Variants of alpha-n-acetylglucosaminidase

Abstract

Provided is a novel hNAGLU variant capable of increasing the expression level of hNAGLU in a host cell into which a gene encoding human α-N-acetylglucosaminidase (hNAGLU) has been introduced, as compared with a case where a gene encoding wild-type hNAGLU has been introduced.SOLUTION: An hNAGLU variant obtained by modifying the amino acid sequence of hNAGLU, the variant having a specific amino acid sequence or an amino acid sequence obtained by adding a mutation to the amino acid sequence.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a mutant of human α-N-acetylglucosaminidase (hNAGLU), and more particularly to a novel hNAGLU mutant that, by adding a mutation to the amino acid sequence of hNAGLU, can increase the expression level of hNAGLU in a host cell into which a gene encoding hNAGLU has been introduced, compared to when a gene encoding wild-type hNAGLU has been introduced. [Background technology]

[0002] Mucopolysaccharidosis type IIIB (MPS-IIIB), a lysosomal disorder also known as Sanfilippo syndrome type B, is a genetic disease caused by a genetic defect in the enzyme α-N-acetylglucosaminidase (NAGLU), which is required for the degradation of heparan sulfate (HS), a type of glycosaminoglycan (GAG), in lysosomes. In severe cases, the accumulation of HS in various organs, including the brain, manifests as cognitive decline, behavioral disorders, and other serious neurological and tissue damage between the ages of 2 and 6. Behavioral problems such as hyperactivity and mental retardation are also observed. Rapid progression of central neurodegenerative symptoms leads to severe mental retardation and motor disorders, with loss of language ability by the age of 7 or 8. By the age of 10, patients exhibit sleep disorders, hepatosplenomegaly, and seizures, becoming immobile and bedridden. Many die of respiratory infections or other causes in their 20s.

[0003] Enzyme replacement therapy is used to replace the enzymes that are deficient or missing in patients with Sanfilippo syndrome type B. The enzyme α-N-acetylglucosaminidase (NAGLU) catalyzes the hydrolysis of the non-reducing terminal α-N-acetylglucosamine residue of heparan sulfate, and when administered to patients, it can degrade HS that has accumulated in the lysosomes of the patient's body.

[0004] The gene encoding wild-type human NAGLU (hNAGLU) was isolated in 1995 (Patent Document 1). The hNAGLU used in enzyme replacement therapy is recombinant hNAGLU, which is produced using cells transformed with an expression vector incorporating the gene encoding it. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2000-500972 Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a novel hNAGLU mutant that can increase the expression level of hNAGLU in a host cell into which a gene encoding hNAGLU has been introduced, compared to when a gene encoding wild-type hNAGLU has been introduced, by introducing a mutation into the amino acid sequence of hNAGLU. [Means for solving the problem]

