and the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 56,and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24, or the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 57, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 25. 70. A heavy chain antibody variable region peptide having affinity for the human transferrin receptor, which has three complementarity determining regions, CDR1, CDR2, and CDR3, and is selected from the group consisting of (1) to (6) below: (1) an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:3, and the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (1) of 69 above; (2) an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:4, and the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (2) of 69 above; (3) an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:5, and the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (3) of 69 above; (4) An antibody heavy chain variable region peptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:6, and wherein the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (4) of 69 above; (5) An antibody heavy chain variable region peptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:7, and wherein the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (5) of 69 above; (6) An antibody heavy chain variable region peptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:8, and wherein the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (6) of 69 above. 71. An antibody heavy chain variable region peptide selected from the group consisting of (1) to (6) of 70 above, wherein the identity is 85% or more. 72. An antibody heavy chain variable region peptide selected from the group consisting of (1) to (6) of 70 above, wherein the identity is 90% or more. 73. The identity is 95% or more, and the sequence is selected from the group consisting of (1) to (6) of 70 above.Heavy chain antibody variable region peptide. 74. A heavy chain antibody variable region peptide having affinity for the human transferrin receptor, which has three complementarity determining regions, CDR1, CDR2, and CDR3, and is selected from the group consisting of (1) to (6) below: (1) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:3, in which 1 to 10 amino acids have been substituted, deleted, or added, and the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (1) of 69 above; (2) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:4, in which 1 to 10 amino acids have been substituted, deleted, or added, and the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (2) of 69 above; (3) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:5, in which 1 to 10 amino acids have been substituted, deleted, or added, and the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (3) of 69 above; (4) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 6, in which 1 to 10 amino acids have been substituted, deleted, or added, and the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (4) of 69 above, (5) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 7, in which 1 to 10 amino acids have been substituted, deleted, or added, and the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (5) of 69 above, (6) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 8, in which 1 to 10 amino acids have been substituted, deleted, or added, and the amino acid sequences of CDR1, CDR1, and CDR3 are those shown in option (6) of 69 above. 75. A heavy chain antibody variable region peptide selected from the group consisting of (1) to (6) of 74 above, in which the number of substituted, deleted, or added amino acids is 1 to 5. 76. 77. A heavy chain antibody variable region peptide selected from the group consisting of (1) to (6) of 74 above, wherein the number of substituted, deleted, or added amino acids is 1 to 3. 78. A heavy chain antibody variable region peptide having affinity for both the extracellular domain of the human transferrin receptor and the extracellular domain of the monkey transferrin receptor,78. A heavy chain antibody variable region peptide according to any one of 69 to 76 above. 79. A heavy chain antibody variable region peptide according to any one of 69 to 76 above, wherein the dissociation constant with the extracellular domain of the human transferrin receptor is 5 x 10, －９ ~1 x 10 －７ 79. The heavy chain antibody variable region peptide according to claim 77, wherein the dissociation constant with the extracellular domain of the human transferrin receptor is 8 x 10 －９ ~2 x 10 －８80. The heavy chain antibody variable region peptide of claim 77, wherein the dissociation constant with the monkey transferrin receptor is 0.5 to 2.5, when the dissociation constant with the extracellular region of the human transferrin receptor is set to 1. 81. The heavy chain antibody variable region peptide of claim 77, wherein the dissociation constant with the monkey transferrin receptor is 0.7 to 1.2, when the dissociation constant with the extracellular region of the human transferrin receptor is set to 1. 82. 82. A heavy chain antibody variable region peptide selected from the group consisting of (1) to (3) below, which is a combination of multiple heavy chain antibody variable region peptides of 69 to 81 above: (1) a combination of 2 to 10 heavy chain antibody variable region peptides of 41 to 53 above, linked directly or via a linker, (2) a combination of 2 or 3 heavy chain antibody variable region peptides of 41 to 53 above, linked directly or via a linker, (3) a combination of two heavy chain antibody variable region peptides of 41 to 53 above, linked directly or via a linker. 83. A heavy chain antibody variable region peptide of 82 above, wherein the linker linking the heavy chain antibody variable region peptides is a peptide linker consisting of 1 to 50 amino acids. 84. 83. The heavy chain antibody variable region peptide of claim 83, wherein the amino acid sequence of the peptide linker connecting the heavy chain antibody variable region peptides is selected from the group consisting of a single amino acid, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 58, and SEQ ID NO: 69. 85. The heavy chain antibody variable region peptide of claim 83, wherein the amino acid sequence of the peptide linker connecting the heavy chain antibody variable region peptides is a tandem amino acid sequence of 2 to 10 amino acid sequences selected from the group consisting of a single amino acid, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 58, and SEQ ID NO: 69.86. A heavy chain antibody variable region peptide-drug conjugate in which a heavy chain antibody variable region peptide is bound to a drug, wherein the heavy chain antibody variable region peptide is any one of those described in 69 to 85 above. 87. The conjugate of 86 above, wherein the drug is any one of other protein (A), nucleic acid, or low molecular weight compound. 88. A fusion protein of a heavy chain antibody variable region peptide and other protein (A), wherein the heavy chain antibody variable region peptide is any one of those described in 69 to 85 above. 89. The fusion protein of 88 above, wherein the heavy chain antibody variable region peptide is bound to the C-terminus of the other protein (A), either directly or via a linker. 90. The fusion protein of 88 above, wherein the heavy chain antibody variable region peptide is bound to the N-terminus of the other protein (A), either directly or via a linker. 91. The fusion protein of 89 or 90 above, wherein the linker is a peptide linker consisting of 1 to 50 amino acids. 92. 92. The fusion protein according to claim 91, wherein the amino acid sequence of the peptide linker is selected from the group consisting of a single amino acid, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 58, and SEQ ID NO: 69. 93. The fusion protein according to claim 91, wherein the amino acid sequence of the peptide linker is a tandem amino acid sequence of 2 to 10 amino acid sequences selected from the group consisting of a single amino acid, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly, the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 58, and SEQ ID NO: 69. 94. The complex or fusion protein according to any of claims 87 to 93, wherein the other protein (A) is of human origin. 95. A complex or fusion protein according to any one of items 87 to 94 above, wherein the other protein (A) is a cytokine, a growth factor, or an antibody drug. 96. A complex or fusion protein according to any one of items 87 to 94 above, wherein the other protein (A) is selected from those shown in item 35 above.97. The complex or fusion protein of any of claims 87 to 94, wherein the other protein (A) is a lysosomal enzyme, and the lysosomal enzyme is selected from those listed in claim 36. 98. The complex or fusion protein of any of claims 87 to 94, wherein serum albumin is bound. 99. The complex or fusion protein of any of claims 87 to 94, wherein a human IgG Fc region or a portion thereof is bound. 100. A nucleic acid encoding the heavy chain antibody variable region peptide of any of claims 69 to 85. 101. A nucleic acid encoding the fusion protein of any of claims 88 to 99. 102. An expression vector incorporating the nucleic acid of claim 100 or 101. 103. A cell transformed with the expression vector of claim 102. 104. The cell of claim 103, wherein the cell is derived from a mammal.

[0010] A peptide having affinity for the human transferrin receptor (hTfR affinity peptide), which is one embodiment of the present invention, can be bound to a substance that can hardly or not at all pass through the blood-brain barrier, thereby allowing the substance to pass through the blood-brain barrier and reach the central nervous system, thereby allowing the substance to exert its function in the central nervous system.

[0011] Photographs showing the results of immunohistochemical staining for anti-hTfR antibodies in mouse cerebellum 6 hours after administration of the hTfR affinity peptide-trastuzumab. (a) Trastuzumab, (b) hTfR affinity peptide-trastuzumab 1, (c) hTfR affinity peptide-trastuzumab 2, (d) hTfR affinity peptide-trastuzumab 3, (e) hTfR affinity peptide-trastuzumab 4, and (f) hTfR affinity peptide-trastuzumab 5 were administered. The bar in the lower right corner of each photograph indicates a 50 μm gauge. Photographs showing the results of immunohistochemical staining for anti-hTfR antibodies in mouse cerebellum 24 hours after administration of the hTfR affinity peptide-trastuzumab. (a) Trastuzumab, (b) hTfR affinity peptide-trastuzumab 1, (c) hTfR affinity peptide-trastuzumab 2, (d) hTfR affinity peptide-trastuzumab 3, and (e) hTfR affinity peptide-trastuzumab 4. The bar at the bottom right of each photograph indicates a 50 μm gauge. Graph showing the quantitative values ​​of these substances in various brain tissues of cynomolgus monkeys 8 hours after administration of hTfR affinity peptide-trastuzumab 5 and trastuzumab. (1) Quantitative values ​​are shown for the cerebral cortex, (2) cerebellum, (3) hippocampus, (4) midbrain, (5) pons, (6) medulla oblongata, (7) caudate nucleus, (8) nucleus pallidus, (9) globus pallidus, (10) thalamus, (11) hypothalamus, (12) cervical spinal cord, (13) thoracic spinal cord, (14) lumbar spinal cord, and (15) retina. The vertical axis shows the amount (μg) of these substances contained per gram of wet weight. The shaded bars show the quantitative values ​​for trastuzumab, and the black bars show the quantitative values ​​for the hTfR affinity peptide-trastuzumab 5. Graph showing the quantitative values ​​of these substances contained in various organs of cynomolgus monkeys 8 hours after administration of the hTfR affinity peptide-trastuzumab 5 and trastuzumab. (1) shows the quantitative values ​​for the liver, (2) the kidney, (3) the heart (left ventricle), (4) the heart (aortic valve), (5) the lung, (6) the bone marrow, (7) the spleen, (8) the thymus, (9) the testis, (10) the prostate, (11) the rectus femoris, (12) the EDL, (13) the soleus, (14) the triceps, and (15) the diaphragm. The vertical axis shows the amount (μg) of each substance contained per gram of wet weight.The shaded bars represent trastuzumab, and the black bars represent the quantitative values ​​for the hTfR affinity peptide-trastuzumab5. Graph showing the measured values ​​of heparan sulfate (HS) concentration in mouse cerebrospinal fluid (CSF) after administration of a fusion protein of hTfR affinity peptide and human iduronate-2-sulfatase (hIDS). The vertical axis represents the heparan sulfate (HS) concentration (μg / mL) in cerebrospinal fluid (CSF). The vertical bars represent SD values. Graph showing the measured values ​​of heparan sulfate (HS) concentration in mouse brain tissue after administration of a fusion protein of hTfR affinity peptide and human iduronate-2-sulfatase (hIDS). The vertical axis represents the heparan sulfate (HS) concentration (μg / mg) per dry weight (mg) of brain tissue. The vertical bars represent SD values. Graphs showing the measured concentrations of heparan sulfate (HS) in mouse cerebrospinal fluid (CSF) and brain tissue after administration of a fusion protein of an hTfR-affinity peptide and human heparan N-sulfatase (hSGSH). (a) shows the measured values ​​of heparan sulfate (HS) in brain tissue, and (b) shows the measured values ​​of heparan sulfate (HS) in cerebrospinal fluid (CSF). In (a), the vertical axis shows the concentration of heparan sulfate (HS) (μg / mg) per dry weight (mg) of brain tissue, and the vertical bars show the SD value. In (b), the vertical axis shows the concentration of heparan sulfate (HS) (μg / mL) in cerebrospinal fluid (CSF), and the vertical bars show the SD value.

[0012] Antibody monomers in many mammals, including humans and mice, form heterotetramers consisting of two heavy chains and two light chains covalently linked by disulfide bonds. At the N-terminus of each chain, there is a region with a different amino acid sequence between different antibodies, called the variable region. The heavy chain variable region contains three complementarity-determining regions (CDRs) that contribute significantly to specific binding to antigens. These three complementarity-determining regions are called CDR1, CDR2, and CDR3 from the N-terminus. Similarly, the light chain also contains three complementarity-determining regions that contribute significantly to specific binding to antigens. These three complementarity-determining regions are called CDR1, CDR2, and CDR3 from the N-terminus.

[0013] One H chain and one L chain pair together to specifically bind to an antigen. In other words, antigen specificity is largely determined by the amino acid sequences of six CDRs: three CDRs in the H chain and three CDRs in the L chain. Many mammalian antibody monomers have two pairs of H and L chains, so each antibody molecule has two regions that can bind to antigens.

[0014] The regions in the variable region other than the CDRs are called framework regions (FRs). There are four FRs in each of the H and L chains, which are called FR1, FR2, FR3, and FR4 from the N-terminus. The CDRs are sandwiched between the FRs in the variable region.

[0015] FR1 is the region from the N-terminus of the variable region of a heavy chain antibody to the amino acid adjacent to the N-terminus of CDR1. FR2 is the region between CDR1 and CDR2. FR3 is the region between CDR2 and CDR3. FR4 is the region from the amino acid adjacent to the C-terminus of CDR3 to the C-terminus of the variable region of a heavy chain antibody.

[0016] In contrast to the monomeric antibodies of many mammals mentioned above, there are antibodies (heavy-chain antibodies) that contain only heavy chains and no light chains. Examples include heavy-chain antibodies (hcIg) produced by camelids and the immunoglobulin new antigen receptor (IgNAR) produced by many cartilaginous fish, including sharks. Camelid heavy-chain antibodies (hcIg) are homodimers in which two heavy chains are covalently linked by disulfide bonds. Each heavy chain has a variable region at its N-terminus, which contains three complementarity-determining regions (CDRs) that contribute significantly to specific binding to antigens. These three complementarity-determining regions are called CDR1, CDR2, and CDR3 from the N-terminus. Each heavy chain constituting an hcIg can specifically bind to an antigen even when it is a monomer, so a single hcIg molecule contains two regions capable of binding to antigens.

[0017] Like hcIg, IgNAR is a homodimer consisting of two heavy chains covalently linked by disulfide bonds. Each heavy chain has a variable region at its N-terminus, which contains three complementarity-determining regions (CDRs) that contribute significantly to specific binding to antigens. These three complementarity-determining regions are called CDR1, CDR2, and CDR3 from the N-terminus. Each heavy chain constituting IgNAR can specifically bind to antigens even when in a monomeric form, meaning that a single IgNAR molecule contains two regions capable of binding to antigens.

[0018] Framework regions in the variable regions of hcIg and IgNAR can be identified from public DNA databases containing germline antibody gene sequences. For example, germline DNA and amino acid sequences of human heavy chain variable region genes can be selected from the "VBase" human germline sequence database (available online at www.mrccpe.cam.ac.uk / vbase). Alternatively, they can be identified from DNA and amino acid sequences described in published literature, such as Kabat EA. Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson IM. J. fol. Biol. 227. 776-98 (1992); and Cox JPL. Eur. J Immunol. 24:827-836 (1994). For example, since the human heavy chain variable region gene is homologous to the hcIg variable region, it is also possible to identify the framework region of hcIg based on information on the human heavy chain variable region gene.

[0019] However, without being limited thereto, in each FR region, (1) a region containing 1 to 5 amino acids from the N-terminus and / or 1 to 5 amino acids at the C-terminus, (2) a region containing 1 to 3 amino acids from the N-terminus and / or 1 to 3 amino acids at the C-terminus, or (3) a region containing 1 or 2 amino acids from the N-terminus and / or 1 or 2 amino acids at the C-terminus may be included in the CDR region without being included in the FR region. Even a region generally considered to be FR may be included in the CDR in the present invention, as long as it is involved in maintaining the CDR structure or binding to an antigen and is considered to have a function of substantially determining antibody complementarity.

[0020] A constant region exists at the C-terminus of the variable region of a heavy chain antibody, via the hinge region. A constant region also exists at the C-terminus of the variable region of IgNAR. These constant regions contribute little to the specific binding of heavy chain antibodies to antigens.

[0021] Heavy chain antibodies are homodimers covalently linked by disulfide bonds at the hinge region, but they do not need to be homodimers to bind to antigens; each heavy chain constituting a heavy chain antibody can bind to antigen independently.

[0022] Heavy chain monomers constituting heavy chain antibodies can be obtained by deleting the cysteine ​​residues that form disulfide bonds in the amino acid sequence of the heavy chain or by substituting them with other amino acids. Such heavy chain monomers can be produced as recombinant proteins using genetic engineering techniques. Herein, such heavy chains are referred to as "single heavy chain antibodies." Single heavy chain antibodies are included in heavy chain antibodies as long as they can bind to the antigen of the original heavy chain antibody. Single heavy chain antibodies can be linked in tandem. The C-terminus of one single heavy chain antibody is linked to the N-terminus of the other single heavy chain antibody, either directly or via a peptide linker. The number of linked single heavy chain antibodies is 2 to 10, e.g., 2. The peptide linker sequences described herein can be used. Linked single heavy chain antibodies also include such single heavy chain antibodies.

[0023] The variable regions of each heavy chain constituting a heavy-chain antibody can bind to an antigen even without the presence of a hinge region and / or constant region. A protein consisting of such variable regions can be produced as a recombinant protein, for example, by constructing a gene encoding only the amino acid sequence of the heavy-chain variable region of a heavy-chain antibody, incorporating this into an expression vector, and introducing it into a host cell.

[0024] For convenience, the term "heavy chain antibody" refers to any antibody that contains a portion or all of the variable region of a heavy chain constituting a heavy chain antibody and has affinity for an antigen. Heavy chain antibodies also include those that contain, in addition to a portion or all of the variable region of a heavy chain constituting a heavy chain antibody, (1) a portion or all of the hinge region, or (2) a portion of the constant region of the heavy chain in addition to the hinge region. Heavy chain antibodies include those derived from hcIg and those derived from IgNAR.

[0025] Heavy chain antibodies derived from hcIg include (1h) those consisting of only the whole or a part of the heavy chain variable region, (2h) those consisting of the whole or a part of the heavy chain variable region and a part of the hinge region, (3h) those consisting of the whole heavy chain variable region and hinge region, and (4h) those comprising a part of the constant region in addition to the heavy chain variable region and hinge region. Here, the part of the constant region contained in the heavy chain antibody is, for example, the C heavy chain. H 2. Heavy chain antibodies derived from IgNAR include (1I) those consisting of all or only a part of the heavy chain variable region, (2I) those consisting of a heavy chain variable region and a part of the hinge region, (3I) those consisting of the heavy chain variable region and the hinge region, and (4I) those comprising a part of the heavy chain constant region in addition to the heavy chain variable region and hinge region. Here, the part of the constant region contained in the heavy chain antibody is, for example, the C heavy chain. H1. In this specification, proteins (1h) to (4h) containing the variable region of a heavy chain antibody derived from hcIg and proteins (1I) to (4I) containing the variable region of a heavy chain antibody derived from IgNAR are collectively referred to as heavy chain antibody variable region peptides. Therefore, heavy chain antibody variable region peptides include those with two antigen-binding sites formed by two peptide chains linked by disulfide bonds, and those with one antigen-binding site formed by a single peptide chain.

[0026] As used herein, the heavy chain antibody recognizes, as an antigen, a molecule present on the surface of vascular endothelial cells, particularly cerebrovascular endothelial cells. While there are no particular limitations on the species of vascular endothelial cells and cerebrovascular endothelial cells, human cells are preferred. The molecules present on the surface of these cells are preferably transferrin receptor (TfR), insulin receptor (IR), leptin receptor, lipoprotein receptor, IGF receptor, OATP-F, organic anion transporter, MCT-8, and monocarboxylate transporter, more preferably TfR or insulin receptor, and even more preferably TfR.