[0007] In the course of research aimed at the above-mentioned object, the present inventors have conducted extensive research and have found that host cells into which a gene encoding an hNAGLU mutant obtained by modifying the amino acid sequence of hNAGLU, as described in detail herein, has been introduced express more hNAGLU than host cells into which a gene encoding wild-type hNAGLU has been introduced, thereby completing the present invention. 1. A mutant of human α-N-acetylglucosaminidase (hNAGLU) selected from the group consisting of the following (1) to (7): (1) an amino acid sequence represented by SEQ ID NO: 3 in which the lysine at position 36 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with glutamic acid and the proline at position 37 is substituted with serine; (2) an amino acid sequence represented by SEQ ID NO: 5 in which serine has been added between leucine at position 44 and glycine at position 45 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1; (3) an amino acid sequence represented by SEQ ID NO: 9 in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with arginine; (4) an amino acid sequence represented by SEQ ID NO: 11 in which glutamic acid at position 228 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with lysine; (5) an amino acid sequence represented by SEQ ID NO: 15 in which the threonine at position 320 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with proline and the glutamic acid at position 321 is substituted with aspartic acid; (6) an amino acid sequence represented by SEQ ID NO: 17 in which the serine at position 505 in the wild-type hNAGLU amino acid sequence represented by SEQ ID NO: 1 is substituted with alanine and the isoleucine at position 506 is substituted with valine; and (7) An amino acid sequence shown in SEQ ID NO: 19 in which the serine at position 526 in the amino acid sequence of wild-type hNAGLU shown in SEQ ID NO: 1 is replaced with asparagine and the alanine at position 528 is replaced with threonine. 2. An hNAGLU mutant obtained by adding mutations to the hNAGLU mutant of item 1 above having the amino acid sequence shown in SEQ ID NO: 3 while preserving glutamic acid at position 36 and serine at position 37 of the amino acid sequence, said hNAGLU mutant being selected from the group consisting of the following (1'-a) to (1'-h): (1'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (1'-b) a polypeptide in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (1'-c) a combination of the substitution in 1'-a and the deletion in 1'-b; (1'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (1'-e) A combination of the substitution of 1'-a and the addition of 1'-d; (1'-f) the combination of the deletion of 1'-b and the addition of 1'-d; (1'-g) the combination of the substitution of 1'-a, the deletion of 1'-b, and the addition of 1'-d; and (1'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 3. An hNAGLU mutant obtained by adding a mutation to the hNAGLU mutant (1) of 1 above having the amino acid sequence shown in SEQ ID NO: 5 while preserving the serine at position 45 of the amino acid sequence, said hNAGLU mutant being selected from the group consisting of the following (2'-a) to (2'-h): (2'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (2'-b) a polypeptide in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (2'-c) a combination of the substitution of 2'-a and the deletion of 2'-b; (2'-d) those in which one or more amino acid residues have been added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (2'-e) A combination of the substitution of 2'-a and the addition of 2'-d; (2'-f) the combination of the deletion of 2'-b and the addition of 2'-d; (2'-g) the combination of the substitution of 2'-a, the deletion of 2'-b, and the addition of 2'-d; and (2'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 4. An hNAGLU mutant obtained by adding a mutation to the hNAGLU mutant of item 1 above having the amino acid sequence shown in SEQ ID NO: 9 while preserving the arginine at position 209 of the amino acid sequence, said hNAGLU mutant being selected from the group consisting of the following (3'-a) to (3'-h): (3'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (3'-b) a peptide in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (3'-c) the combination of the above 3'-a substitution and 3'-b deletion; (3'-d) one in which one or more amino acid residues are added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (3'-e) A combination of the above 3'-a substitution and 3'-d addition; (3'-f) the combination of the deletion of 3'-b and the addition of 3'-d; (3'-g) a combination of the above 3'-a substitution, 3'-b deletion, and 3'-d addition; and (3'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 5. An hNAGLU mutant obtained by adding a mutation to the hNAGLU mutant of item 1 above having the amino acid sequence shown in SEQ ID NO: 11 while preserving the lysine at position 228 of said amino acid sequence, said hNAGLU mutant being selected from the group consisting of the following (4'-a) to (4'-h): (4'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (4'-b) a peptide in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (4'-c) the combination of the above 4'-a substitution and 4'-b deletion; (4'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (4'-e) A combination of the substitution of 4'-a and the addition of 4'-d; (4'-f) the combination of the deletion of 4'-b and the addition of 4'-d; (4'-g) the combination of the substitution of 4'-a above, the deletion of 4'-b, and the addition of 4'-d; and (4'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 6. An hNAGLU mutant obtained by adding mutations to the hNAGLU mutant of 1 above having the amino acid sequence shown in SEQ ID NO: 15 while preserving the proline at position 320 and the aspartic acid at position 321 of the amino acid sequence, wherein the hNAGLU mutant is selected from the group consisting of the following (5'-a) to (5'-h): (5'-a) an amino acid residue constituting the amino acid sequence is substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (5'-b) a sequence in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (5'-c) the above 5'-a substitution combined with the 5'-b deletion; (5'-d) one in which one or more amino acid residues are added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (5'-e) the combination of the above 5'-a substitution and 5'-d addition; (5'-f) the combination of the above 5'-b deletion and 5'-d addition; (5'-g) the above 5'-a substitution, the 5'-b deletion, and the 5'-d addition; and (5'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 7. An hNAGLU mutant obtained by adding mutations to the hNAGLU mutant of 1 above having the amino acid sequence shown in SEQ ID NO: 17 while preserving the alanine at position 505 and the valine at position 506 of the amino acid sequence, wherein the hNAGLU mutant is selected from the group consisting of the following (6'-a) to (6'-h): (6'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (6'-b) a peptide in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (6'-c) the combination of the above 6'-a substitution and 6'-b deletion; (6'-d) one in which one or more amino acid residues are added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (6'-e) A combination of the above 6'-a substitution and 6'-d addition; (6'-f) the combination of the deletion of 6'-b and the addition of 6'-d; (6'-g) the combination of the above 6'-a substitution, 6'-b deletion, and 6'-d addition; and (6'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 8. An hNAGLU mutant obtained by adding mutations to the hNAGLU mutant of 1 above having the amino acid sequence shown in SEQ ID NO: 19 while preserving the asparagine at position 526 and the threonine at position 528 of the amino acid sequence, wherein the hNAGLU mutant is selected from the group consisting of the following (7'-a) to (7'-h): (7'-a) an amino acid residue constituting the amino acid sequence is substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (7'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (7'-c) the combination of the above 7'-a substitution and 7'-b deletion; (7'-d) those in which one or more amino acid residues have been added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (7'-e) A combination of the substitution of 7'-a and the addition of 7'-d; (7'-f) the combination of the deletion of 7'-b and the addition of 7'-d; (7'-g) the combination of the above 7'-a substitution, the 7'-b deletion, and the 7'-d addition; and (7'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 9. The hNAGLU mutant of 4 above, which is selected from the group consisting of the following (8) to (15): (8) an hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, which has been mutated while preserving the arginine at position 209 of the amino acid sequence, and which has the amino acid sequence shown in SEQ ID NO: 25, in which the lysine at position 36 is replaced with glutamic acid and the proline at position 37 is replaced with serine; (9) an hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, which has been mutated while preserving the arginine at position 209 of the amino acid sequence, and which has the amino acid sequence shown in SEQ ID NO: 27, in which serine has been added between the leucine at position 44 and the glycine at position 45; (10) an hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, which has been mutated while preserving the arginine at position 209 of the amino acid sequence, and has the amino acid sequence shown in SEQ ID NO: 29, in which the threonine at position 320 is replaced with proline and the glutamic acid at position 321 is replaced with aspartic acid; (11) An hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, which has been mutated while preserving the arginine at position 209 of the amino acid sequence, in which the lysine at position 36 has been replaced with glutamic acid, the proline at position 37 has been replaced with serine, and serine has been added between the leucine at position 44 and the glycine at position 45, resulting in an amino acid sequence shown in SEQ ID NO: 31; (12) An hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, which has been mutated while preserving the arginine at position 209 of the amino acid sequence, and has the amino acid sequence shown in SEQ ID NO: 33, in which valine at position 54 is substituted with isoleucine and arginine at position 620 is substituted with lysine; (13) An hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, which has been mutated while preserving the arginine at position 209 of the amino acid sequence, and which has the amino acid sequence shown in SEQ ID NO: 35, in which valine at position 54 has been replaced with isoleucine and serine has been added between leucine at position 44 and glycine at position 45; (14) an hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, which has been mutated while preserving the arginine at position 209 of the amino acid sequence, and which has the amino acid sequence shown in SEQ ID NO: 37, in which the arginine at position 620 is replaced with a lysine and a serine is added between the leucine at position 44 and the glycine at position 45; and (15) An hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, in which the valine at position 54 is replaced with isoleucine and the arginine at position 620 is replaced with lysine, while preserving the arginine at position 209 of the amino acid sequence, and which has the amino acid sequence shown in SEQ ID NO: 39, in which serine is added between the leucine at position 44 and the glycine at position 45. 10. An hNAGLU mutant according to 9 above, having the amino acid sequence shown in SEQ ID NO: 25, wherein mutations have been added while preserving arginine at position 209, glutamic acid at position 36, and serine at position 37 of said amino acid sequence, said hNAGLU mutant being selected from the group consisting of the following (8'-a) to (8'-h): (8'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (8'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (8'-c) the above 8'-a substitution combined with 8'-b and deletion; (8'-d) one in which one or more amino acid residues are added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (8'-e) A combination of the substitution of 8'-a and the addition of 8'-d; (8'-f) the combination of the deletion of 8'-b and the addition of 8'-d; (8'-g) the combination of the above 8'-a substitution, 8'-b deletion, and 8'-d addition; (8'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 11. An hNAGLU mutant according to claim 9, which has the amino acid sequence shown in SEQ ID NO: 27, but is mutated while preserving the arginine at position 210 and the serine at position 45 of the amino acid sequence, and is selected from the group consisting of the following (9'-a) to (9'-h): (9'-a) an amino acid residue constituting the amino acid sequence is substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (9'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (9'-c) the combination of the above 9'-a substitution and 9'-b deletion; (9'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (9'-e) A combination of the substitution of 9'-a and the addition of 9'-d; (9'-f) the combination of the deletion of 9'-b and the addition of 9'-d; (9'-g) the combination of the above 9'-a substitution, 9'-b deletion, and 9'-d addition; (9'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 12. An hNAGLU mutant according to 9 above, having the amino acid sequence shown in SEQ ID NO: 29, wherein mutations have been added while preserving arginine at position 209, proline at position 320, and aspartic acid at position 321 of said amino acid sequence, said hNAGLU mutant being selected from the group consisting of the following (10'-a) to (10'-h): (10'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (10'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (10'-c) the combination of the substitution in 10'-a and the deletion in 10'-b; (10'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (10'-e) A combination of the substitution of 10'-a and the addition of 10'-d; (10'-f) the combination of the deletion of 10'-b and the addition of 10'-d; (10'-g) the combination of the substitution of 10'-a, the deletion of 10'-b, and the addition of 10'-d; (10'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 13. An hNAGLU mutant according to 9 above, having the amino acid sequence shown in SEQ ID NO: 31, wherein mutations have been added while preserving arginine at position 210, glutamic acid at position 36, serine at position 37, and serine at position 45 of the amino acid sequence, and wherein the hNAGLU mutant is selected from the group consisting of the following (11'-a) to (11'-h): (11'-a) an amino acid residue constituting the amino acid sequence is substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (11'-b) A compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (11'-c) the combination of the substitution of 11'-a and the deletion of 11'-b; (11'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (11'-e) A combination of the substitution of 11'-a and the addition of 11'-d; (11'-f) the combination of the deletion of 11'-b and the addition of 11'-d; (11'-g) the combination of the above substitution 11'-a, deletion 11'-b, and addition 11'-d; (11'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 14. An hNAGLU mutant according to 9 above, having the amino acid sequence shown in SEQ ID NO: 33, wherein mutations have been added while preserving the arginine at position 209, the isoleucine at position 54, and the lysine at position 620 of said amino acid sequence, said hNAGLU mutant being selected from the group consisting of the following (12'-a) to (12'-h): (12'-a) an amino acid residue constituting the amino acid sequence is substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (12'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (12'-c) the combination of the substitution of 12'-a and the deletion of 12'-b; (12'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (12'-e) A combination of the substitution of 12'-a and the addition of 12'-d; (12'-f) the combination of the deletion of 12'-b and the addition of 12'-d; (12'-g) the combination of the above 12'-a substitution, 12'-b deletion, and 12'-d addition; (12'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 15. An hNAGLU mutant according to 9 above, having the amino acid sequence shown in SEQ ID NO: 35, wherein mutations have been added while preserving arginine at position 210, isoleucine at position 55, and serine at position 45 of said amino acid sequence, said hNAGLU mutant being selected from the group consisting of the following (13'-a) to (13'-h): (13'-a) an amino acid residue constituting the amino acid sequence is substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (13'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (13'-c) the combination of the substitution of 13'-a and the deletion of 13'-b; (13'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (13'-e) A combination of the substitution of 13'-a and the addition of 13'-d; (13'-f) the combination of the deletion of 13'-b and the addition of 13'-d; (13'-g) the combination of the above 13'-a substitution, the deletion of 13'-b, and the addition of 13'-d; (13'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 16. An hNAGLU mutant according to claim 9, which has the amino acid sequence shown in SEQ ID NO: 37, and which is mutated while preserving the arginine at position 210, the lysine at position 621, and the serine at position 45 of said amino acid sequence, and which is selected from the group consisting of the following (14'-a) to (14'-h): (14'-a) an amino acid residue constituting the amino acid sequence is substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (14'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (14'-c) the combination of the substitution of 14'-a and the deletion of 14'-b; (14'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (14'-e) A combination of the substitution of 14'-a and the addition of 14'-d; (14'-f) the combination of the deletion of 14'-b and the addition of 14'-d; (14'-g) the combination of the substitution of 14'-a above, the deletion of 14'-b, and the addition of 14'-d; (14'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 17. An hNAGLU mutant according to claim 9, having the amino acid sequence shown in SEQ ID NO: 39, wherein mutations have been added while preserving the arginine at position 210, the isoleucine at position 55, the lysine at position 621, and the serine at position 45 of said amino acid sequence, said hNAGLU mutant being selected from the group consisting of (15'-a) to (15'-h) below: (15'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (15'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (15'-c) the combination of the substitution of 15'-a and the deletion of 15'-b; (15'-d) those in which one or more amino acid residues have been added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (15'-e) A combination of the substitution of 15'-a and the addition of 15'-d; (15'-f) the combination of the deletion of 15'-b and the addition of 15'-d; (15'-g) the combination of the above 15'-a substitution, the deletion of 15'-b, and the addition of 15'-d; (15'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence. 18. DNA comprising a gene encoding any one of the hNAGLU mutants 1 to 17 above. 19. An expression vector comprising the DNA of 18 above. 20. A mammalian cell transformed with the expression vector of claim 19. 21. A method for producing an hNAGLU mutant, comprising culturing the mammalian cells of the above 20 in a serum-free medium. 22. A fusion protein comprising an hNAGLU mutant described in any one of 1 to 17 above and an antibody, wherein the antibody binds to a receptor on cerebrovascular endothelial cells, thereby enabling the fusion protein to pass through the blood-brain barrier (BBB). 23. The fusion protein according to 22 above, wherein the receptor on the cerebrovascular endothelial cells is selected from the group consisting of insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and IGF receptor. 24. The fusion protein according to 22 above, wherein the receptor on cerebrovascular endothelial cells is a transferrin receptor. 25. The fusion protein according to any one of 22 to 24 above, wherein the antibody is any one of a Fab antibody, a F(ab')2 antibody, a F(ab')2 antibody, a single domain antibody, a single chain antibody, and an Fc antibody. 26. A fusion protein described in any one of 22 to 25 above, wherein the hNAGLU mutant is bound to either the C-terminus or the N-terminus of the light chain of the antibody. 27. A fusion protein described in any one of 22 to 25 above, wherein the hNAGLU mutant is bound to either the C-terminus or the N-terminus of the heavy chain of the antibody. 28. A fusion protein described in any one of items 22 to 27 above, in which the hNAGLU mutant is linked via a linker sequence to either the C-terminal or N-terminal side of the antibody light chain, or to either the C-terminal or N-terminal side of the antibody heavy chain. 29. The fusion protein according to 28 above, wherein the linker sequence consists of 1 to 50 amino acid residues. 30. The fusion protein described in 29 above, wherein the linker sequence comprises an amino acid sequence selected from the group consisting of one glycine, one 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: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and amino acid sequences consisting of 1 to 10 consecutive amino acid sequences of these amino acid sequences. 31. A DNA comprising a gene encoding the fusion protein according to any one of items 22 to 30 above. 32. An expression vector comprising the DNA according to 31 above. 33. A mammalian cell transformed with the expression vector according to 32 above. 34. A method for producing a fusion protein of an hNAGLU variant and an antibody, comprising the step of culturing the mammalian cell according to 33 above in a serum-free medium. [Effects of the Invention]