[0027] Heavy chain antibody variable region peptides that have affinity for molecules present on the surface of vascular endothelial cells, particularly cerebrovascular endothelial cells, are referred to as blood-brain barrier heavy chain antibody variable region peptides (BBB heavy chain antibody variable region peptides). Furthermore, BBB heavy chain antibody variable region peptides, and all peptides derived from BBB heavy chain antibody variable region peptides that have affinity for molecules present on the surface of vascular endothelial cells, are referred to as BBB affinity peptides. Herein, the proteins (1h) or (2h) above, which contain the variable region of a heavy chain antibody derived from hcIg, are referred to as immunoglobulin single variable domain peptides. Immunoglobulin single variable domain peptides contained in BBB heavy chain antibody variable region peptides can also be specifically referred to as BBB immunoglobulin single variable domain peptides. In the present invention, the terms VHH, VHH domain, VHH antibody, nanobody (trademark of Ablynx NV), and Nanobody (trademark of Ablynx NV) are synonymous with immunoglobulin single variable domain peptide and are used interchangeably. Therefore, for example, VHHs with affinity for TfR are included in BBB immunoglobulin single variable domain peptides. Heavy chain antibody variable region peptides with affinity for anti-hTfR are called anti-hTfR heavy chain antibody variable region peptides. Furthermore, anti-hTfR heavy chain antibody variable region peptides, and all peptides with affinity for anti-hTfR derived from anti-hTfR heavy chain antibody variable region peptides, are called hTfR affinity peptides. VHHs with affinity for hTfR are particularly included in hTfR affinity peptides. The same applies to VHHs with affinity for other molecules present on the surface of such cells; for example, VHHs with affinity for hIR are included in hIR affinity peptides. There are no particular limitations on the number of amino acids constituting a BBB affinity peptide, but it is usually a peptide composed of 80 to 200 amino acids, preferably 80 to 150 amino acids, more preferably 100 to 130 amino acids, and even more preferably 110 to 125 amino acids. For example, BBB affinity peptides consist of 113, 115, 119, or 120 amino acids. In this specification, the term "peptide" is synonymous with "polypeptide" or "protein," and can be replaced with these terms as appropriate.Below, the characteristics and the like of a heavy chain antibody variable region peptide having affinity for the human transferrin receptor (hTfR) will be described in detail as an example, but these descriptions can also be generally applied to heavy chain antibody variable region peptides having affinity for other molecules present on the surface of such cells.

[0028] While antibodies typically consist of a light chain and a heavy chain, VHHs consist of a single heavy chain, which simplifies the process of producing a fusion protein bound to another protein (A) as an hTfR affinity peptide. For example, when using a conventional antibody as an hTfR affinity peptide, it is necessary to express two proteins, whereas with VHHs, it is sufficient to express only one protein.

[0029] Heavy chain antibody variable region peptides with affinity for the human transferrin receptor are particularly referred to as anti-hTfR heavy chain antibody variable region peptides. Herein, the proteins (1h) or (2h) described above, which contain the variable region of a heavy chain antibody derived from hcIg, are referred to as immunoglobulin single variable domain peptides. Immunoglobulin single variable domain peptides with affinity for the human transferrin receptor (hTfR) are particularly referred to as hTfR immunoglobulin single variable domain peptides or human transferrin receptor affinity peptides (hTfR affinity peptides). hTfR affinity peptides are preferably peptides consisting of 100 to 130 amino acids, more preferably 110 to 125 amino acids. For example, hTfR affinity peptides consist of 113, 115, or 119 amino acids.

[0030] In addition to the heavy chain antibody variable region peptides described above as (1h) to (4h) and (1I) to (4I), those with mutations such as substitutions, deletions, and additions are also included, as long as they have affinity for the antigens to which the original peptides have affinity. Furthermore, in addition to the immunoglobulin single variable domain peptides described above as (1h) or (2h), those with mutations such as substitutions, deletions, and additions are also included, as long as they have affinity for the antigens to which the original peptides have affinity. Mutations into hTfR immunoglobulin single variable domain peptides are described in detail below.

[0031] In the present invention, the term "human transferrin receptor" or "hTfR" refers to a membrane protein having the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the anti-hTfR antibody of the present invention specifically binds to the portion of the amino acid sequence shown in SEQ ID NO: 1 from the 89th cysteine ​​residue from the N-terminus to the phenylalanine residue at the C-terminus (extracellular domain of hTfR), but is not limited to this. Furthermore, in the present invention, the term "monkey transferrin receptor" or "monkey TfR" particularly refers to a membrane protein derived from cynomolgus monkeys (Macaca fascicularis) and having the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the anti-hTfR antibody of the present invention also binds to the portion of the amino acid sequence shown in SEQ ID NO: 2 from the 89th cysteine ​​residue from the N-terminus to the phenylalanine residue at the C-terminus (extracellular domain of monkey TfR), but is not limited to this.

[0032] hTfR affinity peptides can also be obtained as recombinant proteins using a similar method. (1a) Recombinant human transferrin receptor (rhTfR) is produced using cells transfected with an expression vector incorporating the hTfR gene. (2a) rhTfR is used to immunize camelids capable of producing hcIg or fish, particularly cartilaginous fish such as sharks, capable of producing IgNAR. (3a) Antibody-producing cells are extracted from the immunized organism using rhTfR. Since antibody-producing cells are particularly present in peripheral blood, bone marrow, and spleen, peripheral blood cells, bone marrow-derived cells, splenocytes, etc. are collected as antibody-producing cells. (4a) cDNA is synthesized by reverse transcription from mRNA extracted from antibody-producing cells. (5a) PCR is performed using the cDNA as a template to amplify DNA fragments containing genes encoding the variable regions of heavy chain antibodies. (6a) A DNA fragment encoding the variable region of a heavy chain antibody is inserted into an expression vector, and host cells are transformed with this expression vector to obtain cells expressing the variable region of a heavy chain antibody. (7a) From the cells expressing the variable region of a heavy chain antibody, cells expressing the variable region of a heavy chain antibody that recognizes hTfR (cells expressing the variable region of an anti-hTfR heavy chain antibody) are selected. From the selected cells, the gene encoding the variable region of a heavy chain antibody that recognizes hTfR can also be amplified by PCR and isolated. (8a) Cells expressing the variable region of an anti-hTfR heavy chain antibody are cultured to express the recombinant anti-hTfR heavy chain antibody variable region peptide in the culture medium or in the cells. (9a) The expressed recombinant anti-hTfR heavy chain antibody variable region peptide is purified.

[0033] The cells expressing the heavy chain antibody variable region obtained in step (6a) of obtaining the anti-hTfR heavy chain antibody that recognizes hTfR do not necessarily express the heavy chain antibody variable region that recognizes hTfR. Therefore, a step of selecting cells that produce heavy chain antibody variable regions with the desired properties (affinity for hTfR), i.e., step (7a), from the cells expressing the heavy chain antibody variable regions obtained in step (6a) is required. The method described in detail below is effective for selecting cells that produce anti-hTfR heavy chain antibody variable region peptides.

[0034] The cells expressing the heavy chain antibody variable region obtained in step (6a) above are seeded onto a 96-well plate so that approximately one cell is contained per well and cultured, and then the culture supernatant is recovered from each well. After recombinant hTfR is added to the plate and allowed to retain therein, the recovered culture supernatant is added, and the variable region of the anti-hTfR heavy chain antibody contained in the culture supernatant is allowed to bind to the recombinant hTfR on the plate. The culture supernatant is then removed from the plate, and the plate is further washed to remove antibodies that have not bound to the recombinant hTfR. The amount of antibody retained on the plate is then measured. According to this method, the higher the affinity for hTfR of the antibody contained in the culture supernatant of the antibody-producing cells added to the plate, the greater the amount of antibody retained on the plate. Therefore, the amount of antibody retained on the plate can be measured, and cells corresponding to plates retaining a greater amount of antibody can be selected as cell lines producing anti-hTfR heavy chain antibody variable region peptides with relatively high affinity for hTfR. A PCR reaction can be performed using cDNA obtained by reverse transcription of mRNA extracted from the cell line selected in this manner as a template, and a DNA fragment containing the gene encoding the anti-hTfR heavy chain antibody variable region peptide can be amplified and isolated.

[0035] Another method for obtaining an anti-hTfR heavy chain antibody as a recombinant protein comprises the following steps. This method involves displaying the heavy chain antibody variable region encoded by a gene encoding the heavy chain antibody variable region on phages and selecting phages that display heavy chain antibody variable regions with desired properties. This process is called the phage display method, a well-known technique described in International Publications (WO 1997 / 09436, WO 1995 / 11317, etc.). A method for obtaining an anti-hTfR heavy chain antibody that recognizes hTfR as a recombinant protein using phage display includes, for example, the following steps (1b) to (9b). An hTfR affinity peptide can also be obtained as a recombinant protein using a similar method. (1b) Recombinant human transferrin receptor (rhTfR) is produced using cells transfected with an expression vector incorporating the hTfR gene. (2b) rhTfR is used to immunize camelids capable of producing hcIg or fish, particularly cartilaginous fish such as sharks, capable of producing IgNAR. (3b) Antibody-producing cells are extracted from the immunized organism using rhTfR. Since antibody-producing cells are particularly present in peripheral blood, bone marrow, and spleen, peripheral blood cells, bone marrow-derived cells, splenocytes, etc. are collected as antibody-producing cells. (4b) cDNA is synthesized by reverse transcription from mRNA extracted from the antibody-producing cells. (5b) PCR is performed using the cDNA as a template to amplify DNA fragments containing genes encoding the heavy chain antibody variable regions. (6b) The DNA fragments encoding the heavy chain antibody variable regions are incorporated into phagemids, and the phagemids are then introduced into host cells. The host cells are then cultured to release phages bearing the heavy chain antibody variable regions on their capsid surface into the medium. (7b) A solution containing the recovered phages is loaded onto a column packed with a carrier carrying rhTfR, and phages carrying on their capsid surface the variable region of a heavy chain antibody with affinity for rhTfR are bound to the column. After washing the column, the phages bound to hTfR are eluted from the column. Alternatively, a solution containing the recovered phages is added to a plate carrying rhTfR, and phages carrying on their capsid surface the variable region of a heavy chain antibody with affinity for rhTfR are bound to the plate.(8b) Only the phages displaying the variable region of the heavy chain antibody that recognizes hTfR that are retained on the column or plate are recovered, and the gene encoding the variable region of the heavy chain antibody that recognizes hTfR is amplified by PCR using the phagemid contained in this phage as a template. (9b) The amplified gene is inserted into an expression vector, and host cells are transformed with this expression vector to obtain cells expressing the variable region of the heavy chain antibody that recognizes hTfR. (10b) Cells expressing the anti-hTfR heavy chain antibody variable region peptide are cultured to express the recombinant anti-hTfR heavy chain antibody variable region peptide in the culture medium or in the cells. (11b) The expressed recombinant anti-hTfR heavy chain antibody variable region peptide is purified.

[0036] In steps (1b) to (3b) of the phage display method described above, it is necessary to extract antibody-producing cells from an organism immunized with rhTfR. Alternatively, mRNA obtained in advance from antibody-producing cells of an organism capable of expressing heavy chain antibodies or DNA fragments synthesized using this as a template can be pooled, and DNA fragments containing genes encoding the variable regions of heavy chain antibodies can be amplified using this as a template. According to the phage display method, for example, 1 x 10 １０ Because it is possible to screen phages at once, it may be possible to select phages displaying the variable region of heavy-chain antibodies that recognize hTfR, even from non-immune cells, without the need to immunize the organism or extract antibody-producing cells from it.

[0037] The above step (7b) is intended to select phages displaying variable regions of heavy chain antibodies that recognize rhTfR. The affinity of the selected phages for rhTfR can be adjusted by adjusting the conditions for binding the phages to the column (or plate) and / or the conditions for washing the column (or plate). Furthermore, by repeating step (7b) while gradually increasing the strictness of the conditions, phages with higher affinity for rhTfR are selected with each repetition of the step, thereby efficiently obtaining only phages displaying variable regions of heavy chain antibodies with high affinity for rhTfR.

[0038] Furthermore, during the replication process of the phage genome, mutations may be introduced into the genome, which may change the affinity of the variable region of the heavy chain antibody encoded by the phage genome for rhTfR. Therefore, by repeating step (7) above, random mutations may be introduced into the genome, potentially yielding antibodies with higher affinity for rhTfR.

[0039] The step (7b) may be repeated, for example, as shown in (7b') below. (7b') A culture solution containing the recovered phage is loaded onto a column packed with a carrier carrying rhTfR, and phage displaying the variable region of a heavy chain antibody that recognizes rhTfR are bound to the column. Alternatively, a solution containing the recovered phage is added to a plate carrying rhTfR, and phage carrying the variable region of a heavy chain antibody with affinity for rhTfR on their capsid surface are bound to the plate. After washing the column or plate, phage displaying the variable region of a heavy chain antibody that recognizes hTfR are eluted from the column or plate. Host cells are infected with the eluted phage, and the host cells are cultured. Phage displaying the variable region of a heavy chain antibody on their surface are recovered from the culture solution. The culture solution containing the recovered phage is loaded onto a column chromatography packed with a carrier carrying rhTfR, or added to a plate carrying rhTfR.

[0040] In the above step (7b'), mutations can be actively introduced into the phage genome by contacting the phage with a mutagen or by adding a mutagen to the culture medium of the host cells infected with the phage. Mutations can also be actively introduced into the phage genome by irradiating the phage or the host cells infected with the phage with electromagnetic waves such as ultraviolet rays or gamma rays.

[0041] Any of the above steps (1a) to (9a) and steps (1b) to (11b) can include a step of isolating a gene encoding the variable region of an anti-hTfR heavy chain antibody. An amino acid sequence containing the variable region of an anti-hTfR heavy chain antibody can be translated from the nucleotide sequence of the isolated gene encoding the anti-hTfR heavy chain antibody, and a DNA fragment encoding this amino acid sequence can be artificially synthesized. When artificially synthesizing a DNA fragment, codons suitable for gene expression in a host cell can be selected. Introducing an expression vector incorporating such a DNA fragment into a host cell can increase the expression level of an anti-hTfR heavy chain antibody variable region peptide in the host cell. The expression level of an rhTfR affinity peptide can also be increased in a similar manner.

[0042] The host cells used in the above process are not particularly limited, regardless of whether they are prokaryotic or eukaryotic, as long as they can express peptides containing heavy chain antibody variable regions by introducing an expression vector incorporating a gene encoding the heavy chain antibody variable region. Particularly preferred are Escherichia coli, Chinese hamster ovary-derived CHO cells, and mouse myeloma-derived NS / 0 cells. The expression vector used to incorporate and express the gene encoding the heavy chain antibody variable region can be any expression vector that can express peptides containing the heavy chain antibody variable region when introduced into host cells. The gene incorporated into the expression vector is located downstream of a DNA sequence (gene expression control site) that can regulate the frequency of gene transcription in the host cell. Examples of gene expression control sites that can be used in the present invention include a cytomegalovirus-derived promoter, the SV40 early promoter, the human elongation factor-1α (EF-1α) promoter, and the human ubiquitin C promoter.

[0043] The amino acid sequence of the variable region in the anti-hTfR heavy chain antibody variable region peptide obtained by the above process basically comprises, from the N-terminus, the amino acid sequences of four FRs and three CDRs: FR1, CDR1, FR2, CDR1, FR3, CDR1, and FR4. However, the amino acid sequence of the variable region is not limited to this, and may also be one in which part or all of FR1 is deleted, one in which part or all of FR4 is deleted, or one in which part or all of FR1 and part or all of FR4 are deleted, as long as it can specifically bind to hTfR. These variable region sequences in which part of the FR region is deleted are also included in the variable region of anti-hTfR heavy chain antibodies. The same applies to hTfR affinity peptides.

[0044] In one embodiment of the present invention, preferred anti-hTfR heavy chain antibody variable region peptides include those comprising the amino acid sequences of hTfR affinity peptides 1 to 5. hTfR affinity peptides 1 to 6 each comprise the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, or 55. The amino acid sequences set forth in SEQ ID NO: 3, 4, 5, 6, 7, or 55 are all amino acid sequences of the variable regions. hTfR affinity peptides 1 to 6 each comprise, from the N-terminus, the amino acid sequences of four FRs and three CDRs: FR1, CDR1, FR2, CDR1, FR3, CDR1, and FR4. hTfR affinity peptides 1 to 6 are all included in the hTfR affinity peptides.

[0045] CDR1 of hTfR affinity peptide 1 is a region containing the amino acid sequence of SEQ ID NO: 8 or 9, CDR2 is a region containing the amino acid sequence of SEQ ID NO: 10 or 11, and CDR3 is a region containing the amino acid sequence of SEQ ID NO: 12 or 13. In hTfR affinity peptide 1, the amino acid sequences of the CDRs can be combined in any manner. For example, CDR1 may be a region containing the amino acid sequence of SEQ ID NO: 8, CDR2 may be a region containing the amino acid sequence of SEQ ID NO: 10, and CDR3 may be a region containing the amino acid sequence of SEQ ID NO: 12, or CDR1 may be a region containing the amino acid sequence of SEQ ID NO: 9, CDR2 may be a region containing the amino acid sequence of SEQ ID NO: 11, and CDR3 may be a region containing the amino acid sequence of SEQ ID NO: 13. Furthermore, any of the CDRs may consist of these amino acid sequences.

[0046] CDR1 of hTfR affinity peptide 2 is a region containing the amino acid sequence of SEQ ID NO: 14 or 15, CDR2 is a region containing the amino acid sequence of SEQ ID NO: 16 or 17, and CDR3 is a region containing the amino acid sequence of SEQ ID NO: 18 or 19. In hTfR affinity peptide 2, the amino acid sequences of the CDRs can be combined in any way. For example, CDR1 may be a region containing the amino acid sequence of SEQ ID NO: 14, CDR2 may be a region containing the amino acid sequence of SEQ ID NO: 16, and CDR3 may be a region containing the amino acid sequence of SEQ ID NO: 18, or CDR1 may be a region containing the amino acid sequence of SEQ ID NO: 15, CDR2 may be a region containing the amino acid sequence of SEQ ID NO: 17, and CDR3 may be a region containing the amino acid sequence of SEQ ID NO: 19. Furthermore, any of the CDRs may consist of these amino acid sequences.

[0047] CDR1 of hTfR affinity peptide 3 is a region comprising the amino acid sequence of SEQ ID NO: 20 or 21, CDR2 is a region comprising the amino acid sequence of SEQ ID NO: 22 or 23, and CDR3 is a region comprising the amino acid sequence of SEQ ID NO: 24 or 25. In hTfR affinity peptide 3, the amino acid sequences of the CDRs can be combined in any manner. For example, CDR1 may be a region comprising the amino acid sequence of SEQ ID NO: 20, CDR2 may be a region comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 may be a region comprising the amino acid sequence of SEQ ID NO: 24, or CDR1 may be a region comprising the amino acid sequence of SEQ ID NO: 21, CDR2 may be a region comprising the amino acid sequence of SEQ ID NO: 23, and CDR3 may be a region comprising the amino acid sequence of SEQ ID NO: 25. Furthermore, any of the CDRs may consist of these amino acid sequences.

[0048] CDR1 of hTfR affinity peptide 4 is a region comprising the amino acid sequence of SEQ ID NO: 26 or 27, CDR2 is a region comprising the amino acid sequence of SEQ ID NO: 28 or 29, and CDR3 is a region comprising the amino acid sequence of SEQ ID NO: 30 or 31. In hTfR affinity peptide 4, the amino acid sequences of the CDRs can be combined in any manner. For example, CDR1 may be a region comprising the amino acid sequence of SEQ ID NO: 26, CDR2 may be a region comprising the amino acid sequence of SEQ ID NO: 28, and CDR3 may be a region comprising the amino acid sequence of SEQ ID NO: 30, or CDR1 may be a region comprising the amino acid sequence of SEQ ID NO: 27, CDR2 may be a region comprising the amino acid sequence of SEQ ID NO: 29, and CDR3 may be a region comprising the amino acid sequence of SEQ ID NO: 31. Furthermore, any of the CDRs may consist of these amino acid sequences.

[0049] CDR1 of hTfR affinity peptide 5 is a region comprising the amino acid sequence of SEQ ID NO: 20 or 21, CDR2 is a region comprising the amino acid sequence of SEQ ID NO: 22 or 23, and CDR3 is a region comprising the amino acid sequence of SEQ ID NO: 24 or 25. In hTfR affinity peptide 5, the amino acid sequences of the CDRs can be combined in any manner. For example, CDR1 may be a region comprising the amino acid sequence of SEQ ID NO: 20, CDR2 may be a region comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 may be a region comprising the amino acid sequence of SEQ ID NO: 24, or CDR1 may be a region comprising the amino acid sequence of SEQ ID NO: 21, CDR2 may be a region comprising the amino acid sequence of SEQ ID NO: 23, and CDR3 may be a region comprising the amino acid sequence of SEQ ID NO: 25. Furthermore, any of the CDRs may consist of these amino acid sequences.