[0008] According to the present invention, hNAGLU, which can be administered as enzyme replacement therapy for the treatment of patients with mucopolysaccharidosis type IIIB, can be efficiently produced using genetic recombination technology. [Brief explanation of the drawings]

[0009] [Figure 1]1 shows the results of an experiment to confirm the expression level of hNAGLU mutants by transient expression (Example 6). The vertical axis of the bar graph indicates fluorescence intensity. (a) shows the SDS-page pattern of the bands corresponding to wild-type hNAGLU or hNAGLU mutants, and (b) shows the Western blotting pattern of the bands corresponding to wild-type hNAGLU or hNAGLU mutants. (1) shows the expression data for the K36E / P37S hNAGLU mutant, (2) for the L44_G45insS hNAGLU mutant, (3) for the R129Q hNAGLU mutant, (4) for the Q209R hNAGLU mutant, (5) for the E228K hNAGLU mutant, (6) for the T240V hNAGLU mutant, (7) for the T320P / E321D hNAGLU mutant, (8) for the S505A / I506V hNAGLU mutant, (9) for the S526N / A528T hNAGLU mutant, (10) for the D613Q hNAGLU mutant, (11) for the H204K hNAGLU mutant, and (12) for the wild-type hNAGLU. [Figure 2] 10 shows the results of an experiment to confirm the expression level of hNAGLU mutants in bulk cells (Example 10). The vertical axis of the bar graph shows the enzyme activity of hNAGLU expressed in 1 x 10 cells (nmol / h / 10 cells) for each hNAGLU mutant. (1) shows the expression data for the K36E / P37S hNAGLU mutant, (2) for the L44_G45insS hNAGLU mutant, (3) for the R129Q hNAGLU mutant, (4) for the Q209R hNAGLU mutant, (5) for the E228K hNAGLU mutant, (6) for the T240V hNAGLU mutant, (7) for the T320P / E321D hNAGLU mutant, (8) for the S505A / I506V hNAGLU mutant, (9) for the S526N / A528T hNAGLU mutant, (10) for the D613Q hNAGLU mutant, (11) for the H204K hNAGLU mutant, and (12) for the wild-type hNAGLU. Measurements were performed four times for each of (1) to (4), and the respective values ​​are shown in the figure. [Figure 3]

[0046] Figure 16 shows the results of an experiment to confirm the expression level of hNAGLU mutants by transient expression. The vertical axis of the bar graph represents fluorescence intensity. (a) shows the SDS-page pattern of the bands corresponding to wild-type hNAGLU or hNAGLU mutants, and (b) shows the Western blotting pattern of the bands corresponding to wild-type hNAGLU or hNAGLU mutants. (1) shows the expression level data for wild-type hNAGLU, (2) the Q209R hNAGLU mutant, (3) the K36E / P37S / Q209R hNAGLU mutant, (4) the L44_G45insS / Q209R hNAGLU mutant, (5) the Q209R / T320P / E321D hNAGLU mutant, and (6) the K36E / P37S / L44_G45insS / Q209R hNAGLU mutant. [Figure 4] (a) shows the results of an experiment to confirm the expression level of hNAGLU mutants by transient expression (Example 16). The vertical axis of the bar graph indicates fluorescence intensity. (b) shows the SDS-page pattern of the bands corresponding to wild-type hNAGLU or hNAGLU mutants. (1) shows wild-type hNAGLU, (2) shows Q209R hNAGLU mutant, (3) shows L44_G45insS / Q209R hNAGLU mutant, (4) shows V54I / Q209R / R620K hNAGLU mutant, (5) shows L44_G45insS / V54I / Q209R hNAGLU mutant, (6) shows L44_G45insS / Q209R / R620K hNAGLU mutant, (7) shows L44_G45insS / V54I / Q209R / R620K hNAGLU mutant, and (8) shows expression level data for the negative control. DETAILED DESCRIPTION OF THE INVENTION

[0010] In this specification, the term "human α-N-acetylglucosaminidase" or "hNAGLU" refers not only to the normal wild-type hNAGLU consisting of 720 amino acid residues as shown in SEQ ID NO: 1, but also to mutants of hNAGLU in which one or more amino acid residues have been substituted, deleted, and / or added (as used herein, "addition" of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence as shown in SEQ ID NO: 1, as long as the mutants have the functions of normal wild-type hNAGLU, such as the enzymatic activity to decompose heparan sulfate. Wild-type hNAGLU is encoded, for example, by a gene having the nucleotide sequence as shown in SEQ ID NO: 2. When an amino acid residue is substituted with another amino acid residue, the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. When amino acid residues are deleted, the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. When amino acid residues are deleted, amino acid residues at the N-terminus may be deleted, in which case the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. These substitutions and deletions of amino acid residues may also be combined.

[0011] When amino acid residues are added to the amino acid sequence shown in SEQ ID NO: 1, one or more amino acid residues are added within the amino acid sequence of hNAGLU or to the N-terminal or C-terminal side of the amino acid sequence. The number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. Furthermore, addition and substitution of amino acid residues may be combined, or addition and deletion of amino acid residues may be combined, or addition, substitution, and deletion of amino acid residues may be combined. Ordinary wild-type hNAGLU is biosynthesized as a precursor consisting of 743 amino acid residues, and hNAGLU is obtained by removing a leader peptide consisting of 23 amino acid residues from the N-terminus shown in SEQ ID NO: 67. When an amino acid is added to the N-terminus of hNAGLU, the amino acid may be derived from the leader peptide. In this case, the amino acid added to the N-terminus is Gly when one amino acid is added, Ala-Gly when two amino acids are added, or Ala-Ala-Gly when two amino acids are added.

[0012] Among these three types of mutations, substitution, deletion, and addition, the following hNAGLU mutants (i) to (iv) can be introduced by combining at least two of these mutations: (i) an amino acid sequence obtained by deleting 0 to 10 amino acid residues from the amino acid sequence shown in SEQ ID NO: 1, substituting 0 to 10 amino acid residues with other amino acid residues, and adding 0 to 10 more amino acid residues; (ii) an amino acid sequence obtained by deleting 0 to 5 amino acid residues, substituting 0 to 5 amino acid residues with other amino acid residues, and adding 0 to 5 additional amino acid residues to the amino acid sequence shown in SEQ ID NO: 1; (iii) an amino acid sequence obtained by deleting 0 to 3 amino acid residues, substituting 0 to 3 amino acid residues with other amino acid residues, and adding 0 to 3 additional amino acid residues to the amino acid sequence shown in SEQ ID NO: 1; (iv) An amino acid sequence obtained by deleting 0 to 2 amino acid residues from the amino acid sequence shown in SEQ ID NO: 1, substituting 0 to 2 amino acid residues with other amino acid residues, and adding 0 to 2 additional amino acid residues.

[0013] The wild-type or mutant hNAGLU described above in which the constituent amino acids are modified with sugar chains is also hNAGLU. The wild-type or mutant hNAGLU described above in which the constituent amino acids are modified with phosphate is also hNAGLU. The wild-type or mutant hNAGLU described above in which the constituent amino acids are modified with something other than sugar chains and phosphate is also hNAGLU. The wild-type or mutant hNAGLU described above in which the side chains of the constituent amino acids have been modified by a substitution reaction or the like is also hNAGLU. Such conversions include, but are not limited to, conversion of a cysteine ​​residue to formylglycine.

[0014] In other words, hNAGLU modified with a sugar chain is included in hNAGLU having the amino acid sequence before the modification. Also, hNAGLU modified with phosphate is included in hNAGLU having the original amino acid sequence before the modification with phosphate. Also, hNAGLU modified with something other than a sugar chain or phosphate is included in hNAGLU having the original amino acid sequence before the modification. Also, hNAGLU in which the side chains of the amino acids constituting hNAGLU have been converted by a substitution reaction or the like is included in hNAGLU having the original amino acid sequence before the conversion. Such conversions include, but are not limited to, conversion of a cysteine ​​residue to formylglycine.

[0015] In the present invention, the term "human α-N-acetylglucosaminidase variant" (hNAGLU variant) refers to a variant in which one or more amino acid residues have been substituted, deleted, and / or added (as used herein, "addition" of an amino acid residue means adding a residue to the end or interior of a sequence) to the amino acid sequence of normal wild-type hNAGLU (the amino acid sequence shown in SEQ ID NO: 1), and which retains the functions of normal wild-type hNAGLU, such as possessing enzymatic activity capable of degrading heparan sulfate. Preferred hNAGLU variants in the present invention include those in which one or more amino acid residues have been substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO: 1. When amino acid residues in the amino acid sequence are substituted with other amino acid residues, the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. When amino acid residues are deleted, the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. The hNAGLU variant may also be a combination of substitution and deletion of these amino acid residues.

[0016] When amino acid residues are added to the amino acid sequence shown in SEQ ID NO: 1, one or more amino acid residues are added within the amino acid sequence of hNAGLU or to the N-terminal or C-terminal side of the amino acid sequence. The number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. Furthermore, addition and substitution of amino acid residues may be combined, addition and deletion of amino acid residues may be combined, or addition, substitution, and deletion of amino acid residues may be combined. In other words, the hNAGLU mutant is one in which at least two of the three types of mutations - amino acid substitution, deletion, and addition - have been introduced into the amino acid sequence shown in SEQ ID NO: 1.

[0017] Among these three types of mutations, substitution, deletion, and addition, the following hNAGLU mutants (i) to (iv) can be introduced by combining at least two of these mutations: (i) an amino acid sequence obtained by deleting 0 to 10 amino acid residues from the amino acid sequence shown in SEQ ID NO: 1, substituting 0 to 10 amino acid residues with other amino acid residues, and adding 0 to 10 additional amino acid residues (excluding wild-type hNAGLU); (ii) an amino acid sequence obtained by deleting 0 to 5 amino acid residues, substituting 0 to 5 amino acid residues with other amino acid residues, and adding 0 to 5 additional amino acid residues to the amino acid sequence shown in SEQ ID NO: 1 (excluding wild-type hNAGLU); (iii) an amino acid sequence obtained by deleting 0 to 3 amino acid residues, substituting 0 to 3 amino acid residues with other amino acid residues, and adding 0 to 3 additional amino acid residues to the amino acid sequence shown in SEQ ID NO: 1 (excluding wild-type hNAGLU); (iv) An amino acid sequence obtained by deleting 0 to 2 amino acid residues from the amino acid sequence shown in SEQ ID NO: 1, substituting 0 to 2 amino acid residues with other amino acid residues, and adding 0 to 2 additional amino acid residues (excluding wild-type hNAGLU).

[0018] The position and type (deletion, substitution, and addition) of each mutation in various hNAGLU mutants compared with normal wild-type hNAGLU can be easily confirmed by aligning the amino acid sequences of both hNAGLUs.

[0019] The amino acid sequence of the hNAGLU mutant preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% identity, with the amino acid sequence of the normal wild-type hNAGLU shown in SEQ ID NO: 1.

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

[0021] Substitution of an amino acid in the amino acid sequence of wild-type hNAGLU or an hNAGLU mutant with another amino acid For example, substitutions occur within a family of amino acids that are related by their side chains and chemical properties. Substitutions within such an amino acid family are predicted not to significantly alter the function of the original protein (i.e., they are conservative amino acid substitutions). Examples of such amino acid families include those shown in (1) to (12) below: (1) The acidic amino acids aspartic acid and glutamic acid, (2) Basic amino acids histidine, lysine, and arginine (3) the aromatic amino acids phenylalanine, tyrosine, and tryptophan, (4) Serine and threonine, which are amino acids with hydroxyl groups (hydroxyamino acids), (5) the hydrophobic amino acids methionine, alanine, valine, leucine, and isoleucine, (6) the neutral hydrophilic amino acids cysteine, serine, threonine, asparagine, and glutamine, (7) Glycine and proline, which are amino acids that affect the orientation of peptide chains, (8) Amide amino acids (polar 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) Alanine and glycine, which are amino acids with particularly small side chains, (12) The branched-chain amino acids valine, leucine, and isoleucine.

[0022] In one embodiment of the present invention, a highly expressed hNAGLU mutant refers to an hNAGLU mutant that, when expressed as a recombinant protein in host cells, results in an expression level that is at least 1.1-fold, 1.5-fold, 2-fold, 4-fold, 5-fold, or 6-fold higher, for example, 1.5-4-fold, 2-5-fold, 2-8-fold, etc., compared to when wild-type hNAGLU is expressed as a recombinant protein in host cells under the same conditions. "Under the same conditions" here means that the expression vector, host cells, culture conditions, etc., are the same.

[0023] Preferred embodiments of such highly expressing hNAGLU mutants include the following (1) to (7): (1) an amino acid sequence represented by SEQ ID NO: 3 in which the lysine at position 36 in the wild-type hNAGLU amino acid sequence represented by SEQ ID NO: 1 is replaced with glutamic acid and the proline at position 37 is replaced with serine; (2) an amino acid sequence represented by SEQ ID NO: 5 in which serine has been added between leucine at position 44 and glycine at position 45 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1; (3) an amino acid sequence represented by SEQ ID NO: 9 in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with arginine; (4) an amino acid sequence represented by SEQ ID NO: 11 in which glutamic acid at position 228 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with lysine; (5) an amino acid sequence represented by SEQ ID NO: 15 in which the threonine at position 320 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with proline and the glutamic acid at position 321 is substituted with aspartic acid, (6) an amino acid sequence represented by SEQ ID NO: 17 in which the serine at position 505 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with alanine and the isoleucine at position 506 is substituted with valine, (7) An amino acid sequence shown in SEQ ID NO: 19 in which the serine at position 526 in the amino acid sequence of wild-type hNAGLU shown in SEQ ID NO: 1 is replaced with asparagine and the alanine at position 528 is replaced with threonine.

[0024] Furthermore, preferred embodiments of such highly expressing hNAGLU mutants include the following (1') to (7'). (1') Without mutations at glutamic acid at position 36 and serine at position 37 of the amino acid sequence shown in SEQ ID NO:3: (1'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (1'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (1'-c) a combination of the substitution in 1'-a and the deletion in 1'-b; (1'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (1'-e) A combination of the substitution of 1'-a and the addition of 1'-d; (1'-f) the combination of the deletion of 1'-b and the addition of 1'-d; (1'-g) the combination of the substitution of 1'-a, the deletion of 1'-b, and the addition of 1'-d; (1'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0025] (2') Without mutation at serine at position 45 of the amino acid sequence shown in SEQ ID NO:5: (2'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (2'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (2'-c) a combination of the substitution of 2'-a and the deletion of 2'-b; (2'-d) those in which one or more amino acid residues have been added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (2'-e) A combination of the substitution of 2'-a and the addition of 2'-d; (2'-f) the combination of the deletion of 2'-b and the addition of 2'-d; (2'-g) the combination of the above substitution of 2'-a, deletion of 2'-b, and addition of 2'-d; (2'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0026] (3') Without mutation to arginine at position 209 of the amino acid sequence shown in SEQ ID NO:9: (3'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (3'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (3'-c) the combination of the above 3'-a substitution and 3'-b deletion; (3'-d) one in which one or more amino acid residues are added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (3'-e) A combination of the above 3'-a substitution and 3'-d addition; (3'-f) the combination of the deletion of 3'-b and the addition of 3'-d; (3'-g) the combination of the above 3'-a substitution, 3'-b deletion, and 3'-d addition; (3'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0027] (4') Without mutation of lysine at position 228 of the amino acid sequence shown in SEQ ID NO: 11: (4'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (4'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (4'-c) the combination of the above 4'-a substitution and 4'-b deletion; (4'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (4'-e) A combination of the substitution of 4'-a and the addition of 4'-d; (4'-f) the combination of the deletion of 4'-b and the addition of 4'-d; (4'-g) the combination of the above 4'-a substitution, the deletion of 4'-b, and the addition of 4'-d; (4'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0028] (5') Without mutations at proline at position 320 and aspartic acid at position 321 of the amino acid sequence shown in SEQ ID NO: 15: (5'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (5'-b) a polypeptide obtained by deleting amino acid residues constituting the amino acid sequence, wherein the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (5'-c) the above 5'-a substitution combined with the 5'-b deletion; (5'-d) one in which one or more amino acid residues are added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (5'-e) the combination of the above 5'-a substitution and 5'-d addition; (5'-f) the combination of the above 5'-b deletion and 5'-d addition; (5'-g) A combination of the above 5'-a substitution, 5'-b deletion and 5'-d addition; (5'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0029] (6') Without mutations at alanine at position 505 and valine at position 506 of the amino acid sequence shown in SEQ ID NO: 17: (6'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (6'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (6'-c) the combination of the above 6'-a substitution and 6'-b deletion; (6'-d) one in which one or more amino acid residues are added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (6'-e) A combination of the above 6'-a substitution and 6'-d addition; (6'-f) the combination of the deletion of 6'-b and the addition of 6'-d; (6'-g) the combination of the above 6'-a substitution, 6'-b deletion, and 6'-d addition; (6'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0030] (7') Without mutations at asparagine at position 526 and threonine at position 528 of the amino acid sequence shown in SEQ ID NO: 19: (7'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (7'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (7'-c) the combination of the above 7'-a substitution and 7'-b deletion; (7'-d) those in which one or more amino acid residues have been added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (7'-e) A combination of the substitution of 7'-a and the addition of 7'-d; (7'-f) the combination of the deletion of 7'-b and the addition of 7'-d; (7'-g) the combination of the above 7'-a substitution, 7'-b deletion and 7'-d addition; (7'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0031] Specific embodiments of the high-expression hNAGLU mutant obtained by further introducing a mutation into the high-expression hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9 in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU shown in SEQ ID NO: 1 in (1) above is replaced with arginine include the following (8) to (15): (8) an amino acid sequence represented by SEQ ID NO: 25 in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with arginine, lysine at position 36 with glutamic acid, and proline at position 37 with serine, (9) an amino acid sequence represented by SEQ ID NO: 27 in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is replaced with arginine and serine is added between leucine at position 44 and glycine at position 45; (10) an amino acid sequence represented by SEQ ID NO: 29 in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with arginine, threonine at position 320 with proline, and glutamic acid at position 321 with aspartic acid; (11) An amino acid sequence represented by SEQ ID NO: 31 in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with arginine, lysine at position 36 with glutamic acid, and proline at position 37 with serine, and serine is added between leucine at position 44 and glycine at position 45; (12) An amino acid sequence represented by SEQ ID NO: 33 in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with arginine, valine at position 54 with isoleucine, and arginine at position 620 with lysine, (13) An amino acid sequence represented by SEQ ID NO: 35, in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with arginine, valine at position 54 is substituted with isoleucine, and serine is added between leucine at position 44 and glycine at position 45, (14) An amino acid sequence represented by SEQ ID NO: 37, in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with arginine, arginine at position 620 is substituted with lysine, and serine is added between leucine at position 44 and glycine at position 45; (15) An amino acid sequence represented by SEQ ID NO: 39 in which glutamine at position 209 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is replaced with arginine, valine at position 54 with isoleucine, and arginine at position 620 with lysine, and serine is added between leucine at position 44 and glycine at position 45.