[0050] CDR1 of hTfR affinity peptide 6 is a region comprising the amino acid sequence of SEQ ID NO: 20 or 21, CDR2 is a region comprising the amino acid sequence of SEQ ID NO: 56 or 57, and CDR3 is a region comprising the amino acid sequence of SEQ ID NO: 24 or 25. In hTfR affinity peptide 6, the amino acid sequences of the CDRs can be combined in any manner. For example, CDR1 may be a region comprising the amino acid sequence of SEQ ID NO: 20, CDR2 may be a region comprising the amino acid sequence of SEQ ID NO: 56, and CDR3 may be a region comprising the amino acid sequence of SEQ ID NO: 24, or CDR1 may be a region comprising the amino acid sequence of SEQ ID NO: 21, CDR2 may be a region comprising the amino acid sequence of SEQ ID NO: 57, and CDR3 may be a region comprising the amino acid sequence of SEQ ID NO: 25. Furthermore, any of the CDRs may consist of these amino acid sequences.

[0051] Furthermore, the amino acid sequence of the variable region of the above-mentioned hTfR affinity peptide may be mutated, as long as it is capable of specifically binding to hTfR. When such mutations are added, the amino acid sequence of the variable region of the hTfR affinity peptide after the mutations preferably has an identity of 80% or more, more preferably 85% or more, even more preferably 90% or more, still more preferably 95% or more, for example, 98% or more, to the original amino acid sequence.

[0052] Furthermore, when mutations are introduced into the amino acid sequence of the variable region, mutations can be introduced only in the FR region without mutations in the amino acid sequences of CDR1, CDR2, and CDR3. When such mutations are introduced, the amino acid sequence of the variable region after the mutations preferably has an identity of 85% or more, more preferably 90% or more, even more preferably 95% or more, for example, 98% or more, to the original amino acid sequence. When mutations are introduced into the amino acid sequence of the variable region, mutations can be introduced only in the CDR region without mutations in the amino acid sequence of the FR region. When such mutations are introduced, the amino acid sequence of the variable region after the mutations has an identity of preferably 90% or more, more preferably 95% or more, for example, 98% or more, to the original amino acid sequence.

[0053] The identity between the amino acid sequence of the original variable region and the mutated variable region can be easily calculated using well-known homology calculation algorithms. Examples of such algorithms include BLAST (Altschul SF. J Mol. Biol. 215, 403-10, (1990)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85, 2444 (1988)), and the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2, 482-9 (1981)). Throughout this specification, "identity" refers to the identity calculated using these algorithms. The terms "amino acid sequence homology" and "amino acid sequence identity" are used interchangeably herein.

[0054] The following describes in detail the cases where substitution, deletion, and addition mutations are made to the amino acid sequence of the variable region of the hTfR affinity peptide.

[0055] When amino acids in the amino acid sequence of the variable region of the hTfR affinity peptide are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, and even more preferably 1 to 5, for example, 1, 2, or 3. When amino acids in the amino acid sequence of the variable region are deleted, the number of deleted amino acids is preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, and even more preferably 1 to 5, for example, 1, 2, or 3. Mutations combining these amino acid substitutions and deletions can also be added. When amino acids are added to the variable region, preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids, for example, 1, 2, or 3 amino acids, are added to the amino acid sequence of the variable region or to the N-terminus or C-terminus. Mutations combining these amino acid additions, substitutions, and deletions can also be added.

[0056] When mutations such as substitutions, deletions, and additions are made to the amino acid sequence of the variable region of an hTfR affinity peptide, mutations can be made only in the FR region without making mutations in the amino acid sequence of the CDR region (CDR1, CDR2, and CDR3). When making substitutions only in the FR region, the number of amino acids to be substituted is preferably 1 to 12, more preferably 1 to 10, even more preferably 1 to 8, and even more preferably 1 to 4, for example, 1, 2, or 3. When amino acids are deleted only in the amino acid sequence of the FR region, the number of amino acids to be deleted is preferably 1 to 8, more preferably 1 to 4, even more preferably 1 to 3, and even more preferably 1 to 2, for example, 1 or 2. Mutations that combine these amino acid substitutions and deletions can also be made. Furthermore, when amino acids are added only to the amino acid sequence of the FR region, preferably 1 to 8, more preferably 1 to 4, even more preferably 1 to 3, and even more preferably 1 to 2 amino acids, for example, 1 or 2 amino acids, are added to the amino acid sequence of the variable region or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added.

[0057] When mutations are introduced only in the FR region, a method is known in which amino acid residues in the FR region of an hTfR affinity peptide are replaced with corresponding amino acid residues in the FR region of a human IgG-type antibody variable region. Herein, this method is referred to as humanization of the hTfR affinity peptide. Such a method is disclosed, for example, in Vincle C., et. al., J. Biol. Chem. 284, 3273-84 (2009). If the original antibody is an alpaca antibody, it may be recognized as an antigen when administered to humans. Humanized antibodies are expected to have lower antigenicity than the original antibody. When mutations are introduced only in the FR region, humanization is a preferred embodiment. Herein, the term "hTfR affinity peptide" also includes humanized hTfR affinity peptides.

[0058] When mutations such as substitutions, deletions, and additions are made to the amino acid sequence of the variable region of an hTfR affinity peptide, mutations can be made only in the CDR region without mutations in the amino acid sequence of the FR region. When substitutions are made only in the CDR region, the number of amino acids to be substituted is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2, for example, 1 or 2. When amino acids are deleted only in the amino acid sequence of the CDR region, the number of amino acids to be deleted is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2, for example, 1 or 2. Mutations that combine these amino acid substitutions and deletions can also be made. When amino acids are added only in the amino acid sequence of the FR region, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2 amino acids, for example, 1 or 2 amino acids, are added to the amino acid sequence of the variable region or to the N-terminus or C-terminus. Mutations that combine these amino acid additions, substitutions, and deletions can also be made.

[0059] Substitution of amino acids in the above variable region amino acid sequences with other amino acids occurs within a family of amino acids that are related, for example, by their side chains and chemical properties, and is predicted to not significantly alter the function of the anti-hTfR heavy chain antibody (i.e., is a conservative amino acid substitution). Examples of such amino acid families include: (1) the acidic amino acids aspartic acid and glutamic acid, (2) the basic amino acids histidine, lysine, and arginine, (3) the aromatic amino acids phenylalanine, tyrosine, and tryptophan, (4) the hydroxyl amino acids serine and threonine, (6) the neutral hydrophilic amino acids cysteine, serine, threonine, asparagine, and glutamine, (7) the amino acids that influence the orientation of the peptide chain glycine and proline, (8) the amide amino acids asparagine and glutamine, (9) the aliphatic amino acids alanine, leucine, isoleucine, and valine, (10) the amino acids with small side chains alanine, glycine, serine, and threonine, (11) the amino acids with particularly small side chains alanine and glycine, and (12) the branched-chain amino acids valine, leucine, and isoleucine.

[0060] The above conservative amino acid substitutions also apply to the substitution of an amino acid for another amino acid in the amino acid sequence of another protein such as human serum albumin (HSA).

[0061] In addition, if an amino acid is added to the C-terminus or N-terminus by mutation of the variable region and the added amino acid is located between the hTfR affinity peptide and another protein (A) when the hTfR affinity peptide is fused with the protein (A), the added amino acid is considered to constitute a part of the hTfR affinity peptide. The linker located between the hTfR affinity peptide and the protein (A) in a fusion protein of the hTfR affinity peptide and the protein (A) will be described in detail later. In this specification, the term "other protein (A)" refers to a protein other than the hTfR affinity peptide that is to be administered to a human in a form bound to the hTfR affinity peptide to exert its physiological activity in the human body.

[0062] The hTfR affinity peptide selected by the above steps has a certain affinity for hTfR. By introducing mutations such as substitutions, deletions, and additions into the amino acid sequence of the variable region of this hTfR affinity peptide, it is possible to modify the variable region of an anti-hTfR heavy chain antibody with desired properties. Introduction of mutations into the amino acid sequence of the variable region of an anti-hTfR heavy chain antibody is carried out by making mutations in the gene corresponding to the amino acid sequence.

[0063] The affinity of the variable region of an hTfR affinity peptide for hTfR can be appropriately adjusted by introducing mutations, such as substitutions, deletions, or additions, into the amino acid sequence that constitutes the variable region. For example, if an hTfR affinity peptide has high affinity for an antigen and a significantly low dissociation constant in aqueous solution, the variable region may not dissociate with the antigen when administered to the body, potentially resulting in functional problems. In such cases, by introducing mutations into the variable region, the dissociation constant can be adjusted in stages, such as 2-5 times, 5-10 times, or 10-100 times that of the original variable region, to obtain the most suitable hTfR affinity peptide for the intended purpose. Conversely, the dissociation constant can also be adjusted in stages, such as 1 / 2-1 / 5 times, 1 / 5-1 / 10 times, or 1 / 10-1 / 100 times that of the original variable region.

[0064] The binding affinity between the hTfR affinity peptide and the transferrin receptor, i.e., the dissociation constant, can be measured using, for example, biolayer interferometry (BLI), specifically the Octet system. All or part of the transferrin receptor derived from an appropriate animal is immobilized on a sensor, and a solution containing the variable region of the hTfR affinity peptide is used as a sample to measure the binding rate constant (Kon) and dissociation rate constant (Koff). The dissociation constant can then be calculated from the results. Similarly, methods using surface plasmon resonance (SPR), specifically the Biacore method, can also be used.

[0065] Introduction of mutations such as substitutions, deletions, and additions into the amino acid sequence of the variable region of the hTfR affinity peptide can be carried out, for example, by using a gene encoding the variable region of an anti-hTfR heavy chain antibody as a template and introducing mutations into specific sites in the nucleotide sequence of the gene by a method such as PCR, or by introducing mutations randomly.

[0066] Introduction of mutations into the amino acid sequence of an hTfR affinity peptide for the purpose of adjusting the affinity between the hTfR affinity peptide and hTfR can be achieved, for example, by incorporating a gene encoding the hTfR affinity peptide into a phagemid, using this phagemid to produce a phage that expresses the hTfR affinity peptide on the capsid surface, and then subjecting this phage to a mutagen, etc. The phage is grown while introducing mutations into the gene encoding the hTfR affinity peptide with the mutagen, and from the grown phages, phages that express single-chain antibodies with the desired dissociation constant can be selected by the method described above.

[0067] The hTfR affinity peptide having a relatively high affinity for hTfR obtained by the above method preferably has a dissociation constant (K D ) is preferably 5X10 －８ M or less, and more preferably 2×10 －８ M or less, for example, 1X10 －８ M or less, 5X10 －９M or less, 1X10 －９ For example, a suitable one is one having a dissociation constant of 5×10 －１１ M-1X10 －８ M, 2X10 －１１ M-1X10 －８ M, 1X10 －１０ M-1X10 －８ Examples of such compounds include those in which M is a methyl group.

[0068] From the hTfR affinity peptides obtained as described above, those with affinity for both human and monkey TfR can be selected. hTfR affinity peptides with affinity for monkey TfR can be selected, for example, by ELISA using recombinant monkey TfR produced using genetic engineering techniques. In this ELISA, recombinant monkey TfR is immobilized on a plate, contacted with an hTfR affinity peptide, and then those not bound to the recombinant monkey TfR are removed from the plate, and the amount of hTfR affinity peptide retained on the plate is measured. Since the higher the affinity for recombinant monkey TfR, the greater the amount of hTfR affinity peptide retained on the plate, the hTfR affinity peptide corresponding to the plate retaining a larger amount of hTfR affinity peptide can be selected as an hTfR affinity peptide with affinity for monkey TfR. The term "monkey" as used herein refers preferably to an animal classified as an Anthropoidea (excluding humans), more preferably to an animal classified as a Cercopithecidae, and even more preferably to an animal classified as a Macaca, such as a cynomolgus monkey or a rhesus monkey, and particularly a cynomolgus monkey. Cynomolgus monkeys are convenient for evaluating the efficacy of drugs that utilize hTfR-affinity peptides. Anti-hTfR heavy chain antibody variable region peptides that have affinity for both human and monkey TfRs can also be obtained in a similar manner.

[0069] An hTfR affinity peptide that has affinity for both human and monkey hTfR has the advantageous effect of allowing the pharmacokinetics of the peptide to be observed in humans in vivo when administered to monkeys. For example, when developing a drug using the hTfR affinity peptide of one embodiment of the present invention, some non-clinical studies, such as pharmacokinetic studies, of the drug can be performed using monkeys, significantly facilitating the development of the drug. The same can be said for anti-hTfR heavy chain antibody variable region peptides.

[0070] In the present invention, the hTfR affinity peptide, which has a relatively high affinity for hTfR and also has affinity for monkey TfR, has the following dissociation constants with human and monkey TfR, as measured by the method described in Example 7: (a) the dissociation constants with both hTfR and monkey TfR are 5×10 －９ M-1X10 －７ (b) the dissociation constants with both human TfR and monkey TfR are 5×10 －９ M-5X10 －８ (d) The dissociation constants with both human TfR and monkey TfR are 1×10 －８ M-3X10 －８ (e) the dissociation constants with both human TfR and monkey TfR are 1x10 －９ M-2X10 －７ (f) the dissociation constants with both human TfR and monkey TfR are 5×10 －９ M-5X10 －７ In particular, in the above (a) to (f), the ratio of the dissociation constant with hTfR to the dissociation constant with monkey TfR, where the value of the dissociation constant with hTfR is taken as 1, is preferably such that the value of the dissociation constant with monkey TfR is 0.3 to 3.0, for example, 0.5 to 2.5, 0.8 to 2.5, 0.5 to 2.5, or 0.8 to 1.5.

[0071] In one embodiment of the present invention, the hTfR affinity peptides 1 to 5 have dissociation constants with human and monkey TfRs that fall within any of the above (a) to (e). Therefore, when administered to monkeys, their behavior in the monkey body is similar to that observed when administered to humans. Therefore, for drugs incorporating the hTfR affinity peptides 1 to 5, monkey testing can provide results that more closely reflect their behavior when administered to humans. Therefore, monkey testing can provide more useful information for conducting clinical trials in humans. In particular, the ratio of the dissociation constants with hTfR to the monkey TfR is in the range of 0.5 to 1.5, where the dissociation constant with hTfR is 1. Therefore, the behavior in the human body can be easily predicted from the behavior in the monkey body, making these peptides suitable.

[0072] When administered into the body via intravenous injection or other means, the hTfR affinity peptide of one embodiment of the present invention can efficiently bind to hTfR present on endothelial cells of brain capillaries. The hTfR affinity peptide bound to hTfR passes through the blood-brain barrier and is taken up into the brain via mechanisms such as endocytosis, exocytosis, and transcytosis. Therefore, by binding proteins, low-molecular-weight compounds, and the like that are to function in the brain to the hTfR affinity peptide, these substances can be efficiently passed through the blood-brain barrier and reach the brain. Furthermore, the hTfR affinity peptide of one embodiment of the present invention is predicted to reach the cerebral parenchyma, hippocampal neuronal cells, cerebellar parenchyma, and / or at least any of these tissues or cells after passing through the blood-brain barrier. Therefore, by binding drugs to be applied to these tissues or cells, specifically proteins, low-molecular-weight compounds, nucleic acids, and the like, to the hTfR affinity peptide of the present invention, the drugs can be delivered to these tissues or cells. In this specification, a conjugate of an hTfR affinity peptide and a drug is referred to as an hTfR affinity peptide-drug conjugate. The same can be said for an anti-hTfR heavy chain antibody variable region peptide.

[0073] Forming a complex with the hTfR affinity peptide of one embodiment of the present invention can be an effective means for delivering substances (e.g., proteins, low-molecular-weight compounds, nucleic acids, etc.) from the blood to the brain and allowing them to exert their physiological or pharmacological effects when administered intravenously. In particular, the hTfR affinity peptides of the present invention are predicted to reach the cerebral parenchyma, hippocampal neuronal-like cells, cerebellar parenchyma, etc., after passing through the blood-brain barrier. Therefore, by administering these substances to the blood via intravenous administration or other means in a form conjugated to the hTfR affinity peptide of the present invention, these substances can pass through the blood-brain barrier and reach central nervous tissue, thereby enabling their physiological activity to be exerted or enhanced in tissues or cells of the central nervous system. The same can be said for anti-hTfR heavy chain antibody variable region peptides.

[0074] Methods for linking hTfR affinity peptides to such substances (proteins, small molecules, nucleic acids, etc.) include linking via non-peptide linkers or peptide linkers. The same applies to anti-hTfR heavy chain antibody variable region peptides. Non-peptide linkers include polyethylene glycol (PEG), polypropylene glycol, ethylene glycol / propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid, or derivatives or combinations thereof. A peptide linker is a peptide chain or derivative thereof consisting of 1 to 50 peptide-bonded amino acids. Its N-terminus and C-terminus form covalent bonds with either the hTfR affinity peptide or protein, small molecule, nucleic acid, etc., respectively, thereby linking the anti-hTfR antibody to the protein, small molecule, nucleic acid, etc.

[0075] The hTfR affinity peptide of the present invention conjugated to a desired other protein (A) using PEG as a non-peptide linker is specifically referred to as an hTfR affinity peptide-PEG-protein. The hTfR affinity peptide-PEG-protein can be produced by conjugating the hTfR affinity peptide to PEG to prepare an hTfR affinity peptide-PEG, and then conjugating the hTfR affinity peptide-PEG to the other protein (A). Alternatively, the hTfR affinity peptide-PEG-protein can be produced by conjugating the other protein (A) to PEG to prepare a protein-PEG, and then conjugating the protein-PEG to the hTfR affinity peptide. When conjugating PEG to the hTfR affinity peptide and the other protein (A), PEG modified with a functional group such as carbonate, carbonylimidazole, active ester of carboxylic acid, azlactone, cyclic imidothione, isocyanate, isothiocyanate, imidate, or aldehyde is used. The functional groups introduced into the PEG react primarily with amino groups in the hTfR affinity peptide and other protein (A) molecules, thereby covalently bonding the PEG to the hTfR affinity peptide and other protein (A). While the molecular weight and shape of the PEG used are not particularly limited, its average molecular weight (MW) is preferably 500 to 60,000, and more preferably 500 to 20,000. For example, PEGs with average molecular weights of about 300, about 500, about 1,000, about 2,000, about 4,000, about 10,000, or about 20,000 can be suitably used as non-peptide linkers. The same applies when linking the hTfR affinity peptide to a desired small molecule compound, nucleic acid, or the like.

[0076] For example, hTfR affinity peptide-PEG can be obtained by mixing an hTfR affinity peptide with polyethylene glycol having an aldehyde group as a functional group (ALD-PEG-ALD) so that the molar ratio of ALD-PEG-ALD to the antibody is 1:11, 1:12.5, 1:15, 1:110, 1:120, etc., and then adding a reducing agent such as NaCNBH3 to the mixture to allow reaction. The hTfR affinity peptide-PEG is then reacted with another protein (A) in the presence of a reducing agent such as NaCNBH3 to obtain an hTfR affinity peptide-PEG-protein. Conversely, the hTfR affinity peptide-PEG-protein can also be obtained by first conjugating another protein (A) with ALD-PEG-ALD to prepare a protein-PEG, and then conjugating the protein-PEG to the hTfR affinity peptide.

[0077] The hTfR affinity peptide and the other protein (A) can also be linked by a peptide bond to the C-terminus or N-terminus of the hTfR affinity peptide, either directly or via a peptide linker, at the N-terminus or C-terminus of the other protein (A). Such a fusion protein comprising the hTfR affinity peptide and the other protein (A) can be obtained as a recombinant protein by incorporating a DNA fragment, in which the cDNA encoding the other protein (A) is located in-frame at the 3'- or 5'-end of the cDNA encoding the hTfR affinity peptide, either directly or via a DNA fragment encoding the amino acid sequence of the peptide linker, into an expression vector for mammalian cells, eukaryotes such as yeast, or prokaryote cells such as Escherichia coli, and culturing the host cells transfected with this expression vector. Herein, such a recombinant protein comprising the hTfR affinity peptide and the other protein (A) linked together is referred to as an hTfR affinity peptide-protein (A) fusion protein. Similarly, a recombinant protein in which an immunoglobulin single variable domain peptide is bound to another protein (A) is called an immunoglobulin single variable domain peptide-protein (A) fusion protein, and a recombinant protein in which a heavy chain antibody variable region peptide is bound to another protein (A) is called a heavy chain antibody variable region peptide-protein (A) fusion protein.

[0078] In this case, the peptide linker disposed between the hTfR affinity peptide and the other protein (A) is a peptide chain composed of preferably 1 to 50, more preferably 1 to 17, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids, but the number of amino acids constituting the peptide linker can be appropriately adjusted to 1, 2, 3, 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, 27, etc., depending on the other protein (A) to be bound to the anti-hTfR antibody. The amino acid sequence of such a peptide linker is not limited as long as the hTfR affinity peptide linked thereto has affinity for hTfR and the other protein (A) linked thereto can exhibit the desired physiological activity under physiological conditions, but preferably is composed of glycine and serine.