[0032] Furthermore, preferred embodiments of such highly expressing hNAGLU mutants include the following (8') to (15').

[0033] (8') Without mutations at arginine at position 209, glutamic acid at position 36, and serine at position 37 of the amino acid sequence shown in SEQ ID NO: 25: (8'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (8'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (8'-c) the combination of the above 8'-a substitution and 8'-b deletion; (8'-d) one in which one or more amino acid residues are added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (8'-e) A combination of the substitution of 8'-a and the addition of 8'-d; (8'-f) the combination of the deletion of 8'-b and the addition of 8'-d; (8'-g) the combination of the above 8'-a substitution, 8'-b deletion, and 8'-d addition; (8'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0034] (9') Without mutations at arginine at position 210 and serine at position 45 of the amino acid sequence shown in SEQ ID NO: 27: (9'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (9'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (9'-c) the combination of the above 9'-a substitution and 9'-b deletion; (9'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (9'-e) A combination of the substitution of 9'-a and the addition of 9'-d; (9'-f) the combination of the deletion of 9'-b and the addition of 9'-d; (9'-g) the combination of the above 9'-a substitution, 9'-b deletion, and 9'-d addition; (9'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0035] (10') Without mutations at arginine at position 209, proline at position 320, and aspartic acid at position 321 of the amino acid sequence shown in SEQ ID NO: 29: (10'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (10'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (10'-c) the combination of the substitution in 10'-a and the deletion in 10'-b; (10'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (10'-e) A combination of the substitution of 10'-a and the addition of 10'-d; (10'-f) the combination of the deletion of 10'-b and the addition of 10'-d; (10'-g) the combination of the substitution of 10'-a, the deletion of 10'-b, and the addition of 10'-d; (10'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0036] (11') Without adding mutations to arginine at position 210, glutamic acid at position 36, serine at position 37, and serine at position 45 of the amino acid sequence shown in SEQ ID NO: 31: (11'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (11'-b) A compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (11'-c) the combination of the substitution of 11'-a and the deletion of 11'-b; (11'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (11'-e) A combination of the substitution of 11'-a and the addition of 11'-d; (11'-f) the combination of the deletion of 11'-b and the addition of 11'-d; (11'-g) the combination of the above substitution 11'-a, deletion 11'-b, and addition 11'-d; (11'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0037] (12') Without mutations at arginine at position 209, isoleucine at position 54, and lysine at position 620 of the amino acid sequence shown in SEQ ID NO: 33: (12'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (12'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (12'-c) the combination of the substitution of 12'-a and the deletion of 12'-b; (12'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (12'-e) A combination of the substitution of 12'-a and the addition of 12'-d; (12'-f) the combination of the deletion of 12'-b and the addition of 12'-d; (12'-g) the combination of the above 12'-a substitution, 12'-b deletion, and 12'-d addition; (12'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0038] (13') Without mutations at arginine at position 210, isoleucine at position 55, and serine at position 45 of the amino acid sequence shown in SEQ ID NO: 35: (13'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (13'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (13'-c) the combination of the substitution of 13'-a and the deletion of 13'-b; (13'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (13'-e) A combination of the substitution of 13'-a and the addition of 13'-d; (13'-f) the combination of the deletion of 13'-b and the addition of 13'-d; (13'-g) the combination of the above 13'-a substitution, the deletion of 13'-b, and the addition of 13'-d; (13'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0039] (14') Without mutations at arginine at position 210, lysine at position 621, and serine at position 45 of the amino acid sequence shown in SEQ ID NO: 37: (14'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (14'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (14'-c) the combination of the substitution of 14'-a and the deletion of 14'-b; (14'-d) those in which one or more amino acid residues have been added to the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (14'-e) A combination of the substitution of 14'-a and the addition of 14'-d; (14'-f) the combination of the deletion of 14'-b and the addition of 14'-d; (14'-g) the combination of the substitution of 14'-a above, the deletion of 14'-b, and the addition of 14'-d; (14'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0040] (15') Without mutations at arginine at position 210, isoleucine at position 55, lysine at position 621, and serine at position 45 of the amino acid sequence shown in SEQ ID NO: 39: (15'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence has been substituted with another amino acid residue, and the number of substituted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (15'-b) a compound in which amino acid residues constituting the amino acid sequence have been deleted, and the number of deleted amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (15'-c) the combination of the substitution of 15'-a and the deletion of 15'-b; (15'-d) those in which one or more amino acid residues have been added within the amino acid sequence or to the N-terminal or C-terminal side of the amino acid sequence, and the number of added amino acid residues is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (15'-e) A combination of the substitution of 15'-a and the addition of 15'-d; (15'-f) the combination of the deletion of 15'-b and the addition of 15'-d; (15'-g) the combination of the above 15'-a substitution, the deletion of 15'-b, and the addition of 15'-d; (15'-h) Those that show 80% or more, 85% or more, 90% or more, 95% or more identity, 98% or more, or 99% identity with the amino acid sequence.

[0041] In one embodiment of the present invention, an hNAGLU mutant is characterized by a higher expression level when expressed as a recombinant protein in a host cell compared to when wild-type hNAGLU is expressed as a recombinant protein in a host cell under the same conditions. In this specification, this type of hNAGLU mutant is referred to as a high-expression hNAGLU mutant. When produced as a recombinant protein, a high-expression hNAGLU mutant can increase production efficiency compared to wild-type hNAGLU, thereby reducing production costs. Note that "under the same conditions" here means that the expression vector, host cells, culture conditions, etc. are the same.

[0042] A gene encoding an hNAGLU mutant whose expression level is increased compared to wild-type hNAGLU when expressed as a recombinant protein in host cells can be obtained, for example, by the following method. Genes encoding mutated hNAGLU and wild-type hNAGLU are each inserted into the same type of expression vector, and the same type of host cell is transformed with these vectors to obtain cells into which each expression vector has been introduced. These cells are then cultured under the same conditions, and the hNAGLU obtained in the culture supernatant is quantified. The gene introduced into cells that shows a higher quantification value compared to the quantification value of cells into which the gene encoding wild-type hNAGLU has been introduced is identified as the gene encoding the desired highly expressed hNAGLU mutant. The quantification value here can be the quantification value of hNAGLU as a protein, or the enzymatic activity value of hNAGLU. For example, the total amount of enzymatic activity of hNAGLU contained in the culture supernatant can also be used as the quantification value.

[0043] Mutations can be introduced into wild-type hNAGLU by randomly introducing mutations into the gene encoding wild-type hNAGLU. For example, mutations can be introduced into the gene encoding wild-type hNAGLU by inserting the gene encoding wild-type hNAGLU into a vector, transforming the vector with the vector, and then exposing the transformed host cell to a mutagen (radiation, mutagenic substance, etc.).

[0044] Mutations can also be introduced into wild-type hNAGLU by introducing mutations into a predetermined site in the gene encoding wild-type hNAGLU. For example, such gene mutations can be introduced by chemically synthesizing a gene having a mutation at a predetermined site.

[0045] Highly expressing hNAGLU mutants can be produced as recombinant proteins by culturing host cells transformed with an expression vector incorporating a gene encoding the mutant.

[0046] The host cells used in this case are not particularly limited as long as they are capable of expressing a highly expressed hNAGLU mutant by introducing such an expression vector, and may be any of eukaryotic cells such as mammalian cells, yeast, plant cells, insect cells, etc., or prokaryotic cells such as Escherichia coli and Bacillus subtilis, although mammalian cells are particularly preferred.

[0047] When mammalian cells are used as host cells, there are no particular limitations on the type of mammalian cell, but cells derived from humans, mice, or Chinese hamsters are preferred, with Chinese hamster ovary-derived CHO cells or mouse myeloma-derived NS / 0 cells being particularly preferred. Furthermore, the expression vector used to incorporate and express a DNA fragment encoding a highly expressed hNAGLU mutant can be any expression vector, as long as it results in expression of the gene when introduced into mammalian cells. The gene incorporated into the expression vector is located downstream of a DNA sequence (gene expression control site) that can regulate the frequency of gene transcription in mammalian cells. Examples of gene expression control sites that can be used in the present invention include a cytomegalovirus-derived promoter, the SV40 early promoter, the human elongation factor-1α (EF-1α) promoter, and the human ubiquitin C promoter.

[0048] Expression vectors are known in which glutamine synthetase (GS) is placed as a selection marker downstream of a gene encoding a target protein via an internal ribosome entry site (IRES) (International Patent Publications WO2012 / 063799, WO2013 / 161958). The expression vectors described in these publications are particularly suitable for producing highly expressing hNAGLU mutants.

[0049] For example, an expression vector for expressing a target protein, which comprises a first gene expression control site, a gene encoding the protein downstream thereof, an internal ribosome binding site further downstream thereof, and a gene encoding glutamine synthetase further downstream thereof, and further comprises a dihydrofolate reductase gene or a drug resistance gene downstream of the first gene expression control site or a second gene expression control site different from the first gene expression control site, can be suitably used to produce a highly expressed hNAGLU mutant. In this expression vector, the first or second gene expression control site can be suitably selected from promoters derived from cytomegalovirus, SV40 early promoter, human elongation factor-1α promoter (hEF-1α promoter), and human ubiquitin C promoter, with the hEF-1α promoter being particularly suitable.

[0050] Furthermore, the internal ribosome binding site preferably is derived from the 5' untranslated region of the genome of a virus selected from the group consisting of Picornaviridae viruses, foot-and-mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine enteric virus, Theiler's murine encephalomyelitis virus, and Coxsackie B virus, or a gene selected from the group consisting of the human immunoglobulin heavy chain binding protein gene, the Drosophila antennapedia gene, and the Drosophila ultravithorax gene. An internal ribosome binding site derived from the 5' untranslated region of the murine encephalomyocarditis virus genome is particularly preferred. When using an internal ribosome binding site derived from the 5' untranslated region of the murine encephalomyocarditis virus genome, in addition to the wild-type internal ribosome binding site, one in which some of the multiple initiation codons contained in the wild-type internal ribosome binding site have been disrupted can also be preferably used. Furthermore, the drug resistance gene preferably used in this expression vector is preferably a puromycin or neomycin resistance gene, and more preferably a puromycin resistance gene.

[0051] Furthermore, for example, an expression vector for expressing a target protein, which comprises a human elongation factor-1α promoter, a gene encoding the protein downstream thereof, an internal ribosome binding site derived from the 5' untranslated region of the murine encephalomyocarditis virus genome further downstream thereof, and a gene encoding glutamine synthetase further downstream thereof, and further comprises another gene expression control site and a dihydrofolate reductase gene downstream thereof, wherein the internal ribosome binding site is one in which some of the multiple initiation codons contained in the wild-type internal ribosome binding site have been disrupted, can be suitably used to produce a highly expressing hNAGLU mutant. Examples of such expression vectors include those described in WO2013 / 161958.

[0052] Furthermore, for example, an expression vector for expressing a target protein, which comprises a human elongation factor-1α promoter, a gene encoding the target protein downstream thereof, an internal ribosome binding site derived from the 5' untranslated region of the murine encephalomyocarditis virus genome further downstream thereof, and a gene encoding glutamine synthetase further downstream thereof, and further comprises another gene expression control site and a drug resistance gene downstream thereof, wherein the internal ribosome binding site is one in which some of the multiple initiation codons contained in the wild-type internal ribosome binding site have been disrupted, can be suitably used to produce a highly expressing hNAGLU mutant. Examples of such expression vectors include pE-mIRES-GS-puro described in WO2012 / 063799 and pE-mIRES-GS-mNeo described in WO2013 / 161958.

[0053] The wild-type murine encephalomyocarditis virus genome contains three initiation codons (ATG) at the 3' end of the internal ribosome binding site (IRBS) derived from the 5' untranslated region. The above-mentioned pE-mIRES-GS-puro and pE-mIRES-GS-mNeo are expression vectors with an IRES in which some of the initiation codons have been destroyed.

[0054] hNAGLU mutants (including high-expression hNAGLU mutants) can be expressed in cells or in culture media by culturing host cells into which an expression vector containing a gene encoding the mutant has been introduced. Methods for expressing hNAGLU mutants when mammalian cells are used as host cells are described in detail below.

[0055] Any medium can be used for culturing mammalian cells without any particular limitations as long as it allows mammalian cells to be cultured and grown, but preferably a serum-free medium is used. In the present invention, a serum-free medium used as a medium for producing recombinant proteins is preferably one containing, for example, 3 to 700 mg / L of amino acids, 0.001 to 50 mg / L of vitamins, 0.3 to 10 g / L of monosaccharides, 0.1 to 10,000 mg / L of inorganic salts, 0.001 to 0.1 mg / L of trace elements, 0.1 to 50 mg / L of nucleosides, 0.001 to 10 mg / L of fatty acids, 0.01 to 1 mg / L of biotin, 0.1 to 20 μg / L of hydrocortisone, 0.1 to 20 mg / L of insulin, 0.1 to 10 mg / L of vitamin B12, 0.01 to 1 mg / L of putrescine, 10 to 500 mg / L of sodium pyruvate, and a water-soluble iron compound. If desired, thymidine, hypoxanthine, a conventional pH indicator, an antibiotic, etc. may be added to the medium.

[0056] As a serum-free medium used as a medium for recombinant protein production, DMEM / F12 medium (a mixed medium of DMEM and F12) may be used as a basal medium, and these media are well known to those skilled in the art. Furthermore, as a serum-free medium, DMEM (HG) HAM modified (R5) medium, which contains sodium bicarbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, iron (II) sulfate, asparagine, aspartic acid, serine, and polyvinyl alcohol, may also be used. Furthermore, commercially available serum-free media, such as CD OptiCHO TM Culture medium, CHO-S-SFM II medium or CD CHO medium (Thermo Fisher Scientific, formerly Life Technologies), IS cho-VTM Culture medium (Irvine Scientific), EX-CELL TM 302 medium or EX-CELL TM 325-PF medium (SAFC Biosciences) or the like can also be used as the basal medium.

[0057] A highly expressed hNAGLU variant is characterized in that when mammalian cells transfected with an expression vector incorporating a gene encoding the variant are cultured in the above-mentioned serum-free medium to express the variant as a recombinant protein, the expression level obtained is at least 1.1 times, 1.5 times, 2 times, 4 times, 5 times, or 6 times higher, for example, 1.5 to 4 times, 2 to 5 times, 2 to 8 times, etc., compared to when wild-type hNAGLU is expressed as a recombinant protein under the same conditions. The mammalian cells used in this case include CHO cells, NS / 0 cells, etc., but are particularly CHO cells.

[0058] By culturing host cells encoding the hNAGLU mutant, the recombinant hNAGLU mutant expressed in the cells or culture medium can be separated from impurities and purified by methods such as column chromatography. The purified hNAGLU mutant can be used as a medicine. In particular, the hNAGLU mutant can be used as a medicine targeting mucopolysaccharidosis type IIIB (MPS-IIIB), also known as Sanfilippo syndrome type B.

[0059] Pharmaceuticals containing hNAGLU variants as active ingredients can be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, or intracerebroventricularly as injections. These injections can be supplied as lyophilized preparations or aqueous solutions. In the case of aqueous solutions, they may be supplied in vials or as pre-filled syringes. In the case of lyophilized preparations, they are dissolved in an aqueous medium and reconstituted before use.

[0060] In one embodiment of the present invention, the hNAGLU variant can be conjugated to an antibody. For example, in one embodiment of the present invention, the hNAGLU variant can be conjugated to an antibody capable of specifically binding to a receptor on cerebrovascular endothelial cells. By conjugating the hNAGLU variant to an antibody capable of specifically binding to a receptor on cerebrovascular endothelial cells, the hNAGLU variant can be made to pass through the blood-brain barrier (BBB) ​​and function in the central nervous system (CNS). Examples of such receptors on cerebrovascular endothelial cells include, but are not limited to, insulin receptors, transferrin receptors, leptin receptors, lipoprotein receptors, and IGF receptors. Furthermore, the receptor is preferably derived from a human.

[0061] In the present invention, the term "antibody" primarily refers to human antibodies, mouse antibodies, humanized antibodies, antibodies derived from camelids (including alpacas), chimeric antibodies between human antibodies and antibodies from other mammals, and chimeric antibodies between mouse antibodies and antibodies from other mammals, but is not limited to these as long as it has the property of specifically binding to a specific antigen, and there are also no particular restrictions on the animal species of the antibody.

[0062] In the present invention, the term "human antibody" refers to an antibody whose entire protein is encoded by a gene of human origin. However, "human antibodies" also include antibodies encoded by genes in which mutations have been added to the original human gene without changing the original amino acid sequence, for purposes such as increasing gene expression efficiency. Furthermore, "human antibodies" also include antibodies created by combining two or more genes encoding human antibodies and substituting a portion of one human antibody for a portion of another human antibody. Human antibodies have three complementarity-determining regions (CDRs) in the light chain and three complementarity-determining regions (CDRs) in the heavy chain. The three CDRs in the light chain are referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. The three CDRs in the heavy chain are also referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. Human antibodies also include antibodies in which the antigen specificity, affinity, etc. of a human antibody have been modified by substituting the CDRs of one human antibody with the CDRs of another human antibody.

[0063] In the present invention, the term "human antibody" also includes antibodies in which mutations such as substitutions, deletions, and additions have been added to the amino acid sequence of the original antibody by modifying the gene of the original human antibody. When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of the original antibody are deleted, the number of deleted amino acids is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, antibodies in which mutations combining these amino acid substitutions and deletions have been added are also human antibodies. When amino acids are added, preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of the original antibody. Antibodies in which mutations combining these amino acid additions, substitutions, and deletions have been added are also human antibodies. The amino acid sequence of the mutated antibody preferably exhibits 80% or more identity with the amino acid sequence of the original antibody, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity. When the above mutations are added to a human antibody, the mutations can be added to the variable region of the antibody. When the above mutations are added to the variable region of the antibody, the mutations can be added to either the CDR or framework region of the variable region, but are particularly added to the framework region. In other words, in the present invention, the term "human-derived gene" includes not only the original human-derived gene, but also genes obtained by modifying it.