[0079] Preferred peptide linkers include, for example, those consisting of a single amino acid (e.g., glycine or serine), the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 33, the amino acid sequence of SEQ ID NO: 34, the amino acid sequence of SEQ ID NO: 35, the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 58, and SEQ ID NO: 69, or those containing 2 to 10 or 2 to 5 consecutive amino acids of these amino acid sequences. Such peptide linkers have sequences consisting of 2 to 50 amino acids, or sequences consisting of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, or 27 amino acids. For example, those containing the amino acid sequence Gly-Ser can be suitably used as peptide linkers. Furthermore, those containing a sequence of 25 consecutive amino acids consisting of five consecutive amino acids of the amino acid sequence of SEQ ID NO: 32 can also be suitably used as peptide linkers.

[0080] The hTfR affinity peptide can also be conjugated to the Fc region of human IgG (hIgG Fc region). Conjugating the hIgG Fc region to the hTfR affinity peptide can enhance the in vivo stability of the hTfR affinity peptide, e.g., its blood retention. Examples of such hTfR affinity peptides with the hIgG Fc region conjugated include those in which the hIgG Fc region is conjugated to the C-terminus of the hTfR affinity peptide, either directly or via a peptide linker, or those in which the hIgG Fc region is conjugated to the N-terminus of the hTfR affinity peptide, either directly or via a peptide linker. Conjugates in which the Fc region is conjugated to the hTfR affinity peptide can be further conjugated to substances (e.g., proteins, low-molecular-weight compounds, nucleic acids, etc.) that normally cannot cross the blood-brain barrier and therefore have little or no physiological or pharmacological effect in the brain when administered intravenously to form complexes. Such substances can be conjugated to the Fc region-hTfR affinity peptide conjugates using the above-mentioned techniques, e.g., PEG-based techniques. When administered intravenously or otherwise, the complex is predicted to penetrate the blood-brain barrier and reach the cerebral parenchyma, hippocampal neuronal cells, cerebellar parenchyma, etc. Therefore, by forming the complex into a living body, the physiological activity of these substances can be exerted in these central nervous system tissues or cells. Furthermore, since the complex has increased stability in blood compared to a complex lacking the hIgG Fc region, it can exert its medicinal effects for a longer period of time when administered in vivo. Proteins in which an hTfR affinity peptide is bound to the Fc region of human IgG can be obtained as recombinant proteins, similar to the hTfR affinity peptide-protein (A) fusion protein.

[0081] An hTfR affinity peptide, another protein (A), and an hIgG Fc region can also be conjugated. The conjugation of the hIgG Fc region can enhance the in vivo stability of the conjugate, for example, its blood retention. Examples of such proteins with an Fc region conjugated include those in which an hIgG Fc region is conjugated to the C-terminus of another protein (A), either directly or via a peptide linker, and an hTfR affinity peptide is conjugated to the C-terminus of the hIgG Fc region, either directly or via a peptide linker. Other proteins include those in which an hIgG Fc region is conjugated to the C-terminus of an hTfR affinity peptide, either directly or via a peptide linker, and another protein (A) is conjugated to the C-terminus of the hIgG Fc region, either directly or via a linker sequence. Still other proteins include those in which another protein (A) is conjugated to the C-terminus of an hTfR affinity peptide, either directly or via a peptide linker, and an hIgG Fc region is conjugated to the C-terminus of the other protein (A), either directly or via a linker sequence. Other examples include an hTfR affinity peptide bound to the C-terminus of another protein (A) directly or via a peptide linker, and an hIgG Fc region bound to the C-terminus of the hTfR affinity peptide directly or via a linker sequence. Other examples include an hTfR affinity peptide bound to the C-terminus of an hIgG Fc region directly or via a peptide linker, and another protein (A) bound to the C-terminus of the hTfR affinity peptide directly or via a linker sequence. Other examples include an hIgG Fc region bound to the C-terminus of another protein (A) directly or via a peptide linker, and an hTfR affinity peptide bound to the C-terminus of the other protein (A) directly or via a linker sequence. Proteins comprising an hTfR affinity peptide, another protein (A), and an hIgG Fc region bound together can be obtained as recombinant proteins, similar to hTfR affinity peptide-protein (A) fusion proteins. Recombinant proteins obtained in this manner are also included in the hTfR affinity peptide-protein (A) fusion proteins.

[0082] The type of IgG for the hIgG Fc region to be conjugated is not particularly limited, and may be any of IgG1 to IgG5. Furthermore, the hIgG Fc region to be introduced may be the entire Fc region or a portion thereof. A preferred embodiment of such an hIgG Fc region is one having the amino acid sequence shown in SEQ ID NO: 37, which is the entire Fc region of human IgG1.

[0083] The hTfR affinity peptide can also be conjugated to human serum albumin (HSA). Conjugating HSA to the hTfR affinity peptide can increase the in vivo stability of the hTfR affinity peptide, e.g., its blood retention. Examples of such HSA-conjugated hTfR affinity peptides include those in which HSA is conjugated to the C-terminus of the hTfR affinity peptide directly or via a peptide linker, or those in which HSA is conjugated to the N-terminus of the hTfR affinity peptide directly or via a peptide linker. The conjugate formed by conjugating HSA to the hTfR affinity peptide can be further conjugated to a substance (e.g., a protein, a low molecular weight compound, a nucleic acid, etc.) that would normally be unable to cross the blood-brain barrier and therefore would have little or no physiological or pharmacological effect in the brain upon intravenous administration to form a complex. Such substances can be conjugated to the HSA-hTfR affinity peptide conjugate using the above-mentioned methods, e.g., PEG-based methods. When administered intravenously or otherwise, the complex is predicted to penetrate the blood-brain barrier and reach the cerebral parenchyma, hippocampal neuronal cells, cerebellar parenchyma, etc. Therefore, by forming the complex into a body, the physiological activity of these substances can be exerted in these central nervous system tissues or cells. Furthermore, since the complex has increased stability in blood compared to those without HSA, it can exert its medicinal effect for a longer period of time when administered to a body. Proteins in which an hTfR affinity peptide is bound to HSA can be obtained as recombinant proteins, similar to the hTfR affinity peptide-protein (A) fusion protein.

[0084] HSA can also be bound to the hTfR affinity peptide-protein (A) fusion protein. Examples of such HSA-bound proteins include those in which HSA is bound to the C-terminus of another protein (A), either directly or via a peptide linker, and an hTfR affinity peptide is further bound to the C-terminus of the HSA, either directly or via a peptide linker. Other proteins include those in which HSA is bound to the C-terminus of an hTfR affinity peptide, either directly or via a peptide linker, and another protein (A) is further bound to the C-terminus of the HSA, either directly or via a linker sequence. Other proteins include those in which another protein (A) is bound to the C-terminus of an hTfR affinity peptide, either directly or via a peptide linker, and HSA is further bound to the C-terminus of the other protein (A), either directly or via a linker sequence. Still other proteins include those in which an hTfR affinity peptide is bound to the C-terminus of another protein (A), either directly or via a peptide linker, and HSA is further bound to the C-terminus of the hTfR affinity peptide, either directly or via a linker sequence. Also, there are those in which an hTfR affinity peptide is bound to the C-terminus of HSA, either directly or via a peptide linker, and another protein (A) is further bound to the C-terminus of the hTfR affinity peptide, either directly or via a linker sequence. Also, there are those in which another protein (A) is bound to the C-terminus of HSA, either directly or via a peptide linker, and another hTfR affinity peptide is further bound to the C-terminus of the other protein (A), either directly or via a linker sequence. Proteins in which an hTfR immunoglobulin single variable domain peptide, another protein (A), and HSA are bound can be obtained as recombinant proteins, similar to hTfR immunoglobulin single variable domain peptide-protein (A) fusion proteins. Recombinant proteins obtained in this manner are also included in the hTfR affinity peptide-protein (A) fusion proteins.

[0085] The above-mentioned HSA is preferably a wild-type HSA having the amino acid sequence shown in SEQ ID NO: 38, but is not limited to this and may also be a mutant HSA in which substitutions, deletions, or additions have been made to the amino acid sequence of wild-type HSA, as long as it can improve the stability in blood of a substance bound to it.

[0086] Substances with affinity for albumin can also be used instead of HSA. Substances with affinity for albumin bind to albumin in the blood, and thus, similar to the use of HSA, can increase the half-life in the blood of fusion proteins of anti-hTfR heavy chain antibodies and other proteins (A) administered to the body. Examples of substances with affinity for albumin include peptides obtained by modifying the albumin-binding domain of a protein derived from Streptococcus strain G418, which has the amino acid sequence shown in SEQ ID NO: 39 (Alm T. Biotechnol J. 5. 605-17 (2010)), to exhibit alkali resistance.

[0087] The binding affinity of the hTfR affinity peptide-protein (A) fusion protein to albumin, into which the albumin affinity peptide has been introduced, is preferably 1×10 when measured by the biolayer interferometry described in Example 7. －７ M or less, more preferably 5X10 －７ M or less, more preferably 1X10 －８ M or less, and even more preferably 1X10 －９ It is below M.

[0088] Multiple hTfR affinity peptides can also be linked directly or via a peptide linker. The multiple hTfR affinity peptides may all have the same amino acid sequence, or they may have different amino acid sequences. The number of hTfR affinity peptides to be linked is not particularly limited, and may be, for example, 2 to 10, 2 to 5, 2, or 3. Linking multiple hTfR affinity peptides can confer properties such as improved affinity to anti-hTfR. In this specification, such linked multiple hTfR affinity peptides are referred to as tandem hTfR affinity peptides. Tandem hTfR affinity peptides are included in the term "hTfR affinity peptides." The same applies to anti-hTfR heavy chain antibody variable region peptides.

[0089] The peptide linker used to link the above-mentioned hTfR affinity peptide to the hIgG Fc region or HSA preferably consists of 1 to 50 amino acids. The number of amino acids is appropriately adjusted to 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, 27, etc. The amino acid sequence of such a peptide linker is not limited, so long as the hTfR affinity peptide linked thereto has affinity for hTfR and the hIgG Fc region or HSA linked thereto can exert the desired function under physiological conditions. However, a peptide linker preferably consists of glycine and serine. The same applies to peptide linkers used to link the hTfR affinity peptide, other protein (A), and hIgG Fc region. The same applies to peptide linkers used to link the hTfR affinity peptide, other protein (A), and HSA. The same also applies to peptide linkers used to link multiple hTfR affinity peptides.

[0090] Preferred peptide linkers include, for example, those consisting of a single amino acid, either glycine or serine, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequences set forth in SEQ ID NOs: 32, 33, 34, 35, 36, 58, and 69, or those containing 2 to 10 or 2 to 5 consecutive amino acids of these amino acid sequences. Such peptide linkers have sequences consisting of 2 to 50 amino acids, 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, or 27 amino acids. For example, those containing the amino acid sequence Gly-Ser can be suitably used as peptide linkers. Furthermore, those containing a sequence of 25 consecutive amino acids, consisting of five consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 32, can also be suitably used as peptide linkers.

[0091] hTfR affinity peptides and hTfR affinity peptide-protein (A) fusion proteins can also be stabilized in blood by methods other than those described above. For example, these proteins can be modified with PEG to increase their stability in blood. This method is commonly used in the field of protein medicine, and PEGylated erythropoietin, interferon, and other drugs have been commercialized. hTfR affinity peptides can also be stabilized by introducing mutations into them.

[0092] There are no particular limitations on the other protein (A) to be bound to the hTfR affinity peptide, but it should be a protein that can exert physiological activity in the body, and in particular, a protein that should reach the brain to exert its function, but cannot pass through the blood-brain barrier as is and is therefore not expected to function in the brain when administered intravenously. Examples of such proteins include iduronate-2-sulfatase (IDS), glucocerebrosidase (GBA), β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase (LAMAN), β-mannosidase, galactosylceramidase (GALC), saposin C, arylsulfatase A (ARSA), α-L-fucosidase (FUCA1), aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase (ASM), α-galactosylceramidase (GALC), and saposin C. These include lysosomal enzymes such as acetylsidase A, β-glucuronidase (GUSB), heparan N-sulfatase (SGSH), α-N-acetylglucosaminidase (NAGLU), acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase (AC), amylo-1,6-glucosidase, sialidase, aspartylglucosaminidase, palmitoyl protein thioesterase-1 (PPT-1), tripeptidyl peptidase-1 (TPP-1), hyaluronidase-1, acid α-glucosidase (GAA), CLN1, and CLN2. The same can be said for anti-hTfR heavy chain antibody variable region peptides.

[0093] hTfR affinity peptides conjugated to α-L-iduronidase (IDUA) are used to treat Hurler syndrome or Hurler-Scheie syndrome, those conjugated to iduronate-2-sulfatase (IDS) are used to treat Hunter syndrome, those conjugated to glucocerebrosidase (GBA) are used to treat Gaucher disease, those conjugated to β-galactosidase are used to treat GM1-gangliosidosis types 1-3, and those conjugated to GM2 activator protein are used to treat GM2-gangliosidosis AB variant. When bound to β-hexosaminidase A, it is used as a therapeutic agent for Sandhoff disease and Tisachs disease; when bound to β-hexosaminidase B, it is used as a therapeutic agent for Sandhoff disease; when bound to N-acetylglucosamine-1-phosphotransferase, it is used as a therapeutic agent for I-Cell disease; when bound to α-mannosidase (LAMAN), it is used as a therapeutic agent for α-mannosidosis; when bound to β-mannosidase, it is used as a therapeutic agent for β-mannosidosis; and when bound to galactosylceramidase ( When bound to α-L-fucosidase (FUCA1), it is used as a treatment for Krabbe disease; when bound to saposin C, it is used as a treatment for Gaucher-like storage disease; when bound to arylsulfatase A (ARSA), it is used as a treatment for metachromatic leukodystrophy; when bound to α-L-fucosidase (FUCA1), it is used as a treatment for fucosidosis; when bound to aspartylglucosaminidase, it is used as a treatment for aspartylglucosaminuria; and when bound to α-N-acetylgalactosaminidase, it is used as a treatment for Schindler disease. and Kawasaki disease, when bound to acid sphingomyelinase (ASM) it is used as a treatment for Niemann-Pick disease, when bound to α-galactosidase A it is used as a treatment for Fabry disease, when bound to β-glucuronidase (GUSB) it is used as a treatment for Sly syndrome, when bound to heparan N-sulfatase (SGSH) it is used as a treatment for Sanfilippo syndrome A, and when bound to α-N-acetylglucosaminidase (NAGLU) it is used as a treatment for Sanfilippo syndrome B,When combined with acetyl-CoA α-glucosaminide N-acetyltransferase and N-acetylglucosamine-6-sulfate sulfatase, they are used as a therapeutic agent for Sanfilippo syndrome. When combined with acid ceramidase (AC), they are used as a therapeutic agent for Farber disease. When combined with amylo-1,6-glucosidase, they are used as a therapeutic agent for Cori's disease (Forbes-Coli disease). When combined with sialidase, they are used as a therapeutic agent for central nervous system disorders in sialidase deficiency. Palmitoyl protein thioesterase-1 (PPT-1) Those bound to tripeptidyl peptidase-1 (TPP-1) can be used as a therapeutic agent for neuronal ceroid lipofuscinosis or Santavuori-Haltia disease, those bound to tripeptidyl peptidase-1 (TPP-1) can be used as a therapeutic agent for neuronal ceroid lipofuscinosis or Jansky-Bielschowsky disease, those bound to hyaluronidase-1 can be used as a therapeutic agent for hyaluronidase deficiency, those bound to acid α-glucosidase (GAA) can be used as a therapeutic agent for Pompe disease, and those bound to CLN1 and 2 can be used as a therapeutic agent for Batten disease. The hTfR affinity peptides bound to the above lysosomal enzymes can be used, in particular, as a therapeutic agent for central nervous system disorders in lysosomal diseases. For example, hTfR affinity peptides bound to α-L-iduronidase (IDUA) can be used as a therapeutic agent for central nervous system disorders in Hurler syndrome or Hurler-Scheie syndrome. Furthermore, the pharmaceutical uses are not limited to these diseases.

[0094] Other proteins that can exert their therapeutic effects when bound to hTfR affinity peptides include growth factors, antibody drugs, cytokines, nerve growth factor (NGF), brain-derived nerve growth factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and interleukin (IL-1). Examples of such antibodies include 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), tumor necrosis factor α receptor (TNF-αR), PD-1 ligand, PD-L1, PD-L2, enzymes with the activity of degrading beta-amyloid, anti-beta-amyloid antibodies, anti-BACE antibodies, anti-EGFR antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-HER2 antibodies, anti-TNF-α antibodies, and anti-CTLA-4 antibodies.

[0095] The hTfR affinity peptide bound to nerve growth factor (NGF) is used as a therapeutic agent for Alzheimer's disease, the one bound to brain-derived growth factor (BDNF) is used as a therapeutic agent for Huntington's disease, Alzheimer's disease, etc., the one bound to CNTF is used as a therapeutic agent for amyotrophic lateral sclerosis, the one bound to GDNF, neurotrophin 3, or neurotrophin 4 / 5 is used as a therapeutic agent for cerebral ischemia, the one bound to GDNF is used as a therapeutic agent for Parkinson's disease, the one bound to neuregulin 1 is used as a therapeutic agent for schizophrenia, and the one bound to erythropoietin or darbepoietin is used as a therapeutic agent for schizophrenia. those bound to bFGF or FGF2 can be used as a therapeutic agent for cerebral ischemia, respectively; those bound to an enzyme having beta-amyloid degrading activity, anti-beta-amyloid antibody or anti-BACE antibody can be used as a therapeutic agent for Alzheimer's disease; those bound to anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-HER2 antibody or anti-CTLA-4 antibody can be used as a therapeutic agent for central nervous system tumors including brain tumors; and those bound to TNFαR can be used as a therapeutic agent for cerebral ischemia and cerebral inflammatory diseases.

[0096] Other proteins (A) that can be fused to hTfR affinity peptides generally include therapeutic agents for diseases such as neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease), psychiatric disorders (e.g., schizophrenia, depression), multiple sclerosis, amyotrophic lateral sclerosis, central nervous system tumors (including brain tumors), lysosomal diseases associated with encephalopathy, glycogen storage diseases, muscular dystrophies, cerebral ischemia, cerebral inflammatory diseases, prion diseases, and traumatic central nervous system disorders. Furthermore, therapeutic agents for viral and bacterial central nervous system diseases generally can be other proteins (A) that can be fused to hTfR affinity peptides. Furthermore, drugs that can be used for recovery after brain surgery or spinal surgery generally can be other proteins (A) that can be fused to hTfR affinity peptides.

[0097] In addition, the therapeutic agents used in the present invention can also be used to prevent the onset of diseases.

[0098] The proteins listed above as other proteins (A) to be bound to the hTfR affinity peptide are usually wild-type proteins. However, mutants in which one or more amino acids constituting these wild-type proteins have been substituted with other amino acids or deleted, etc., are also included in these proteins as long as they retain the original physiological activity of the protein. Here, "a protein having the original physiological activity" means that the protein has 20% or more of the physiological activity of the wild-type protein. The physiological activity of the protein is preferably 40% or more, more preferably 50% or more, even more preferably 80% or more, and even more preferably 90% or more of the physiological activity of the wild-type protein.

[0099] The amino acid sequence of the mutant protein preferably has an identity of 80% or more, more preferably 85% or more, even more preferably 90% or more, still more preferably 95% or more, for example, 98% or more, to the amino acid sequence of the corresponding wild-type other protein (A).

[0100] When the amino acid sequence of the wild-type other protein (A) is substituted with other amino acids to create a mutant, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids are deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, a desired mutant can be created by combining these amino acid substitutions and deletions. Furthermore, proteins in which one or more amino acids have been added to the amino acid sequence or to the N-terminus or C-terminus of the wild-type other protein (A) or its mutant are also included as long as they retain the functions of these proteins completely or partially. The number of added amino acids in this case is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. A desired mutant can also be created by combining these amino acid additions, substitutions, and deletions.

[0101] In addition, if a mutation is made into these other proteins (A) to add an amino acid to the C-terminus or N-terminus, and the added amino acid is located between these proteins and the hTfR affinity peptide when these proteins are fused with the hTfR affinity peptide, the added amino acid will be considered to constitute part of the other protein (A).

[0102] When the other protein (A) is human IDS, suitable fusion proteins include the following (1) to (4): (1) a fusion protein in which the N-terminus of hIDS is bound to the C-terminus of an hTfR affinity peptide consisting of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:55 via a peptide linker having the amino acid sequence of SEQ ID NO:59; (2) a fusion protein consisting of the amino acid sequence of SEQ ID NO:62 in which the N-terminus of hIDS is bound to the C-terminus of hTfR affinity peptide 5 consisting of the amino acid sequence of SEQ ID NO:7 via a peptide linker having the amino acid sequence of SEQ ID NO:59; (3) a fusion protein consisting of the amino acid sequence of SEQ ID NO:60 in which the N-terminus of hIDS is bound to the C-terminus of hTfR affinity peptide 6 consisting of the amino acid sequence of SEQ ID NO:55 via a peptide linker having the amino acid sequence of SEQ ID NO:59; (4) A fusion protein consisting of the amino acid sequence of SEQ ID NO: 64, in which the N-terminus of hTfR affinity peptide 5 consisting of the amino acid sequence of SEQ ID NO: 7 is bound to the C-terminus of hTfR affinity peptide 5 consisting of the amino acid sequence of SEQ ID NO: 7 via a peptide linker having the amino acid sequence of SEQ ID NO: 59, and the N-terminus of hIDS is further bound to the C-terminus of the N-terminus of hTfR affinity peptide 5 via a peptide linker having the amino acid sequence GS.

[0103] When the other protein (A) is human SGSH, suitable fusion proteins include the following: (1) a fusion protein in which the N-terminus of hSGSH is linked to the C-terminus of an hTfR affinity peptide consisting of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:55 via a peptide linker having the amino acid sequence of SEQ ID NO:59, (2) a fusion protein in which the N-terminus of hSGSH consisting of the amino acid sequence of SEQ ID NO:66 is linked to the C-terminus of an hTfR affinity peptide 3 consisting of the amino acid sequence of SEQ ID NO:5 via a peptide linker having the amino acid sequence of SEQ ID NO:59.

[0104] Trastuzumab is an antibody drug that exerts antitumor effects by specifically binding to HER2, which is encoded by the human oncogene HER2 / neu (c-erbB-2). Trastuzumab consists of a heavy chain and a light chain, the amino acid sequences of which are shown in SEQ ID NOs: 40 and 41, respectively.

[0105] A specific example of a fusion protein of an hTfR affinity peptide and another protein (A) in the present invention is a fusion protein of an hTfR affinity peptide and trastuzumab, in which hTfR affinity peptide 1 is linked to the C-terminus of the trastuzumab heavy chain via a linker. The fusion protein of hTfR affinity peptide 1 and trastuzumab can be produced by culturing host cells into which an expression vector for a protein having the amino acid sequence of SEQ ID NO: 42 (hTfR affinity peptide 1-trastuzumab heavy chain fusion protein) in which hTfR affinity peptide 1 having the amino acid sequence of SEQ ID NO: 3 is linked to the C-terminus of the trastuzumab heavy chain via a peptide linker having the amino acid sequence of SEQ ID NO: 69, and an expression vector for a trastuzumab light chain protein have been introduced. Fusion proteins of other embodiments of hTfR affinity peptides and trastuzumab can also be produced by using, instead of an expression vector for the hTfR affinity peptide 1-trastuzumab heavy chain fusion protein, an expression vector for an hTfR affinity peptide 2-trastuzumab heavy chain fusion protein having the amino acid sequence of SEQ ID NO: 43, an hTfR affinity peptide 3-trastuzumab heavy chain fusion protein having the amino acid sequence of SEQ ID NO: 44, an hTfR affinity peptide 4-trastuzumab heavy chain fusion protein having the amino acid sequence of SEQ ID NO: 45, or an hTfR affinity peptide 5-trastuzumab heavy chain fusion protein having the amino acid sequence of SEQ ID NO: 46. An expression vector for the hTfR affinity peptide 6-trastuzumab heavy chain fusion protein can also be used.

[0106] Trastuzumab is primarily used as a treatment for breast cancer, but because it cannot cross the BBB when administered intravenously, it is ineffective against cancers of the central nervous system. However, by combining trastuzumab with an anti-hTfR heavy chain antibody variable region peptide, it becomes able to cross the BBB, enabling it to act against cancers of the central nervous system that express HER2. Because breast cancer can metastasize to the central nervous system, trastuzumab is expected to be effective against cancers of the central nervous system that have metastasized from breast cancer.

[0107] Antibody drugs other than trastuzumab can also be produced as fusion proteins with anti-hTfR heavy chain antibody variable region peptides in the same manner as trastuzumab. In this case, the anti-hTfR heavy chain antibody variable region peptide may be bound to the C-terminus of the antibody heavy chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody light chain, or the N-terminus of the antibody light chain.

[0108] The drug to be bound to the hTfR affinity peptide to form a drug conjugate may be a low molecular weight compound. The low molecular weight compound is preferably a compound that passes through the BBB and acts on the central nervous system, but is not limited thereto and may also be a contrast agent, a fluorescent dye, a phospholipid that can be a component of liposomes, a functional lipid that can be a component of lipid nanoparticles, or the like. The molecular weight of the low molecular weight compound is, for example, 1 to 50 kD, 0.5 to 20 kD, or 1 to 5 kD. Peptides consisting of 5 to 20 amino acids and polysaccharides with a structure formed by dehydration condensation of 5 to 20 monosaccharides are also included in the low molecular weight compound.

[0109] The hTfR affinity peptide and the low molecular weight compound are covalently bonded directly or via a linker. The binding mode between the hTfR affinity peptide and the low molecular weight compound is not particularly limited, as long as the binding affinity of the hTfR affinity peptide to hTfR is maintained and the drug exerts its medicinal effect in the central nervous system when the hTfR affinity peptide-drug conjugate is administered in vivo. Suitable linkers include, for example, linkers containing valine-citrulline or valine-alanine, which are cleaved by cathepsin B, and linkers containing cysteine-cysteine. When administered in vivo, such linkers can be cleaved by in vivo metabolic mechanisms, such as enzymes, allowing the low molecular weight compound to cleave from the hTfR affinity peptide and exert its medicinal effect independently. In this specification, such linkers that can be cleaved in vivo are referred to as biodegradable linkers.

[0110] When the hTfR affinity peptide and the low molecular weight compound are linked via a biodegradable linker, a spacer can be placed between them in addition to the linker. The placement of the spacer improves the accessibility of the enzyme complex that cleaves the linker. Examples of such spacers include, but are not limited to, p-aminocarbamate / carbamate groups.

[0111] The drug in the hTfR affinity peptide-drug conjugate may be a nucleic acid. Here, the nucleic acid may be any of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA. To prevent in vivo nuclease degradation of nucleic acids, the nucleotides constituting these nucleic acids may be modified to make them nuclease-resistant. The length of the nucleic acid is not particularly limited, but is, for example, 10 bp to 1000 bp, or 10 bp to 100 bp. In this conjugate, the hTfR affinity peptide and nucleic acid are covalently linked directly or via a linker. The binding mode between the immunoglobulin single variable domain and the low-molecular-weight compound is not particularly limited, as long as the binding affinity of the hTfR affinity peptide to hTfR is maintained and the drug exerts its pharmacological effect in the central nervous system when the hTfR affinity peptide-nucleic acid conjugate is administered in vivo. A biodegradable linker is preferably used as the linker.

[0112] When the hTfR affinity peptide and nucleic acid are linked via a biodegradable linker, a spacer can be placed between them in addition to the linker. The placement of the spacer increases the accessibility of the nucleic acid complex to cleave the linker. Suitable examples of such spacers include, but are not limited to, a p-aminocarbamate group / carbamate group.

[0113] The nucleic acid to be bound to the hTfR affinity peptide is, for example, a nucleic acid used in the treatment of central nervous system diseases. Such a nucleic acid is an antisense nucleic acid against an mRNA whose expression is abnormally increased in the central nervous system. Such an antisense nucleic acid binds to the abnormally expressed mRNA and inhibits its translation into protein.

[0114] The hTfR affinity peptide-drug conjugate (including the hTfR affinity peptide-protein (A) fusion protein) can be supplied to medical institutions as a drug (pharmaceutical composition) containing the conjugate as an active ingredient in the form of a lyophilized product or aqueous liquid. In the case of aqueous liquid, the hTfR affinity peptide-drug conjugate can be pre-dissolved in a solution containing a stabilizer, buffer, and isotonicity agent and then sealed in a vial or syringe. Preparations sealed in a syringe are generally called pre-filled syringe preparations. Pre-filled syringe preparations can simplify self-administration of the drug by patients. Drug administration is performed parenterally, for example, by intravenous injection. The administered drug can pass through the blood-brain barrier and exert its medicinal effects in the central nervous system, for example, in the cerebral cortex, cerebellum, hippocampus, midbrain, pons, medulla oblongata, caudate nucleus, nucleus pallidus, thalamus, and hypothalamus, and in particular, in the cerebral cortex, cerebellum, hippocampus, and midbrain.

[0115] hTfR affinity peptides can be used to modify the surface of vesicles that can encapsulate drugs. Examples of vesicles include liposomes and lipid nanoparticles (LNPs). Liposomes are spherical vesicles with a lipid bilayer and generally contain phospholipids, particularly phosphatidylcholine, as their constituent components. Lipid nanoparticles may also contain other lipids, such as egg yolk phosphatidylethanolamine. Lipid nanoparticles are lipid-based particles with diameters ranging from 10 nm to 1000 nm, typically less than approximately 200 nm. They can encapsulate hydrophobic (lipophilic) molecules and are primarily composed of biocompatible lipids, such as triglycerides, diglycerides, monoglycerides, fatty acids, and steroids. Drugs can be encapsulated in these vesicles.

[0116] When drug-encapsulated vesicles are administered into the body, the membranes of the vesicles fuse with the cell membrane, releasing the drug into the cells. In other words, these vesicles can be used as drug carriers. When an hTfR-affinity peptide is attached to the surface of such vesicles, the vesicles bind to hTfR on cerebral vascular endothelial cells that make up the blood-brain barrier via the peptide. The vesicles then fuse with the cell membrane of the cerebral vascular endothelial cells, or are taken up by the cells via endocytosis, releasing the encapsulated drug into the brain parenchyma, such as the cerebellar parenchyma.

[0117] In this specification, the term "vesicles" includes not only the above-mentioned liposomes and lipid nanoparticles, but also polymer nanoparticles, micelles, emulsions, nanoemulsions, microspheres, nanospheres, microcapsules, nanocapsules, dendrimers, nanogels, metal nanoparticles, and any other nano- or microparticles that can be used as a drug delivery system (DDS).

[0118] The present invention will be described in more detail below with reference to examples, but it is not intended that the present invention be limited to these examples.

[0119] Example 1: Construction of a vector for hTfR expression A gene fragment encoding the human transferrin receptor (hTfR) was amplified by PCR using human spleen Quick Clone cDNA (Clontech) as a template and primers hTfR5' (SEQ ID NO: 49) and hTfR3' (SEQ ID NO: 50). The amplified gene fragment encoding hTfR was digested with MluI and NotI and inserted between the MluI and NotI sites of the pCI-neo vector (Promega). The resulting vector was designated pCI-neo(hTfR). Next, this vector was digested with MluI and NotI to excise the gene fragment encoding hTfR. The excised fragment was then inserted between the MluI and NotI sites of pE-mIRES-GS-puro, an expression vector described in International Publication WO 2012 / 063799, to construct pE-mIRES-GS-puro(hTfR), an hTfR expression vector.

[0120] [Example 2] Preparation of recombinant hTfR pE-mIRES-GS-puro(hTfR) was introduced into CHO-K1 cells by electroporation, and then the cells were transfected with CD OptiCHO containing methionine sulfoximine (MSX) and puromycin. ＴＭ The cells were selectively cultured using a medium (Invitrogen) to obtain recombinant hTfR-expressing cells, which were then cultured to prepare recombinant hTfR (rhTfR).

[0121] Example 3: Construction of a phage library. Alpacas were immunized using the rhTfR prepared in Example 2 as an antigen. Blood was collected from the immunized alpacas, and peripheral blood lymphocytes were isolated from the blood. RNA was extracted from the peripheral blood lymphocytes and reverse-transcribed to obtain cDNA. Using the obtained cDNA as a template, DNA fragments containing the base sequence encoding the variable region of the heavy chain antibody were amplified by PCR. The amplified DNA fragments were incorporated into phagemids so that the variable region of the heavy chain antibody would be displayed on the surface of the phage when packaged into the phage. These phagemids were then introduced into Escherichia coli by electroporation. The phagemid-introduced Escherichia coli was further infected with helper phage (M13K07), and the Escherichia coli were then cultured to release the phage into the culture medium. After the culture was completed, the medium was centrifuged to recover the culture supernatant containing the phage. This recovered culture supernatant containing the phage was used as a phage library. The concentration of the phage library was 3.36 × 10 13 PFU / mL.

[0122] Example 4: Isolation of anti-hTfR heavy chain antibody-expressing phage clones Phages expressing the variable region of a heavy chain antibody that specifically binds to hTfR (anti-hTfR heavy chain antibody-expressing phage) were cloned from the phage library prepared in Example 3 by biopanning. Cloning was generally performed as follows: A solution containing rhTfR prepared in Example 2 was added to each well of a microtiter plate to immobilize rhTfR to the plate. Each well was then washed three times with 0.05% PBST solution, after which a blocking agent was added to each well to block each well. Each well was then washed three times with 0.05% PBST solution, and the phage library diluted with the blocking agent was added to each well. The plate was then left to stand, allowing phages bearing anti-hTfR heavy chain antibodies on their surface to bind to rhTfR. Each well was then washed five times with 0.05% PBST solution to remove phages that did not bind to rhTfR. Next, the phage bound to rhTfR was dissociated from rhTfR by adding an acidic elution solution and collected as a solution.

[0123] The collected solution was diluted and added to a solution containing E. coli, and the phage was allowed to infect the E. coli. The phage-infected E. coli was then further infected with helper phage (M13K07), and the E. coli was then cultured to release the phage into the culture medium. The phage was purified using a PEG solution.

[0124] E. coli was infected with the purified phages and then plated on an agar medium. E. coli that formed single colonies on the medium were collected and further cultured in the medium, after which they were infected with helper phage (M13K07) to release the phages into the culture medium. The phages released into the culture medium were collected as anti-hTfR heavy chain antibody-expressing phage clones in the culture supernatant obtained by centrifugation of the culture medium. Each phage clone contained in the culture supernatant was purified using PEG solution.

[0125] Example 5: Determination of the amino acid sequence of the variable region of the anti-hTfR heavy chain antibody expressed by each anti-hTfR heavy chain antibody-expressing phage clone. Five clones were selected from the anti-hTfR heavy chain antibody-expressing phage isolated in Example 4 and designated clones 1 to 5. Phagemids were purified from each clone, and the nucleotide sequence of the variable region of the anti-hTfR heavy chain antibody encoded by each purified phagemid was determined. Based on this, the amino acid sequence of the variable region of the anti-hTfR heavy chain antibody expressed by each clone was determined (Table 1).

[0126]

[0127] Here, the protein having the amino acid sequence shown in SEQ ID NO: 3 was designated hTfR affinity peptide 1, the protein having the amino acid sequence shown in SEQ ID NO: 4 was designated hTfR affinity peptide 2, the protein having the amino acid sequence shown in SEQ ID NO: 5 was designated hTfR affinity peptide 3, the protein having the amino acid sequence shown in SEQ ID NO: 6 was designated hTfR affinity peptide 4, and the protein having the amino acid sequence shown in SEQ ID NO: 7 was designated hTfR affinity peptide 5. hTfR affinity peptides 1 to 5 each contain CDRs 1 to 3. The amino acid sequences of each CDR are shown in Table 2. The amino acid sequence of the CDR of hTfR affinity peptide 5 shown in Table 2 is the same as that of hTfR affinity peptide 3. Table 2 also shows the amino acid sequences of each CDR of hTfR affinity peptide 6, which will be described later in Example 6.

[0128]

[0129] Example 6: Preparation of hTfR affinity peptides 1 to 6 A DNA fragment containing a gene encoding hTfR affinity peptide 1 with a peptide tag shown in SEQ ID NO:54 attached to the N-terminus was artificially synthesized and inserted between the MluI and NotI sites of the pCI-neo Mammalian Expression Vector to create an hTfR affinity peptide 1 expression vector. This expression vector was introduced into CHO-S cells using the ExpiCHO Expression System (ThermoFisher Scientific) according to the attached protocol, and hTfR affinity peptide 1 was expressed in the CHO-S cells. The cells expressed hTfR affinity peptide 1 and secreted it into the culture medium. The cells were cultured in the presence of neomycin according to the attached protocol. Specifically, at a concentration of 6 x 10 6 CHO-S cells were suspended in 15 mL of medium at a concentration of 1 / mL and transferred to a 150 mL Erlenmeyer flask. The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air at a stirring speed of approximately 120 rpm for 1 day. The feed and enhancer solutions included with the ExpiCHO Expression System were added, and the cells were cultured at 32°C in a humidified atmosphere of 5% CO2 and 95% air at a stirring speed of approximately 120 rpm for 9 days to express hTfR affinity peptide 1. After the culture was completed, the culture medium was centrifuged (3000 g, 5 minutes), and the resulting supernatant was filtered through a 0.22 μm filter (Millipore) and collected as the culture supernatant. hTfR affinity peptide 1 was purified from the culture supernatant using the following method.

[0130] The collected culture supernatant was passed through an Anti-FLAG M1 Agarose Affinity Gel column (Sigma-Aldrich) using 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl and 1 mM CaCl2 (5 column volumes) as an equilibration solution. The culture supernatant was then loaded onto the column, and hTfR affinity peptide 1 was adsorbed onto the affinity column. The column was then washed with 5 column volumes of equilibration solution. hTfR affinity peptide 1 was eluted by passing the eluent (50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl) through the column. The eluted hTfR affinity peptide 1 was used as a purified product in the following experiments. Purified products of hTfR affinity peptides 2 to 5 were also obtained in the same manner as for hTfR affinity peptide 1. Furthermore, an expression vector encoding hTfR affinity peptide 6, which has the amino acid sequence shown in SEQ ID NO: 55, in which one amino acid in CDR2 of hTfR affinity peptide 5 was replaced, was prepared by standard methods, and a purified product of hTfR affinity peptide 6 was obtained using this vector in the same manner as hTfR affinity peptides 1 to 5.

[0131] Example 7: Evaluation of the binding activity of hTfR affinity peptides 1-6 to human TfR and monkey TfR. The binding activity of hTfR affinity peptides 1-5 to human TfR (hTfR) and monkey TfR was measured using the OctetRED96 (ForteBio, a division of Pall Corporation), a biomolecular interaction analysis system that employs biolayer interferometry (BLI). The basic principle of biolayer interferometry is briefly explained below. When light of a specific wavelength is projected onto a layer of biomolecules immobilized on the surface of a sensor chip, light is reflected from both the biomolecular layer and an internal reference layer, generating an optical interference wave. When molecules in the measurement sample bind to the biomolecules on the sensor chip surface, the thickness of the layer at the sensor tip increases, resulting in a wavelength shift in the interference wave. By measuring this change in wavelength shift, the number of molecules binding to the biomolecules immobilized on the sensor chip surface can be quantified and kinetically analyzed in real time. Measurements were performed generally according to the operating manual provided with the OctetRED96. The human TfR used was recombinant hTfR (rhTfR, Sino Biological Co., Ltd.) with an N-terminal histidine tag and the amino acid sequence of the extracellular domain of hTfR, which is from the 89th cysteine ​​residue from the N-terminus to the C-terminal phenylalanine in the amino acid sequence shown in SEQ ID NO: 1. The monkey TfR used was recombinant monkey TfR (rmonkey TfR, Sino Biological Co., Ltd.) with an N-terminal histidine tag and the amino acid sequence of the extracellular domain of cynomolgus monkey TfR, which is from the 89th cysteine ​​residue from the N-terminus to the C-terminal phenylalanine in the amino acid sequence shown in SEQ ID NO: 2.