[0064] In the present invention, the term "humanized antibody" refers to an antibody in which the amino acid sequence of a portion of the variable region (e.g., all or part of the CDRs in particular) is derived from a mammal other than human, and the remaining regions are derived from humans. For example, a humanized antibody may be produced by replacing three complementarity-determining regions (CDRs) of the light chain and three complementarity-determining regions (CDRs) of the heavy chain constituting a human antibody with CDRs from another mammal. The other mammalian species from which the CDRs are grafted to appropriate positions in a human antibody is not particularly limited as long as it is a mammal other than human, but is preferably a mouse, rat, rabbit, horse, or non-human primate, more preferably a mouse or rat, and even more preferably a mouse. Furthermore, antibodies in which mutations similar to those that can be added to the amino acid sequence of the original humanized antibody have been added are also included in the "humanized antibody."

[0065] In the present invention, the term "chimeric antibody" refers to an antibody formed by linking fragments of two or more different antibodies derived from two or more different species.

[0066] A chimeric antibody between a human antibody and an antibody from another mammal is an antibody in which parts of a human antibody are replaced with parts of an antibody from a mammal other than a human. The antibody consists of an Fc region, Fab region, and hinge region, as explained below. A specific example of such a chimeric antibody is a chimeric antibody in which the Fc region is derived from a human antibody while the Fab region is derived from an antibody from another mammal. The hinge region is derived from either a human antibody or an antibody from another mammal. Conversely, a chimeric antibody in which the Fc region is derived from another mammal while the Fab region is derived from a human antibody is also included. The hinge region is derived from either a human antibody or an antibody from another mammal.

[0067] Alternatively, an antibody can be said to consist of a variable region and a constant region. Another specific example of a chimeric antibody is a heavy chain constant region (C H ) and the light chain constant region (C L ) is derived from a human antibody, while the variable region of the heavy chain (V H ) and the light chain variable region (V L) derived from antibodies of other mammals, conversely, the heavy chain constant region (C H ) and the light chain constant region (C L ) are derived from antibodies of other mammals, while the variable region of the heavy chain (V H ) and the light chain variable region (V L ) derived from a human antibody. Here, the other mammalian species is not particularly limited as long as it is a mammal other than a human, but is preferably a mouse, rat, rabbit, horse, or non-human primate, such as a mouse.

[0068] An antibody in one embodiment of the present invention has a basic structure consisting of two immunoglobulin light chains (or simply "light chains") and two immunoglobulin heavy chains (or simply "heavy chains"), a total of four polypeptide chains. However, in the present invention, the term "antibody" includes those having this basic structure as well as: (1) Consists of two polypeptide chains, one light chain and one heavy chain, (2) Those consisting of an Fab region, which is the basic structure of an antibody in the original sense, with the Fc region deleted, and those consisting of an Fab region and all or part of the hinge region (including Fab, F(ab') and F(ab')2), (3) A single-chain antibody, which is composed of a light chain with a linker sequence at the C-terminus thereof and a heavy chain at the C-terminus thereof. (4) A single-chain antibody, which is composed of a heavy chain with a linker sequence at the C-terminus thereof and a light chain bound to the C-terminus thereof. (5) An antibody consisting of an Fc region in which the Fab region has been deleted from the basic structure of the original antibody, and the amino acid sequence of the Fc region has been modified so that it has the property of specifically binding to a specific antigen (Fc antibody). (6) Single domain antibodies, which will be described later, are also included in the "antibodies" of the present invention.

[0069] An antibody in one embodiment of the present invention is an antibody derived from a camelid (including alpaca). Some camelid antibodies consist of two heavy chains linked by a disulfide bond. An antibody consisting of these two heavy chains is called a heavy-chain antibody. A VHH is an antibody consisting of a single heavy chain including the variable region of the heavy chain that constitutes a heavy-chain antibody, or an antibody consisting of a single heavy chain lacking the constant region (CH) that constitutes a heavy-chain antibody. A VHH is also an antibody in an embodiment of the present invention. Another antibody in an embodiment of the present invention is an antibody consisting of two light chains linked by a disulfide bond. An antibody consisting of these two light chains is called a light-chain antibody. An antibody in one embodiment of the present invention is one in which mutations have been made to the amino acid sequence of a camelid antibody (including VHH) in order to reduce the antigenicity when the camelid-derived antibody (including VHH) is administered to humans. When mutations are made to the amino acids of a camelid antibody, the same mutations that can be made to the antibodies described herein can be made.

[0070] An antibody in one embodiment of the present invention is a shark-derived antibody. Shark antibodies consist of two heavy chains linked by a disulfide bond. An antibody consisting of these two heavy chains is called a heavy-chain antibody. VNAR is an antibody consisting of a single heavy chain that includes the variable region of the heavy chain that constitutes a heavy-chain antibody, or an antibody consisting of a single heavy chain that lacks the constant region (CH) that constitutes a heavy-chain antibody. VNAR is also one of the antibodies in an embodiment of the present invention. Shark-derived antibodies (including VNAR) with mutations added to the amino acid sequence to reduce antigenicity when administered to humans are also antibodies in one embodiment of the present invention. When mutations are added to the amino acids of shark antibodies, mutations similar to those that can be added to antibodies described herein can be added. Humanized shark antibodies are also one of the antibodies in an embodiment of the present invention.

[0071] An antibody has a basic structure consisting of four polypeptide chains: two light chains and two heavy chains. L ) contains three complementarity-determining regions (CDRs) and a heavy chain variable region (V H) have three complementarity determining regions (CDRs). The three CDRs of the light chain are called CDR1, CDR2, and CDR3, starting from the N-terminus. The three CDRs of the heavy chain are also called CDR1, CDR2, and CDR3, starting from the N-terminus. However, even if some or all of these CDRs are incomplete or absent, they are included in the antibody as long as they have the property of specifically binding to a specific antigen. The variable regions (V L and V H The regions other than the CDRs of a protein are called framework regions (FRs). The FRs are called FR1, FR2, FR3, and FR4, starting from the N-terminus. Usually, the CDRs and FRs are present in the following order from the N-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0072] In one embodiment of the present invention, antibodies include antibodies in which mutations such as substitution, deletion, and addition have been added to the amino acid sequence of the original antibody. When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of the original antibody are deleted, the number of deleted amino acids is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, antibodies in which mutations combining these amino acid substitutions and deletions have been added are also antibodies. When amino acids are added, preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of the original antibody. Antibodies in which mutations combining these amino acid additions, substitutions, and deletions have been added are also antibodies. The amino acid sequence of the mutated antibody preferably exhibits 80% or more identity to the amino acid sequence of the original antibody, more preferably 85% or more identity, and even more preferably 90% or more, 95% or more, or 98% or more identity.

[0073] In one embodiment of the present invention, antibodies include antibodies in which mutations such as substitution, deletion, and addition have been added to the amino acid sequence of the variable region of the original antibody. When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of the original antibody are deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, antibodies in which mutations combining these amino acid substitutions and deletions have been added are also antibodies. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of the original antibody. Antibodies in which mutations combining these amino acid additions, substitutions, and deletions have been added are also antibodies. The amino acid sequence of the mutated antibody preferably exhibits 80% or more identity with the amino acid sequence of the original antibody, more preferably 85% or more identity, and even more preferably 90% or more, 95% or more, or 98% or more identity. When mutations are added to the variable region of an antibody, the mutations may be added to either the CDR or framework region of the variable region, but are particularly added to the framework region.

[0074] In one embodiment of the present invention, antibodies include those in which mutations such as substitution, deletion, or addition have been added to the amino acid sequence of the framework region of the variable region of the original antibody. When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of the original antibody are deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, antibodies in which mutations combining these amino acid substitutions and deletions have been added are also antibodies. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of the original antibody. Antibodies in which mutations combining these amino acid additions, substitutions, and deletions have been added are also antibodies. The amino acid sequence of the mutated antibody preferably exhibits 80% or more identity to the amino acid sequence of the original antibody, more preferably 85% or more identity, and even more preferably 90% or more, 95% or more, or 98% or more identity.

[0075] In one embodiment of the present invention, antibodies include those in which mutations such as substitution, deletion, or addition have been added to the amino acid sequence of the CDR region of the variable region of the original antibody. When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2. When amino acids in the amino acid sequence of the original antibody are deleted, the number of deleted amino acids is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2. Furthermore, antibodies in which mutations combining these amino acid substitutions and deletions have been added are also antibodies. When amino acids are added, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of the original antibody. Antibodies in which mutations combining these amino acid additions, substitutions, and deletions have been added are also antibodies. The amino acid sequence of the mutated antibody preferably exhibits 80% or more identity to the amino acid sequence of the original antibody, more preferably 85% or more identity, and even more preferably 90% or more, 95% or more, or 98% or more identity.

[0076] The identity between the amino acid sequence of the original antibody and the amino acid sequence of the mutated antibody can be easily calculated using well-known homology calculation algorithms, such as BLAST (Altschul SF. J Mol. Biol. 215. 403-10, (1990)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), and the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2. 482-9 (1981)).