[0132] The purified hTfR affinity peptides 1 to 6 obtained in Example 6 were each diluted with HBS-P+ (10 mM HEPES containing 150 mM NaCl, 1% BSA, 50 μM EDTA, and 0.05% Surfactant P20) to prepare solutions with three concentrations ranging from 10 to 40 nM. These solutions were used as sample solutions. rhTfR and rmonkey TfR were each diluted with HBS-P+ to prepare 15 μg / mL solutions, which were used as hTfR-ECD (Histag) solutions and monkey TfR-ECD (Histag) solutions, respectively.

[0133] The sample solution was dispensed into a 96-well black plate (Greiner Bio-One) at 200 μL per well. The prepared hTfR-ECD (Histag) solution and monkey TfR-ECD (Histag) solution were dispensed into the designated wells at 200 μL per well. HBS-P+ was dispensed into the baseline, dissociation, and washing wells at 200 μL per well. 10 mM Glycine-HCl (pH 1.7) was dispensed into the regeneration wells at 200 μL per well. 0.5 mM NiCl2 solution containing 1% BSA was dispensed into the activation wells at 200 μL per well. The plate and a biosensor (Biosensor / Ni-NTA: ForteBio, a division of Pall Corporation) were placed in the designated positions on the OctetRED96.

[0134] After acquiring data by operating OctetRED96 under the conditions shown in Table 3 below, the binding reaction curves were fitted to a 1:1 binding model or a 2:1 binding model using the analysis software provided with OctetRED96. The association rate constants (k) and dissociation rate constants (k) for each of the hTfR affinity peptides 1 to 6, hTfR, and monkey TfR were measured, and the dissociation constants (K D The measurements were carried out at a temperature of 25 to 30°C.

[0135]

[0136] Table 4 shows the association rate constants (k) and dissociation rate constants (koff) of hTfR affinity peptides 1 to 6 for hTfR and monkey TfR, as well as the dissociation constants (K D For hTfR affinity peptides 1 and 2, KD values ​​for monkey TfR could not be obtained. For these peptides, KD values ​​were separately measured using Biacore using the hTfR-ECD (Histag) solution and monkey TfR-ECD (Histag) solution prepared in Example 2. D As a result, the K values ​​of hTfR affinity peptide 1 for monkey TfR and human TfR were D The values ​​are 1.76×10 -8 and 1.65 x 10 -8 The KD values ​​of hTfR affinity peptide 2 for monkey TfR and human TfR were measured to be 1.76 × 10 -8 and 1.65 x 10 -8 These results indicate that the KD value of each hTfR-affinity peptide with monkey TfR is approximately 1.0 × 10 -8 ~5.0×10 -５ The KD value with human TfR is approximately 1.0 × 10 -8 ~1.0×10 -7 This indicates that

[0137]

[0138] Based on the above results, the relative dissociation constants of each hTfR affinity peptide with monkey TfR were calculated, assuming the dissociation constant with hTfR of each peptide to be 1 (Table 5). The relative values ​​for all hTfR affinity peptides were in the range of 0.8 to 2.5. These results indicate that the affinity for hTfR is similar to that for monkey TfR. In particular, the relative values ​​for all hTfR affinity peptides, except for hTfR affinity peptide 2, were in the range of 0.8 to 1.3, indicating that the affinity for hTfR is very similar to that for monkey TfR. Therefore, it is reasonable to expect that the pharmacokinetics of all these hTfR affinity peptides (especially hTfR affinity peptides 1 and 3-5) when administered to monkeys will be very similar to that when administered to humans. Therefore, when applying these peptides to drug development, some nonclinical studies, such as pharmacokinetic studies, can be performed in monkeys, significantly facilitating the development of such drugs.

[0139]

[0140] Example 8: Preparation of hTfR affinity peptide-trastuzumab expression vector pCI-neo Mammalian Expression Vector (Promega) was digested with MluI and NotI. A DNA fragment was artificially synthesized containing an MluI site on the 5' end and a NotI site on the 3' end of a gene having the nucleotide sequence shown in SEQ ID NO:47 encoding a protein having the amino acid sequence shown in SEQ ID NO:42 in which hTfR affinity peptide 1 was linked to the C-terminus of a trastuzumab heavy chain having the amino acid sequence shown in SEQ ID NO:38 via a peptide linker having the amino acid sequence shown in SEQ ID NO:69. This DNA fragment was digested with MluI and NotI and inserted between the MluI and NotI sites of the pCI-neo Mammalian Expression Vector to prepare an expression vector for a protein in which hTfR affinity peptide 1 and trastuzumab heavy chain are linked. This expression vector was designated pCI-neo-Trastuzumab(HC)-VHH(1). Similarly, DNA fragments containing genes encoding proteins in which hTfR affinity peptides 2 to 5 are bound to the trastuzumab heavy chain were artificially synthesized. These DNA fragments were digested with MluI and NotI and inserted into the MluI and NotI sites of the pCI-neo Mammalian Expression Vector to create expression vectors for proteins in which one of hTfR affinity peptides 2 to 5 is bound to the trastuzumab heavy chain. The resulting expression vectors were named pCI-neo-Trastuzumab(HC)-VHH(2) to pCI-neo-Trastuzumab(HC)-VHH(5), respectively.

[0141] Furthermore, a DNA fragment having an MluI site on the 5' side and a NotI site on the 3' side of a gene having the nucleotide sequence shown in SEQ ID NO:48, which encodes the light chain of trastuzumab having the amino acid sequence shown in SEQ ID NO:41, was artificially synthesized, digested with MluI and NotI, and ligated with the previously prepared pCI-neo Mammalian Expression Vector (Promega) digested with MluI and NotI to construct an expression vector for the trastuzumab light chain. This expression vector was designated pCI-neo-Trastuzumab(LC).

[0142] Example 9: Preparation of hTfR affinity peptide-Trastuzumab The pCI-neo-Trastuzumab(HC)-VHH(1) and pCI-neo-Trastuzumab(LC) prepared in Example 8 were simultaneously transfected into CHO-S cells using the ExpiCHO Expression System (ThermoFisher Scientific) according to the attached protocol, and a protein composed of the hTfR affinity peptide 1 and trastuzumab heavy chain bound thereto and the trastuzumab light chain were co-expressed in the CHO-S cells. These cells express a protein composed of the hTfR affinity peptide and trastuzumab fused thereto, which is secreted into the culture medium. Hereinafter, this fusion protein will be referred to as hTfR affinity peptide-Trastuzumab 1. These cells were cultured in the presence of neomycin according to the attached protocol. Specifically, at a concentration of 6 × 10 6 CHO-S cells were suspended in 15 mL of medium at a density of cells / mL and transferred to a 150 mL Erlenmeyer flask. The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air at a stirring speed of approximately 120 rpm for one day. The feed and enhancer solutions included with the ExpiCHO Expression System were added, and the cells were cultured at 32°C in a humidified atmosphere of 5% CO2 and 95% air at a stirring speed of approximately 120 rpm for nine days to express the hTfR-affinity peptide, trastuzumab 1. After culturing, the culture medium was centrifuged (3000 g, 5 minutes), and the resulting supernatant was filtered through a 0.22 μm filter (Millipore) and collected as the culture supernatant.

[0143] An open column packed with 1 mL of MabSelect SuRe LX (Cytiva) was equilibrated by passing through 5 column volumes of 25 mM HEPES buffer (pH 7.5) containing 50 mM NaCl. The culture supernatant was then loaded onto the column, and the hTfR affinity peptide-trastuzumab 1 was bound to the resin. The column was washed with 5 column volumes of 25 mM HEPES buffer (pH 7.5) containing 50 mM NaCl, and then the hTfR affinity peptide-trastuzumab 1 was eluted with 150 mM glycine (pH 3.0) containing 100 mM NaCl. The pH of the eluate containing the hTfR affinity peptide-trastuzumab 1 was adjusted to neutral by adding 5% of 1 M Tris-HCl (pH 8.0). This was subjected to buffer exchange using a PD-10 column (Cyteva) and the solvent was replaced with PBS(-).The resulting solution was used in the following experiments as a purified product of the hTfR affinity peptide-trastuzumab 1.

[0144] As with pCI-neo-Trastuzumab(HC)-VHH(1), pCI-neo-Trastuzumab(HC)-VHH(2) to pCI-neo-Trastuzumab(HC)-VHH(5) were co-transfected with pCI-neo-Trastuzumab(LC) into CHO-S cells, and proteins composed of hTfR affinity peptides 2 to 5 bound to the trastuzumab heavy chain and the trastuzumab light chain were co-expressed in the CHO-S cells. These cells express proteins composed of trastuzumab fused to one of hTfR affinity peptides 2 to 4, and secrete these proteins into the culture medium. Hereinafter, the fusion proteins expressed by these cells are referred to as hTfR affinity peptide-trastuzumab 2 to 5. Furthermore, hTfR affinity peptide-trastuzumab 1 to 5 are collectively referred to as hTfR affinity peptide-trastuzumab. For the hTfR affinity peptide-trastuzumab 2 to 5, purified products were obtained in the same manner as for the hTfR affinity peptide-trastuzumab 1.

[0145] Example 10 Evaluation of the binding activity of hTfR affinity peptide-trastuzumab to human TfR and monkey TfR The affinity of the hTfR affinity peptides-trastuzumab 1 to 5 prepared in Example 9 to hTfR and monkey TfR was examined according to the method described in Example 7. Table 6 shows the measurement results of the association rate constant (k) and dissociation rate constant (koff) of the hTfR affinity peptides-trastuzumab 1 to 5 to hTfR and monkey TfR, as well as the dissociation constant (K D ) is shown.

[0146]

[0147] Measurement of the binding activity of hTfR affinity peptide-trastuzumab to hTfR showed that all hTfR affinity peptide-trastuzumab had a dissociation constant (K D ) is 1X10 -9 M to 1X10 -10 The dissociation constant with monkey TfR was also 1x10 -9 M to 1X10 -10 These results indicate that these hTfR-affinity peptide-trastuzumab maintain their affinity for both hTfR and monkey TfR even when fused to other proteins.

[0148] From the above results, the relative dissociation constants of each hTfR affinity peptide-trastuzumab with monkey TfR were calculated, assuming the dissociation constant with hTfR of each hTfR affinity peptide-trastuzumab was 1 (Table 7). For all hTfR affinity peptide-trastuzumab, the relative values ​​ranged from 0.9 to 2.5. These results indicate that the affinity of each hTfR affinity peptide-trastuzumab for hTfR is similar to that for monkey TfR. In particular, for all hTfR affinity peptides except for each hTfR affinity peptide-trastuzumab 2, the relative values ​​ranged from 0.8 to 1.9, indicating that the affinity for hTfR is very similar to that for monkey TfR. Therefore, it is naturally predicted that the pharmacokinetics of each of these hTfR affinity peptide-trastuzumabs (especially hTfR affinity peptide-trastuzumabs 1 and 3-5) when administered to monkeys will be extremely similar to the pharmacokinetics when administered to humans. Therefore, when these are applied to develop a drug, some of the non-clinical tests, such as pharmacokinetic tests, of the drug can be conducted using monkeys, which will significantly accelerate the development of the drug.

[0149]

[0150] Example 11 Evaluation of brain delivery of hTfR affinity peptide-trastuzumab using hTfR knock-in mice 1 Next, whether hTfR affinity peptide-trastuzumab 1 to 5 cross the BBB and deliver to the brain when intravenously injected was evaluated using hTfR knock-in mice (hTfR-KI mice) in which the gene encoding the extracellular domain of the mouse transferrin receptor was replaced with a gene encoding the extracellular domain of the human transferrin receptor gene. The hTfR-KI mice were generated roughly by the method described below.

[0151] A DNA fragment having the nucleotide sequence shown in SEQ ID NO:51 was chemically synthesized. The cDNA encoding a chimeric hTfR, whose intracellular domain is the amino acid sequence of mouse hTfR and whose extracellular domain is the amino acid sequence of human hTfR, contained a neomycin resistance gene flanked by loxP sequences at the 3' end. This DNA fragment was incorporated into a targeting vector having the nucleotide sequence shown in SEQ ID NO:52 as the 5' arm sequence and the nucleotide sequence shown in SEQ ID NO:53 as the 3' arm sequence, using standard methods, and then introduced into mouse ES cells by electroporation. After gene transfer, the mouse ES cells were selectively cultured in the presence of neomycin, and mouse ES cells in which the targeting vector had been integrated into the chromosome by homologous recombination were selected. The resulting genetically modified mouse ES cells were injected into 8-cell ICR mouse embryos (host embryos) and transplanted into pseudopregnant mice (recipient mice) obtained by mating with vasoligated mice. The resulting offspring (chimeric mice) were judged for coat color, and individuals in which ES cells contributed highly efficiently to the formation of the organism, i.e., individuals with a high proportion of white hair relative to their total hair, were selected. These chimeric mice were crossed with ICR mice to obtain F1 mice. White F1 mice were selected, and DNA extracted from tail tissue was analyzed. Mice in which the mouse transferrin receptor gene on the chromosome had been replaced with the chimeric hTfR were designated hTfR-KI mice.

[0152] The purified hTfR affinity peptide-trastuzumab 1 to 5 products obtained in Example 9 were intravenously administered to two male hTfR-KI mice at 5 mg / kg each. As a negative control, trastuzumab was intravenously administered to two male hTfR-KI mice at 5 mg / kg. Six and 24 hours after administration, the mice were euthanized, perfused whole body with saline, and their brains (including the cerebrum and cerebellum) were collected. The weights (wet weights) of the excised brains were measured, and the brains were divided in the sagittal plane. One portion was used to measure the concentrations of hTfR affinity peptide-trastuzumab 1 to 5 in the tissue, and the other portion was used for immunohistochemical staining.

[0153] Brain tissue samples for concentration measurement were homogenized in T-PER (Thermo Fisher Scientific) containing Protease Inhibitor Cocktail (Sigma-Aldrich). The resulting homogenate was centrifuged (3000 g, 5 min) to collect the supernatant. The amounts of hTfR affinity peptide-trastuzumab and trastuzumab in the homogenate supernatant were measured as follows: First, equal volumes of SULFO-Goat Anti-Human IgG (h+l) (Bethyl) (0.5 μg / mL) and Goat Anti-Human Kappa Light Chain-biotin (IBL) (0.5 μg / mL) were mixed, and 25 μL of each mixture was dispensed onto a sampling plate and shaken at room temperature for 1 hour on a microplate mixer to form a reaction solution. 150 μL of SuperBlock blocking buffer in PBS was added to a Streptavidin Gold plate (Meso Scale Diagnostics) and shaken at room temperature for 1 hour on a microplate mixer to block the plate. After washing with PBST, 25 μL of reaction solution was added to each well and the plate was shaken at room temperature for 1 hour on a microplate mixer. Next, 150 μL of Read buffer T (Meso Scale Diagnostics) was added to each well, and the plate was then incubated at room temperature for 1 hour. ＴＭ The amount of luminescence from each well was measured using an Imager 6000 reader. A calibration curve was created from the measurements of standard samples of hTfR affinity peptide-trastuzumab of known concentrations, and the amount of hTfR affinity peptide-trastuzumab contained per gram weight (wet weight) of brain (concentration of hTfR affinity peptide-trastuzumab in brain tissue) was calculated by interpolating the measurements of each sample. The results are shown in Table 8.

[0154]

[0155] Both hTfR affinity peptide-trastuzumab groups showed higher concentrations in brain tissue compared to the negative control group at both 6 and 24 hours after administration, demonstrating that hTfR affinity peptide-trastuzumab has the property of accumulating in brain tissue via the BBB.

[0156] Example 12: Evaluation of brain delivery of hTfR affinity peptide-trastuzumab using hTfR knock-in mice 2 Immunohistochemical staining of hTfR affinity peptide in brain tissue was performed generally as follows. Brain tissue collected for immunohistochemical staining was immersed in OCT compound (Sakura Finetech Japan Co., Ltd.) and rapidly frozen to -80°C using a Histotech Pino (Sakura Finetech Japan Co., Ltd.) to prepare frozen tissue blocks. These frozen blocks were sectioned at 4 μm and attached to MAS-coated glass slides (Matsunami Glass Co., Ltd.). The slides were immersed in 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.) at 4°C for 5 minutes to fix the specimens. The slides were then immersed in PBS containing 1% BSA for blocking. An appropriately diluted Goat Anti-hIgG-heavy and light chain antibody (Bethyl Laboratories) was then added dropwise to the tissue slices and allowed to react for 1 hour. Next, the Fluorescein Amplification Working Solution included in the TSA-Plus Fluorescence Kit (PerkinElmer) was added dropwise to the tissue slices and incubated for 15 minutes in the dark. Next, the Anti-Fluorescein-HRP included in the CSA II Biotin-FRee Tyramide Signal Amplification System (Dako) was added dropwise to the tissue slices and incubated for 15 minutes. The tissue slices were then reacted with DAB substrate (3,3'-diaminobenzidine, Vector Laboratories) to develop color. The slices were then counterstained with Mayer's hematoxylin (Merck), dehydrated, cleared, mounted, and observed under a light microscope.

[0157] Figure 1 shows optical photographs of tissue sections stained 6 hours after administration of the hTfR affinity peptide-trastuzumab, and Figure 2 shows optical photographs of tissue sections stained 24 hours after administration of the hTfR affinity peptide-trastuzumab. In the cerebellar tissue of mice administered trastuzumab as a negative control, no staining was observed at either 6 or 24 hours after administration, demonstrating that intravenously injected trastuzumab did not reach the cerebellar brain parenchyma and that there was almost no background staining (Figures 1(a) and 2(a)). In contrast, in the cerebellar tissue of mice administered the hTfR affinity peptides-trastuzumab 1 to 5, staining was observed in the brain parenchyma at either 6 or 24 hours after administration, demonstrating that intravenously injected trastuzumab reached the cerebellar brain parenchyma (Figures 1(b)-(f) and 2(b)-(f)). In particular, staining was observed in the cerebellar tissue of mice administered the hTfR affinity peptide-trastuzumab 3 at both 6 and 24 hours after administration, demonstrating that the intravenously injected hTfR affinity peptide-trastuzumab 3 efficiently reached the cerebellar brain parenchyma (Figure 1(e) and Figure 2(e)).

[0158] Example 13 Evaluation of Brain Delivery of hTfR Affinity Peptide-Trastuzumab Using Cynomolgus Monkeys The purified hTfR affinity peptide-trastuzumab 5 product obtained in Example 9 and trastuzumab were each administered intravenously in a single dose of 5 mg / kg to one male cynomolgus monkey (Macaca fascicularis). As a control, the same volume of saline was administered intravenously to one male cynomolgus monkey. Peripheral blood samples were collected 20 minutes, 1 hour, 2 hours, 4 hours, and 8 hours after administration, and the concentrations of hTfR affinity peptide-trastuzumab 5 and trastuzumab in the peripheral blood were measured using the method described in Example 11. Cmax (μg / mL), AUC0-8hr (μg / hr / mL), and t1 / 2β (hr) were calculated from the measured values. The Cmax (μg / mL), AUC0-8hr (μg / hr / mL), and t1 / 2β (hr) of the hTfR affinity peptide-trastuzumab 5 were 142, 816, and 25.1, respectively. On the other hand, the Cmax (μg / mL), AUC0-8hr (μg / hr / mL), and t1 / 2β (hr) of trastuzumab were 110, 509, and 2.27, respectively. These results indicate that the hTfR affinity peptide-trastuzumab 5 is relatively stable in the blood after intravenous administration to cynomolgus monkeys.

[0159] Eight hours after administration, cynomolgus monkeys were subjected to whole-body perfusion with physiological saline. After perfusion, brain tissues including the medulla oblongata and various organs were removed. The concentrations of the hTfR affinity peptide-trastuzumab 5 and trastuzumab in the removed brain tissues and various organs were measured using the method described in Example 11.

[0160] Brain tissue was divided into the cerebral cortex, cerebellum, hippocampus, midbrain, pons, medulla oblongata, caudate nucleus, nucleus pallidus, thalamus, hypothalamus, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord, and measurements were taken for each of the following organs: retina, liver, kidney, heart (left ventricle), heart (aortic valve), lung, bone marrow, spleen, thymus, testis, prostate, rectus femoris muscle, EDL, soleus muscle, triceps muscle, and diaphragm.

[0161] Figure 3 shows the results of brain tissue analysis. Trastuzumab was barely detected in the cerebral cortex, cerebellum, hippocampus, midbrain, pons, medulla oblongata, caudate nucleus, nucleus pallidus, and thalamus, but hTfR affinity peptide-trastuzumab 5 was detected at significantly higher concentrations than trastuzumab in these organs. Furthermore, hTfR affinity peptide-trastuzumab 5 was detected at concentrations more than twice those of trastuzumab in the hypothalamus. Meanwhile, hTfR affinity peptide-trastuzumab 5 was detected at lower concentrations than trastuzumab in the cervical, thoracic, and lumbar spinal cord. In the retina, hTfR affinity peptide-trastuzumab 5 and trastuzumab were detected at approximately the same concentrations. These results indicate that the hTfR-affinity peptide-trastuzumab 5, administered intravenously to cynomolgus monkeys, crosses the blood-brain barrier by binding to transferrin present on cerebral vascular endothelial cells, and reaches central nervous system tissues such as the cerebral cortex, cerebellum, hippocampus, midbrain, pons, medulla oblongata, caudate nucleus, nucleus pallidus, and thalamus.

[0162] Figure 4 shows the measurement results for various organs. Trastuzumab was detected at high concentrations in the heart, where HER2 protein, the target molecule of trastuzumab, is expressed, and the hTfR affinity peptide-trastuzumab 5 was detected at high concentrations in the bone marrow, where transferrin receptor expression levels are high. This indicates that the hTfR affinity peptide-trastuzumab 5 administered intravenously to cynomolgus monkeys reaches many organs, including the liver, kidneys, heart, lungs, bone marrow, spleen, testes, and prostate.

[0163] Example 14: Preparation of hTfR affinity peptide-hIDS expression vector pD2535nt-HDP_v2 (HORIZON Discovery) was digested with PacI and PmeI. A DNA fragment was artificially synthesized containing a PacI site on the 5' end and a PmeI site on the 3' end of a gene having the nucleotide sequence shown in SEQ ID NO:61 encoding a protein having the amino acid sequence shown in SEQ ID NO:60, in which hTFR affinity peptide 6 having the amino acid sequence shown in SEQ ID NO:55 was linked to the N-terminus of hIDS having the amino acid sequence shown in SEQ ID NO:58 via a peptide linker having the amino acid sequence shown in SEQ ID NO:59. This DNA fragment was digested with PacI and PmeI and inserted between the PacI and PmeI sites of pD2535nt-HDP_v2. The resulting vector was designated the VHH6-GS3-hIDS expression vector. The protein having the amino acid sequence shown in SEQ ID NO:60 encoded by this expression vector was designated VHH6-GS3-hIDS.

[0164] pD2535nt-HDP_v2 (HORIZON Discovery) was digested with PacI and PmeI. A DNA fragment was artificially synthesized containing a PacI site on the 5' end and a PmeI site on the 3' end of a gene having the nucleotide sequence shown in SEQ ID NO:63 encoding a protein having the amino acid sequence shown in SEQ ID NO:62, in which hTFR affinity peptide 5 having the amino acid sequence shown in SEQ ID NO:7 was linked to the N-terminus of hIDS having the amino acid sequence shown in SEQ ID NO:58 via a peptide linker having the amino acid sequence shown in SEQ ID NO:59. This DNA fragment was digested with PacI and PmeI and inserted between the PacI and PmeI sites of pD2535nt-HDP_v2. The resulting vector was designated the VHH5-GS3-hIDS expression vector. The protein having the amino acid sequence shown in SEQ ID NO:62 encoded by this expression vector was designated VHH5-GS3-hIDS.

[0165] pD2535nt-HDP_v2 (HORIZON Discovery) was digested with PacI and PmeI. A DNA fragment was artificially synthesized containing a PacI site on the 5' end and a PmeI site on the 3' end of a gene having the nucleotide sequence shown in SEQ ID NO: 65 encoding a protein having the amino acid sequence shown in SEQ ID NO: 64, in which hTFR affinity peptide 5 having the amino acid sequence shown in SEQ ID NO: 7 was bound to the N-terminus of hIDS having the amino acid sequence shown in SEQ ID NO: 58 via a peptide linker having the amino acid sequence shown in SEQ ID NO: 59, and further hTFR affinity peptide 5 was bound to the N-terminus of this hTFR affinity peptide 5 via a peptide linker having the amino acid sequence shown in SEQ ID NO: 59. This DNA fragment was digested with PacI and PmeI and inserted between the PacI and PmeI sites of pD2535nt-HDP_v2. The resulting vector was designated the VHH5-Tandem-GS3-hIDS expression vector. The protein encoded by this expression vector and having the amino acid sequence shown in SEQ ID NO: 64 was designated VHH5-Tandem-GS3-hIDS.

[0166] Example 15: Preparation of hTfR-affinity peptide-hIDS The VHH5-GS3-hIDS expression vector prepared in Example 14 was introduced into GS knockout CHO cells (HD-BIOP3 cells) using a glutamine synthetase (GS) knockout CHO expression platform (HORIZON Discovery) according to the attached protocol, and VHH5-GS3-hIDS was expressed in HD-BIOP3 cells. pD2535nt-HDP_v2 incorporates the GS gene as a selection marker, allowing for gene introduction into HD-BIOP3 cells and selection in glutamine-free medium. Furthermore, higher selection pressure can be applied to cells by using a medium containing a low concentration (up to 50 μmol / L) of MSX. Cell transformation was generally performed as follows. At a concentration of 1 × 10 6HD-BIOP3 cells were suspended at a cell density of 1 / mL in 15 mL of Dynamis medium (ThermoFisher) supplemented with 4 mM L-glutamine and transferred to a 150 mL Erlenmeyer flask. A solution of 37.5 μL of FreeStyle MAX Reagent (ThermoFisher) in 600 μL of OptoPro SFM (ThermoFisher) was mixed with 37.5 μg of the VHH5-GS3-hIDS expression vector in 600 μL of OptoPro SFM, and the resulting mixture was added to the cell suspension. After culturing at 37°C in a humidified atmosphere of 5% CO2 and 95% air at an agitation speed of approximately 120 rpm for 2 days, the cells were selectively cultured in Dynamis medium containing MSX at 25 μM or 35 μM. The HD-BIOP3 cells obtained by selective culture express VHH5-GS3-hIDS and secrete it into the culture medium. These cells were cultured at a concentration of 3 × 10 5 The cells were suspended in 200 mL of Dynamis medium at a concentration of 1000 cells / mL, transferred to a 1000 mL Erlenmeyer flask, and cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air at an agitation speed of approximately 120 rpm for 7 days to allow expression of VHH5-GS3-hIDS. On the 3rd and 5th days after the start of culture, 10 mL of EfficientFeed was added. TM C+ 2X Supplement (ThermoFisher) was added. After incubation, the culture medium was centrifuged (3000 g, 5 minutes), and the resulting supernatant was filtered through a 0.22 μm filter (Millipore). This was then collected as the culture supernatant.

[0167] An open column packed with 1 mL of MabSelect Xtra (Cytiva) was equilibrated by passing through 5 column volumes of 25 mM HEPES buffer (pH 7.5) containing 50 mM NaCl. The culture supernatant was then loaded onto the column, and VHH5-GS3-hIDS was bound to the resin. The column was washed with 5 column volumes of 25 mM HEPES buffer (pH 7.5) containing 50 mM NaCl, and VHH5-GS3-hIDS was eluted with 150 mM glycine (pH 3.0) containing 100 mM NaCl. The pH of the eluate containing VHH5-GS3-hIDS was adjusted to neutral by adding 5% of 1 M Tris-HCl (pH 8.0). The solution was then ultrafiltered using an Ultracel-30 regenerated cellulose membrane (pore size: 30 kDa, Merck) and concentrated to a volume of approximately 1 mL.

[0168] The concentrate was loaded onto a size-exclusion column, Superdex 200 increase 10 / 300 GL (Cytiva) (column volume: 24 mL, bed height: 30 cm), equilibrated with 1.5 column volumes of 20 mM Tris buffer (pH 7.4) containing 300 mM NaCl at a constant flow rate of 0.5 mL / min. The same buffer was then supplied at the same flow rate. An absorption spectrophotometer was placed in the eluate flow path from the size-exclusion column to continuously measure the absorbance of the eluate at 280 nm. The fractions showing an absorption peak at 280 nm were collected as fractions containing VHH5-GS3-hIDS. The concentrate was then buffer-exchanged into 20 mM Tris buffer (pH 7.4) containing 50 mM NaCl using an Ultracel-30 regenerated cellulose membrane, and the resulting concentrate was used as the purified VHH5-GS3-hIDS preparation.

[0169] VHH6-GS3-hIDS was purified from the culture supernatant of HD-BIOP3 cells transfected with the VHH6-GS3-hIDS expression vector by the same method as for VHH5-GS3-hIDS. VHH5-Tandem-GS3-hIDS was purified from the culture supernatant of HD-BIOP3 cells transfected with the VHH5-Tandem-GS3-hIDS expression vector by the same method as for VHH5-GS3-hIDS. The three proteins, VHH5-GS3-hIDS, VHH6-GS3-hIDS, and VHH5-Tandem-GS3-hIDS, are collectively referred to as hTfR affinity peptide-hIDS.

[0170] Example 16: Evaluation of the binding activity of hTfR-affinity peptide-hIDS to human TfR and monkey TfR. The binding activity of VHH5-GS3-hIDS, VHH6-GS3-hIDS, and VHH5-Tandem-GS3-hIDS proteins obtained by standard methods using CHO cells was measured for their binding activity to the human transferrin receptor (hTfR) by ELISA. ELISA was performed roughly as follows: 100 μL of recombinant hTfR containing the extracellular domain of hTfR consisting of the amino acid sequence from the N-terminus to the 89th amino acid in the amino acid sequence set forth in SEQ ID NO: 1, diluted with 0.05 M bicarbonate buffer (pH 9.6) to a concentration of 10 μg / mL, was added to each well of a 96-well microtiter plate (Nunc). The plate was then left to stand at room temperature for at least 1 hour to allow the antibody to adsorb to the plate. Next, 300 μL of TBS containing 1% BSA and 0.05% Tween 20 was added to each well, and the plate was left to stand at room temperature for 1 hour. After washing each well three times with TBS containing 0.05% Tween 20 (TBS-T), VHH5-GS3-hIDS was diluted to 100, 33.33, 11.11, 3.71, 1.24, 0.41, and 0.14 nM in TBS containing 0.1% BSA and 0.05% Tween 20; VHH6-GS3-hIDS was diluted to 333, 111, 37, 12.33, 4.11, 1.37, and 0.46 nM in TBS; and VHH5-Tandem-GS3-hIDS was diluted to 1, 0.33, 0.11, 0.037, 0.012, 0.041, and 0.014 nM in TBS containing 0.1% BSA and 0.05% Tween 20; 100 μL of each diluted solution was added to each well. The plate was then incubated at room temperature for at least 1 hour. After washing the plate three times with TBS-T, 100 μL of anti-hIDS monoclonal antibody diluted to 0.5 μg / mL in TBS containing 0.1% BSA and 0.05% Tween 20 was added to each well and the plate was left to stand at room temperature for at least 1 hour. After washing three times with TBS-T, 100 μL of HRP-labeled anti-mouse IgG polyclonal antibody (Bethyl) diluted to 50 ng / mL in TBS containing 0.1% BSA and 0.05% Tween 20 was added to each well and the plate was left to stand at room temperature for at least 1 hour.After washing each well three times with TBS-T, 50 μL of TMB Stabilized Substrate for Horseradish Peroxidase (Promega) was added to each well and allowed to stand at room temperature for 5-10 minutes. Next, 100 μL of stop solution (1N hydrochloric acid) was added to each well, and the absorbance at 450 nm of each well was measured using a plate reader (MOLECULAR DEVICES). The EC was calculated from the measured values. 50 The EC values ​​of VHH5-GS3-hIDS, VHH6-GS3-hIDS, and VHH5-Tandem-GS3-hIDS were calculated. 50 The affinities of these three hTfR-affinity peptide-hIDSs were 1.087, 22.67, and 0.077 nM, respectively. These results indicate that all three hTfR-affinity peptide-hIDSs have relatively high affinity for hTfR.

[0171] Example 17: Generation of IDS-KO / hTfR-KI Mice IDS-KO / hTfR-KI mice are mice that are hemi-deficient in the iduronate-2-sulfatase (IDS) gene and heterozygous for a chimeric TfR gene. IDS-KO / hTfR-KI mice were generated generally by the following method. A DNA fragment was chemically synthesized containing a neomycin resistance gene flanked by loxP sequences at the 3' end of a cDNA encoding a chimeric TfR whose intracellular domain is the amino acid sequence of mouse TfR and whose extracellular domain is the amino acid sequence of human TfR. This DNA fragment was incorporated into a targeting vector containing 5' and 3' arm sequences by standard methods, and then introduced into mouse ES cells by electroporation. After gene introduction, the mouse ES cells were selectively cultured in the presence of neomycin, and mouse ES cells in which the targeting vector had been integrated into the chromosome by homologous recombination were selected. The resulting recombinant mouse ES cells were injected into 8-cell ICR mouse embryos (host embryos) and then transplanted into pseudopregnant recipient mice obtained by mating with vasectomized mice. The resulting offspring (chimeric mice) were assessed for coat color, and individuals in which ES cells contributed highly efficiently to the formation of the organism, i.e., individuals with a high proportion of white hair relative to their total hair, were selected. These chimeric mice were then crossed with ICR mice to generate F1 mice. White F1 mice were selected, and DNA extracted from their tail tissue was analyzed. Mice in which the mouse hTfR gene was replaced by the chimeric TfR on the chromosome were designated hTfR-KI mice. Based on these mice, mice with a hemi-deficient IDS gene and a heterozygous chimeric TfR gene (IDS-KO / hTfR-KI mice) were generated. The IDS-KO / hTfR-KI mice were produced in accordance with the method described in patent document (WO2016 / 208695).

[0172] Example 17: Evaluation of the efficacy of each protein using IDS-KO / hTfR-KI mice The efficacy of the hTfR affinity peptide-hIDS was evaluated by measuring the concentration of heparan sulfate (HS), which is known to accumulate in the organs of patients with Hunter syndrome, a condition in which hIDS activity is genetically deleted. The method is described in detail below.

[0173] The purified products of the three proteins, VHH5-GS3-hIDS, VHH6-GS3-hIDS, and VHH5-Tandem-GS3-hIDS obtained in Example 15, were diluted with saline to prepare solutions containing each protein at a concentration of 0.4 mg / mL.

[0174] Diluted VHH5-GS3-hIDS, VHH6-GS3-hIDS, and VHH5-Tandem-GS3-hIDS were administered to 50-week-old IDS-KO / hTfR KI mice at a dose of 2 mg / kg via tail vein injection once per week for a total of three doses. Wild-type IDS-wildtype mice were used as wild-type controls, and hemi-IDS-KO mice were used as pathological controls. These control mice were administered saline once per week for a total of three doses. The volume of fluid administered to each group was adjusted to be approximately the same. The genotypes of the mice used are shown in Table 10. The number of mice in each group is also shown in Table 10.

[0175]

[0176] One week after the third administration, CSF was collected from the cisterna magna of each mouse under a triple anesthesia regimen consisting of Vetorfal (Meiji Seika Pharma), midazolam (Sandoz), and Domitor (Nippon Zenyaku Kogyo). After CSF collection, the mice were euthanized by exsanguination. The brains were then removed and flash-frozen.

[0177] Example 18 Quantification of Heparan Sulfate in Brain Tissue and CSF Quantification of heparan sulfate (HS) in the brain was carried out generally by the following method. Note that heparan sulfate is a substrate for hIDS.

[0178] The solutions (a) to (l) used in the test were prepared as follows: (a) MeCN / water: 2 mL of water for injection (Otsuka Pharmaceutical Co., Ltd.) and 18 mL of acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed to prepare MeCN / water. (b) Deuterium-labeled solvent: 240 μL of acetyl chloride (Sigma-Aldrich Co., Ltd.) was added dropwise to 1.5 mL of methanol-d4 (Sigma-Aldrich Co., Ltd.) in an ice bath to prepare the deuterium-labeled solvent. (c) Mobile phase A: 475 mL of water for injection and 25 mL of 1 M ammonium formate aqueous solution (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed to prepare mobile phase A. (d) Mobile phase B: A 93:7 (v / v) mixture of acetonitrile and mobile phase A was used to prepare mobile phase B. (e) Heparan sulfate standard stock solution (HS standard stock solution): Heparan sulfate (Iduron) was dissolved in water for injection to prepare a 5.0 mg / mL solution. This solution was used as the HS standard stock solution. (f) Heparan sulfate internal standard solution (HS internal standard solution): 40 μL of the HS standard stock solution was measured and placed in a borosilicate screw-cap test tube, and the solvent was evaporated under a nitrogen stream. 400 μL of deuterium-labeled solvent was added to the dried product, stirred, and then reacted at 65°C for 75 minutes to perform deuterium methanolysis. After the reaction, the solvent was evaporated under a nitrogen stream. 500 μL of MeCN / water was added to the dried product, and the mixture was sonicated for 30 minutes. This solution was used as the heparan sulfate internal standard solution (HS internal standard solution). (i) Internal standard solution: 1 μL of HS internal standard solution was added to 1 mL of methanol, stirred, and then sonicated for 30 minutes. This solution was used as the internal standard solution. (j) Calibration curve sample: 980 μL of water for injection was measured out, and 10 μL of HS standard stock solution was added to each to prepare a solution containing 50 μg / mL of heparan sulfate. This solution was diluted with water for injection to prepare a solution containing 5000 ng / mL of heparan sulfate. This solution was serially diluted with water for injection to prepare solutions containing heparan sulfate at concentrations of 25, 50, 100, 250, 500, 1000, 2500, and 5000 ng / mL.20 μL of this was measured out and dispensed into borosilicate screw-cap test tubes. This solution was used as the calibration curve sample. (k) Tissue extraction solution: 1 mL of polyoxyethylene (10) octylphenyl ether was added to physiological saline to make a total volume of 500 mL. This solution was used as the tissue extraction solution. (l) 10% ammonium carbonate solution: 5 g of ammonium carbonate was dissolved in 50 mL of water for injection. This solution was used as the 10% ammonium carbonate solution.

[0179] 20 μL of the CSF collected in Example 17 was measured and dispensed into a borosilicate screw-cap test tube, which was used as a CSF sample solution.

[0180] The brain tissue extracted in Example 17 was freeze-dried and its dry weight was measured. The freeze-dried tissue was crushed in tissue extraction solution, and the supernatant was centrifuged. 20 μL of this was measured and dispensed into a borosilicate screw-cap test tube. This was used as the sample solution.

[0181] The solvent in each sample solution and calibration curve sample was evaporated under a nitrogen stream. 20 μL of 2,2-dimethoxypropane (Tokyo Chemical Industry Co., Ltd.) and 200 μL of 3 mol / L HCl-methanol (Sigma-Aldrich) were added to the dried product and stirred. The methanolysis reaction was carried out at 70°C for 90 minutes. The reaction was stopped by cooling on ice, and 200 μL of 10% ammonium carbonate solution and 50 μL of the internal standard solution were added. After the solvent was evaporated under a nitrogen stream, 250 μL of water for injection was added to the dried product. The resulting mixture was loaded onto a solid-phase cartridge (OASIS HLB (1 cc, 30 mg, Waters)) previously conditioned with methanol and water for injection. After washing with water for injection, 500 μL of methanol was added and eluted by centrifugation. After distilling off the solvent under a nitrogen stream, 2 mL of water for injection and 18 mL of acetonitrile were added and redissolved by ultrasonic treatment. After centrifuging, the supernatant was filled into an LC vial.

[0182] LC / MS / MS analysis was performed using a combination of hydrophilic interaction ultra-high performance liquid chromatography and a tandem quadrupole mass spectrometer. The mass spectrometer (MS / MS device) was a QTRAP5500 (AB Sciex), and the HPLC device was a Nexera X2 (Shimadzu Corporation). The LC column was an Acquity UPLC. TM BEH Amide 1.7 μm (2.1 × 150 mm, Waters) was used. Mobile phases A and B were used. The column temperature was set to 60°C.

[0183] After equilibrating the column with mobile phase B, 5 μL of sample was injected, and chromatography was performed under the mobile phase gradient conditions shown in Table 11. The flow rate of the mobile phase was 0.4 mL / min.

[0184]

[0185] The ion source parameters of the MS / MS instrument were set as shown in Table 12 according to the instruction manual for QTRAP5500 (AB Sciex).

[0186]

[0187] Table 13 shows the MS internal parameters.

[0188]

[0189] LC / MS / MS analysis was performed on each calibration curve sample, and the area of ​​the peak on the chromatogram chart corresponding to the product ions derived from heparan sulfate in the calibration curve sample (HS detection peak area) was calculated. In addition, the area of ​​the detection peak corresponding to the product ions derived from the HS internal standard solution (HS-IS detection peak area) was calculated.

[0190] For each calibration curve sample, the area of ​​the detection peak derived from heparan sulfate relative to the detection peak area derived from the HS internal standard solution (HS detection peak area / HS-IS detection peak area) was plotted on the vertical axis, and the heparan sulfate concentration of each calibration curve sample was plotted on the horizontal axis. A regression equation was obtained using quadratic discriminant analysis.

[0191] The brain tissue sample solution was subjected to LC / MS / MS analysis, and the heparan sulfate contained in the sample solution was quantified by interpolation into a regression equation.

[0192] Figure 5 shows the results of measuring HS in CSF. In the VHH5-GS3-hIDS, VHH6-GS3-hIDS, and VHH5-Tandem-GS3-hIDS groups, the HS concentration in CSF was reduced compared to the pathological control group, indicating that all of these drugs crossed the BBB, reached the central nervous system, and degraded the HS that had accumulated there. In the VHH5-Tandem-GS3-hIDS group, the HS concentration in CSF was slightly less than 40% of that in the pathological control group, demonstrating that linking two hTfR-affinity peptides in tandem is an effective means of allowing fusion proteins to cross the BBB.

[0193] Figure 6 shows the results of measuring HS in brain tissue. In the VHH5-GS3-hIDS, VHH6-GS3-hIDS, and VHH5-Tandem-GS3-hIDS groups, the HS concentrations in brain tissue were all reduced compared to pathological controls, indicating that all of these drugs crossed the BBB, reached brain tissue, and degraded accumulated HS. The HS concentration in brain tissue of VHH5-Tandem-GS3-hIDS was reduced by almost half compared to pathological controls, demonstrating that linking two hTfR affinity peptides in tandem is an effective means of allowing the fusion protein to cross the BBB. These results also suggest that intravenous injection of hIDS fused to an hTfR affinity peptide into Hunter syndrome patients will cross the BBB, reach the central nervous system, and degrade accumulated HS, thereby exerting its therapeutic effect.

[0194] Example 18: Preparation of hTfR affinity peptide-hSGSH expression vector pCI-neo Mammalian Expression Vector (Promega) was digested with MluI and NotI. A DNA fragment was artificially synthesized containing an MluI site on the 5' end and a NotI site on the 3' end of a gene having the nucleotide sequence shown in SEQ ID NO:68 encoding a protein having the amino acid sequence shown in SEQ ID NO:67, in which heparan N-sulfatase (hSGSH) shown in SEQ ID NO:66, with a DYKDDDDK tag attached to its N-terminus, was linked to the C-terminus of hTfR affinity peptide 3 having the amino acid sequence shown in SEQ ID NO:5 via a peptide linker having the amino acid sequence shown in SEQ ID NO:59. This gene was digested with MluI and NotI and inserted between the MluI and NotI sites of the pCI-neo Mammalian Expression Vector to prepare an expression vector for VHH3-GS3-hSGSH. This expression vector was designated pCI-neo-VHH3-GS3-hSGSH.

[0195] Example 19: Preparation of hTfR affinity peptide-hSGSH The pCI-neo-VHH3-GS3-hSGSH prepared in Example 18 was transfected into CHO-S cells using the ExpiCHO Expression System (ThermoFisher Scientific) according to the attached protocol, and a protein consisting of hTfR affinity peptide 3 and hSGSH (VHH3-GS3-hSGSH) was expressed in the CHO-S cells. These cells expressed VHH3-GS3-hSGSH and secreted it into the culture medium. Here, the CHO-S cells transfected with pCI-neo-VHH3-GS3-hSGSH were designated pCI-neo-VHH3-GS3-hSGSH-expressing cells. The fusion protein of the hTfR affinity peptide and hSGSH is referred to as hTfR affinity peptide-hSGSH.

[0196] pCI-neo-VHH3-GS3-hSGSH expressing cells were cultured according to the protocol attached to the ExpiCHO Expression System. Specifically, the concentration was 6 × 10 6The cells were suspended in 15 mL of medium at a density of 1000 cells / mL and transferred to a 150 mL Erlenmeyer flask. They were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air at a stirring speed of approximately 120 rpm for one day. The feed and enhancer solutions included with the ExpiCHO Expression System were added, and the cells were cultured at 32°C in a humidified atmosphere of 5% CO2 and 95% air at a stirring speed of approximately 120 rpm for an additional 9 days. After culturing, the culture medium was centrifuged (3000 g, 5 minutes), and the resulting supernatant was filtered through a 0.22 μm filter (Millipore) and collected as the culture supernatant.

[0197] An open column packed with 1 mL of anti-FLAG M1 antibody-agarose affinity gel (MERCK) was equilibrated with 5 column volumes of 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl and 1 mM CaCl2. The culture supernatant was adjusted to 1 mM CaCl2 by adding 1 M CaCl2 at a ratio of 1 / 1000. The culture supernatant was then loaded onto the column, and VHH3-GS3-hSGSH was bound to the resin. The column was washed with 5 column volumes of 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl and 1 mM CaCl2. VHH3-GS3-hSGSH was then eluted with 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl and 2 mM EDTA. The resulting solution was buffer exchanged using a PD-10 column (Cyteva) to replace the solvent with 20 mM phosphate buffer containing 7.5 mg / mL sucrose and 0.8 mg / mL NaCl, and the resulting solution was used in the following experiments as a purified preparation of VHH3-GS3-hSGSH.

[0198] Example 20: Evaluation of binding activity of hTfR affinity peptide-hSGSH to human TfR and monkey TfR The affinity of VHH-GS3-hSGSH prepared in Example 19 to hTfR and monkey TfR was examined according to the method described in Example 7. Table 13 shows the measurement results of the association rate constant (k) and dissociation rate constant (koff) of VHH3-GS3-hSGSH to hTfR and monkey TfR (cynomolgus monkey TfR), as well as the dissociation constant (K D The cynomolgus monkey TfR used was a recombinant monkey TfR (rMonkey TfR, Sino Biological Co., Ltd.) with an N-terminal histidine tag and the amino acid sequence of the extracellular domain of the cynomolgus monkey TfR, spanning from the 89th cysteine ​​residue from the N-terminus to the C-terminal phenylalanine in the amino acid sequence shown in SEQ ID NO: 2.

[0199]

[0200] Example 21: Evaluation of the efficacy of the hTfR affinity peptide-hSGSH The efficacy of the hTfR affinity peptide-hSGSH was evaluated by measuring the concentration of heparan sulfate (HS), which is known to accumulate in the organs of patients with Sanfilippo syndrome A, who genetically lack hSGSH activity. The method is described in detail below.

[0201] The hTfR-KI mice prepared by the method described in Example 17 were crossed with SGSH gene knockout mice (SGSH-KO mice), a model mouse for Sanfilippo syndrome A, to obtain SGSH-KO / hTfR KI mice.

[0202] VHH3-GS3-hSGSH obtained in Example 21 was diluted with physiological saline to prepare solutions containing each protein at a concentration of 0.4 mg / mL.

[0203] Diluted VHH3-GS3-hSGSH was administered at a dose of 2 mg / kg to 16- to 19-week-old SGSH-KO / hTfR KI mice via tail vein injection once a week for a total of four doses. Wild-type SGSH gene wild-type mice were used as wild-type controls, and homozygous SGSH-KO mice were used as pathological controls. These control mice were administered saline once a week for a total of four doses. The volume of fluid administered to each group was adjusted to be approximately the same. The genotypes of the SGSH-KO / hTfR KI mice used are shown in Table 12. The number of mice in each group is also shown in Table 14.

[0204]

[0205] One week after the fourth administration, CSF was collected from the cisterna magna of each mouse under a triple anesthesia regimen using Betolfar (Meiji Seika Pharma), midazolam (Sandoz), and Domitor (Nippon Zenyaku Kogyo). After CSF collection, the mice were euthanized by exsanguination. Then, the mouse brains were removed and flash-frozen. The amount of HS in the CSF and brain tissue was measured using the method described in Example 18.

[0206] Figure 7 shows the results of measuring HS in brain tissue and CSF. In the VHH3-GS3-hSGSH-treated group, HS concentrations in brain tissue were reduced by approximately 37% compared to pathological controls, indicating that VHH3-GS3-hSGSH crossed the BBB, reached the central nervous system, and degraded HS accumulated there (Figure 7(a)). Furthermore, in the VHH3-GS3-hSGSH-treated group, HS concentrations in CSF were reduced by approximately 13% compared to pathological controls, indicating that VHH3-GS3-hSGSH crossed the BBB, reached the central nervous system, and degraded HS accumulated there (Figure 7(b)). These results suggest that hSGSH fused to an hTfR-affinity peptide, when administered intravenously to patients with Sanfilippo syndrome A, can cross the BBB, reach the central nervous system, and degrade HS accumulated there, thereby exerting its therapeutic effect.

[0207] According to one embodiment of the present invention, it is possible to provide a drug that combines a human transferrin receptor affinity peptide with a pharmacologically active substance and that can pass through the blood-brain barrier and exert its effect in the central nervous system.

[0208] 1. Blood vessels 2. Brain parenchyma

[0209] SEQ ID NO: 1: Amino acid sequence of human transferrin receptor SEQ ID NO: 2: Amino acid sequence of cynomolgus monkey transferrin receptor SEQ ID NO: 3: Amino acid sequence of hTfR affinity peptide 1 SEQ ID NO: 4: Amino acid sequence of hTfR affinity peptide 2 SEQ ID NO: 5: Amino acid sequence of hTfR affinity peptide 3 SEQ ID NO: 6: Amino acid sequence of hTfR affinity peptide 4 SEQ ID NO: 7: Amino acid sequence of hTfR affinity peptide 5 SEQ ID NO: 8: Amino acid sequence 1 of CDR1 of hTfR affinity peptide 1 SEQ ID NO: 9: Amino acid sequence 2 of CDR1 of hTfR affinity peptide 1 SEQ ID NO: 10: Amino acid sequence 1 of CDR2 of hTfR affinity peptide 1 SEQ ID NO: 11: Amino acid sequence 2 of CDR2 of hTfR affinity peptide 1 SEQ ID NO: 12: Amino acid sequence 1 of CDR3 of hTfR affinity peptide 1 SEQ ID NO: 13: Amino acid sequence 2 of CDR3 of hTfR affinity peptide 1 SEQ ID NO: 14: Amino acid sequence 1 of CDR1 of hTfR affinity peptide 2 SEQ ID NO: 15: Amino acid sequence 2 of CDR1 of hTfR affinity peptide 2 SEQ ID NO: 16: Amino acid sequence 1 of CDR2 of hTfR affinity peptide 2 SEQ ID NO: 17: Amino acid sequence 2 of CDR2 of hTfR affinity peptide 2 SEQ ID NO: 18: Amino acid sequence 1 of CDR3 of hTfR affinity peptide 2 SEQ ID NO: 19: Amino acid sequence 2 of CDR3 of hTfR affinity peptide 2 SEQ ID NO: 20: Amino acid sequence 1 of CDR1 of hTfR affinity peptide 3 SEQ ID NO: 21: Amino acid sequence 2 of CDR1 of hTfR affinity peptide 3 SEQ ID NO: 22: Amino acid sequence 1 of CDR2 of hTfR affinity peptide 3 SEQ ID NO: 23: Amino acid sequence 2 of CDR2 of hTfR affinity peptide 3 SEQ ID NO: 24: Amino acid sequence 1 of CDR3 of hTfR affinity peptide 3 SEQ ID NO: 25: Amino acid sequence 2 of CDR3 of hTfR affinity peptide 3 SEQ ID NO: 26: Amino acid sequence 1 of CDR1 of hTfR affinity peptide 4 SEQ ID NO: 27: Amino acid sequence 2 of CDR1 of hTfR affinity peptide 4 SEQ ID NO: 28: Amino acid sequence 1 of CDR2 of hTfR affinity peptide 4 SEQ ID NO: 29: Amino acid sequence 2 of CDR2 of hTfR affinity peptide 4 SEQ ID NO: 30: Amino acid sequence 1 of CDR3 of hTfR affinity peptide 4 SEQ ID NO: 31: Amino acid sequence 2 of CDR3 of hTfR affinity peptide 4SEQ ID NO: 32: Amino acid sequence of peptide linker 1 SEQ ID NO: 33: Amino acid sequence of peptide linker 2 SEQ ID NO: 34: Amino acid sequence of peptide linker 3 SEQ ID NO: 35: Amino acid sequence of peptide linker 4 SEQ ID NO: 36: Amino acid sequence of peptide linker 5 SEQ ID NO: 37: An example of the amino acid sequence of the Fc region of human IgG SEQ ID NO: 38: Amino acid sequence of human serum albumin SEQ ID NO: 39: Albumin-binding domain of a protein derived from Streptococcus strain G418 SEQ ID NO: 40: Amino acid sequence of the heavy chain of trastuzumab SEQ ID NO: 41: Amino acid sequence of the light chain of trastuzumab SEQ ID NO: 42: Amino acid sequence of a fusion protein of hTfR affinity peptide 1 and the heavy chain of trastuzumab SEQ ID NO: 43: Amino acid sequence of a fusion protein of hTfR affinity peptide 2 and the heavy chain of trastuzumab SEQ ID NO: 44: Amino acid sequence of a fusion protein of hTfR affinity peptide 3 and the heavy chain of trastuzumab SEQ ID NO: 45: Amino acid sequence of the fusion protein of hTfR affinity peptide 4 and the heavy chain of trastuzumab SEQ ID NO: 46: Amino acid sequence of the fusion protein of hTfR affinity peptide 5 and the heavy chain of trastuzumab SEQ ID NO: 47: Nucleotide sequence of DNA encoding the fusion protein of hTfR affinity peptide 1 and the heavy chain of trastuzumab, synthetic sequence SEQ ID NO: 48: Nucleotide sequence of DNA encoding the light chain of trastuzumab, synthetic sequence SEQ ID NO: 49: Nucleotide sequence of primer hTfR5', synthetic sequence SEQ ID NO: 50: Nucleotide sequence of primer hTfR3', synthetic sequence SEQ ID NO: 51: Nucleotide sequence of DNA in which a neomycin resistance gene flanked by loxP sequences is located on the 3' side of cDNA encoding chimeric hTfR, synthetic sequence SEQ ID NO: 52: Nucleotide sequence of the 5' arm of the targeting vector, synthetic sequence SEQ ID NO: 53: Nucleotide sequence of the 3' arm of the targeting vector, synthetic sequence SEQ ID NO: 54: Peptide tag SEQ ID NO: 55: Amino acid sequence of hTfR affinity peptide 6 SEQ ID NO: 56: Amino acid sequence 1 of CDR2 of hTfR affinity peptide 6 SEQ ID NO: 57: Amino acid sequence 2 of CDR2 of hTfR affinity peptide 6 SEQ ID NO: 58: Amino acid sequence of peptide linker 6 SEQ ID NO: 59: Amino acid sequence of human I2S SEQ ID NO: 60: Amino acid sequence of VHH6-GS3-hI2S SEQ ID NO: 61: Nucleotide sequence of VHH6-GS3-hI2SSEQ ID NO: 62: Amino acid sequence of VHH5-GS3-hI2S SEQ ID NO: 63: Nucleotide sequence of VHH5-GS3-hI2S SEQ ID NO: 64: Amino acid sequence of VHH5-Tandem-GS3-hI2S SEQ ID NO: 65: Nucleotide sequence of VHH5-Tandem-GS3-hI2S SEQ ID NO: 66: Amino acid sequence of human SGSH SEQ ID NO: 67: Amino acid sequence of VHH3-GS3-hSGSH SEQ ID NO: 68: Nucleotide sequence of VHH3-GS3-hSGSH SEQ ID NO: 69: Amino acid sequence of peptide linker 7

Claims

1. A peptide having affinity for the human transferrin receptor (human transferrin receptor affinity peptide), which comprises a heavy chain antibody variable region having three complementarity determining regions, CDR1, CDR2, and CDR3, and is selected from the group consisting of (1) to (5) below; (1) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; (2) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19; (3) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25; (4) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31; (5) The amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO:

25.

2. A human transferrin receptor affinity peptide selected from the group consisting of the following (1) to (6): (1) A polypeptide having an amino acid sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 3 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (1) of claim 1; (2) A peptide having an amino acid sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 4 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequences shown in (2) of claim 1; (3) A polypeptide having an amino acid sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 5 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (3) of claim 1; (4) A polypeptide having an amino acid sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 6 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (4) of claim 1; (5) A polypeptide having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 7 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequence shown in (3) of claim 1; and (6) A polypeptide having an amino acid sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 55 and having three complementarity determining regions, CDR1, CDR2, and CDR3, having the amino acid sequences shown in (5) of claim 1.

3. The peptide having affinity for the human transferrin receptor according to claim 2, wherein the amino acid sequence has an identity of 95% or more.

4. The peptide with affinity for human transferrin receptor according to claim 1, which comprises an amino acid sequence selected from the group consisting of the following (1) to (6): (1) An amino acid sequence in which 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 3 have been substituted, deleted, or added, and the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; (2) An amino acid sequence in which 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 4 are substituted, deleted, or added, and the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:

19. (3) An amino acid sequence in which 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 5 are substituted, deleted, or added, and the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO:

25. (4) An amino acid sequence in which 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 6 are substituted, deleted, or added, and the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO:

31. (5) An amino acid sequence in which 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 7 are substituted, deleted, or added, and the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO:

25. (6) An amino acid sequence in which 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 55 have been substituted, deleted, or added, and in which the amino acid sequence of CDR1 is the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, the amino acid sequence of CDR2 is the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 56, and the amino acid sequence of CDR3 is the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO:

25.

5. The human transferrin receptor affinity peptide according to claim 1, which has affinity for both the extracellular domain of the human transferrin receptor and the extracellular domain of the monkey transferrin receptor.

6. The dissociation constant with the extracellular domain of the human transferrin receptor is 5 x 10 －９ ~1 x 10 －７ The peptide with affinity for human transferrin receptor according to claim 5,

7. The peptide having affinity for the human transferrin receptor according to claim 5, wherein the dissociation constant with the monkey transferrin receptor is 0.5 to 2.5, when the dissociation constant with the extracellular domain of the human transferrin receptor is set to 1.

8. A human transferrin receptor affinity peptide-drug conjugate, comprising the human transferrin receptor affinity peptide according to any one of claims 1 to 7 and a drug bound thereto.

9. The complex according to claim 8, wherein the drug is any one of another protein (A), a nucleic acid, or a low molecular weight compound.

10. A fusion protein of the peptide having affinity for human transferrin receptor according to any one of claims 1 to 7 and another protein (A).

11. The fusion protein according to claim 10, wherein the other protein (A) is a cytokine, a growth factor, or an antibody drug.

12. The other protein (A) is selected from the group consisting of brain-derived nerve growth factor (BDNF), 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 (IL-1), interleukin (IL-2), interleukin (IL-3), interleukin (IL-4), interleukin (IL-5), interleukin (IL-6), interleukin (IL-7), interleukin (IL-8), interleukin (IL-1), interleukin (IL-1), interleukin (IL-1), interleukin (IL-1), interleukin (IL-1), interleukin (IL-2), interleukin (IL-3), interleukin (IL-1 ...

11. The fusion protein according to claim 10, wherein the antibody is selected from the group consisting of phenylalanine 6, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage-colony-stimulating factor (M-CSF), tumor necrosis factor α receptor (TNF-α receptor), PD-1 ligand, PD-L1, PD-L2, an enzyme having an activity of degrading beta-amyloid, an anti-beta-amyloid antibody, an anti-BACE antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-HER2 antibody, an anti-TNF-α antibody, and an anti-CTLA-4 antibody.

13. The other protein (A) is a lysosomal enzyme, and the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, acid α-glucosidase, glucocerebrosidase, β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase.

11. The fusion protein of claim 10, wherein the fusion protein is selected from the group consisting of A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2.

14. The fusion protein of claim 10, which is bound to serum albumin.

15. The fusion protein according to claim 10, to which a human IgG Fc region or a part thereof is bound.

16. A nucleic acid encoding the peptide having affinity for the human transferrin receptor according to any one of claims 1 to 7.

17. A nucleic acid encoding the fusion protein of claim 10.