[0077] In one embodiment of the present invention, Fab is a fragment of a variable region and a C L One light chain containing the constant region (the light chain constant region) and one variable region and C HFab is a molecule in which one heavy chain containing one region (the constant region of the heavy chain) is bound by disulfide bonds between the cysteine ​​residues present in each heavy chain. In Fab, the heavy chain consists of a variable region and a C H In addition to the region 1 (part 1 of the heavy chain constant region), a portion of the hinge region may also be included, but in this case the hinge region lacks the cysteine ​​residues present in the hinge region that bind the heavy chains of an antibody. In Fab, the light chain and the heavy chain are connected by the light chain constant region (C L The cysteine ​​residues in the heavy chain constant region (C H The heavy chains that make up Fab are called Fab heavy chains. Fab lacks the cysteine ​​residues that connect the heavy chains of antibodies in the hinge region, so it consists of one light chain and one heavy chain. The light chains that make up Fab consist of a variable region and a C L The heavy chain that makes up Fab contains the variable region and C H It may consist of one domain, a variable domain, C H It may also contain a portion of the hinge region in addition to the variable region. In this case, however, the hinge region is selected so as not to contain cysteine ​​residues that connect the heavy chains, so that disulfide bonds are not formed between the two heavy chains at the hinge region. In F(ab'), the heavy chain consists of a variable region and a C HIn addition to the first region, the heavy chain includes all or part of the hinge region containing the cysteine ​​residues that link the heavy chains. F(ab')2 refers to a molecule in which two F(ab')s are bonded by disulfide bonds between cysteine ​​residues present in their hinge regions. The heavy chain that forms F(ab') or F(ab')2 is called an Fab' heavy chain. Furthermore, polymers such as dimers and trimers formed by multiple antibodies linked directly or via linkers are also antibodies. Furthermore, the term "antibody" as used herein includes any entity that contains a portion of an antibody molecule and has the property of specifically binding to an antigen. In other words, the term "light chain" as used herein includes those derived from a light chain and having all or part of the amino acid sequence of its variable region. Furthermore, the term "heavy chain" includes those derived from a heavy chain and having all or part of the amino acid sequence of its variable region. Therefore, as long as it has all or part of the amino acid sequence of the variable region, even those lacking the Fc region, for example, are heavy chains.

[0078] Also, here, Fc or Fc region refers to the C region in an antibody molecule. H 2 region (part 2 of the heavy chain constant region), and C H It refers to a region containing a fragment consisting of three regions (part 3 of the heavy chain constant region).

[0079] Furthermore, the antibody in one embodiment of the present invention is (7) Also included are scFab, scF(ab'), and scF(ab')2, which are single-chain antibodies formed by linking the light chain and heavy chain constituting the Fab, F(ab'), or F(ab')2 shown in (2) above via a linker sequence. Here, scFab, scF(ab'), and scF(ab')2 may be formed by linking a linker sequence to the C-terminus of the light chain and then linking a heavy chain to that C-terminus, or by linking a linker sequence to the C-terminus of the heavy chain and then linking a light chain to that C-terminus. Furthermore, the antibodies of the present invention also include scFv, which are single-chain antibodies formed by linking the variable region of the light chain and the variable region of the heavy chain via a linker sequence. In the case of scFv, it may be composed of a linker sequence attached to the C-terminus of the light chain variable region, and then the heavy chain variable region attached to the C-terminus of that, or it may be composed of a linker sequence attached to the C-terminus of the heavy chain variable region, and then the light chain variable region attached to the C-terminus of that.

[0080] Furthermore, the term "antibody" as used herein includes not only full-length antibodies but also antigen-binding fragments (antibody fragments) in which a portion of a full-length antibody is deleted, which is a broader concept that includes (1) to (7), in addition to full-length antibodies and those shown in (1) to (7). Antigen-binding fragments also include heavy chain antibodies, light chain antibodies, VHHs, VNARs, and those in which a portion of these is deleted.

[0081] The term "antigen-binding fragment" refers to a fragment of an antibody that retains at least a portion of its specific binding activity to an antigen. Examples of binding fragments include Fab, Fab', F(ab')2, variable region (Fv), heavy chain variable region (V), and so on. H ) and the light chain variable region (V L ) linked with an appropriate linker, a single-chain antibody (scFv), a heavy chain variable region (V H ) and the light chain variable region (V L ) and scFv heavy chain (H chain) containing a portion of the constant region (C H 3) includes minibodies, which are dimers of the combined antibody, and other low molecular weight antibodies, etc. However, the present invention is not limited to these molecules as long as they have the ability to bind to antigens.

[0082] In one embodiment of the present invention, the term "single-chain antibody" refers to a protein that can specifically bind to a specific antigen, comprising an amino acid sequence containing all or part of a light-chain variable region, to which a linker sequence is attached at the C-terminus, and to which an amino acid sequence containing all or part of a heavy-chain variable region is further attached at the C-terminus of the linker sequence. A protein that can specifically bind to a specific antigen, comprising an amino acid sequence containing all or part of a heavy-chain variable region, to which a linker sequence is attached at the C-terminus of the linker sequence, and to which an amino acid sequence containing all or part of a light-chain variable region is further attached at the C-terminus of the linker sequence, is also a "single-chain antibody" in the present invention. In a single-chain antibody in which a light chain is attached to the C-terminus of a heavy chain via a linker sequence, the heavy chain typically lacks an Fc region. The light-chain variable region has three complementarity-determining regions (CDRs) that are involved in the antigen specificity of the antibody. Similarly, the heavy-chain variable region also has three CDRs. These CDRs are the main regions that determine the antigen specificity of an antibody. Therefore, it is preferable that a single-chain antibody contains all three CDRs of the heavy chain and all three CDRs of the light chain. However, as long as the antigen-specific affinity of the antibody is maintained, the single-chain antibody may also be one in which one or more CDRs are deleted.

[0083] In single-chain antibodies, the linker sequence disposed between the light and heavy chains of the antibody is a peptide chain composed of preferably 2 to 50, more preferably 8 to 50, even more preferably 10 to 30, and even more preferably 12 to 18 or 15 to 25 amino acid residues, for example, 15 or 25. The amino acid sequence of such a linker sequence is not limited as long as the anti-hTfR antibody linking both chains retains affinity for hTfR, but preferably is composed of only glycine or glycine and serine, such as the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence in which any of these amino acid sequences is repeated 2 to 10 times or 2 to 5 times. For example, when linking the light chain variable region to the C-terminus of an amino acid sequence consisting of the entire heavy chain variable region via a linker sequence, a linker sequence containing a total of 15 amino acids corresponding to three consecutive amino acids of the amino acid sequence Gly-Gly-Gly-Gly-Ser (sequence number 3) is preferred.

[0084] In one embodiment of the present invention, a single-domain antibody refers to an antibody that has the property of specifically binding to an antigen through a single variable region. Single-domain antibodies include antibodies whose variable region consists only of the variable region of a heavy chain (heavy-chain single-domain antibodies) and antibodies whose variable region consists only of the variable region of a light chain (light-chain single-domain antibodies). VHH and VNAR are types of single-domain antibodies.

[0085] In one embodiment of the present invention, the term "human transferrin receptor" refers to a membrane protein having the amino acid sequence shown in SEQ ID NO: 57. In one embodiment, the antibody of the present invention specifically binds to the portion of the amino acid sequence shown in SEQ ID NO: 57 from the 89th cysteine ​​residue from the N-terminus to the phenylalanine at the C-terminus (extracellular domain of the transferrin receptor), but is not limited to this.

[0086] A common method for producing antibodies against a desired protein is to produce a recombinant protein using cells into which an expression vector incorporating a gene encoding the protein has been introduced, and then immunize an animal such as a mouse with this recombinant protein. After immunization, antibody-producing cells against the recombinant protein are extracted from the animal, and these are fused with myeloma cells to produce hybridoma cells capable of producing antibodies against the recombinant protein.

[0087] Furthermore, cells that produce antibodies against a desired protein can also be obtained by immunizing immune system cells obtained from animals such as mice with the desired protein using in vitro immunization. When using in vitro immunization, there are no particular limitations on the animal species from which the immune system cells are derived, but preferred are mice, rats, rabbits, guinea pigs, dogs, cats, horses, and primates including humans, more preferably mice, rats, and humans, and even more preferably mice and humans. For example, splenocytes prepared from mouse spleens can be used as mouse immune system cells. For human immune system cells, cells prepared from human peripheral blood, bone marrow, spleen, etc. can be used. When human immune system cells are immunized using in vitro immunization, human antibodies against the recombinant protein can be obtained.

[0088] Hybridoma cells capable of producing antibodies can be produced by immunizing immune system cells using in vitro immunization and then fusing the cells with myeloma cells. Alternatively, mRNA can be extracted from the immunized cells, cDNA can be synthesized, and DNA fragments containing genes encoding the light and heavy chains of immunoglobulins can be amplified by PCR using this cDNA as a template, and these can then be used to artificially reconstruct antibody genes.

[0089] The hybridoma cells obtained as they are by the above method include cells that produce antibodies that recognize undesired proteins as antigens. Furthermore, not all hybridoma cells that produce antibodies against a desired protein necessarily produce antibodies that exhibit the desired properties, such as high affinity for that protein.

[0090] Similarly, artificially reconstructed antibody genes include genes that encode antibodies that recognize undesired proteins as antigens. Furthermore, not all genes that encode antibodies against a desired protein necessarily encode antibodies that exhibit desired properties, such as high affinity for that protein.

[0091] Therefore, a step of selecting hybridoma cells that produce antibodies with desired properties from the hybridoma cells obtained as described above is required. Furthermore, in the case of artificially reconstructed antibody genes, a step of selecting a gene that encodes an antibody with desired properties from the antibody genes is required. For example, the method described in detail below is effective as a method of selecting hybridoma cells that produce antibodies that exhibit high affinity for a desired protein (high-affinity antibodies), or a gene that encodes a high-affinity antibody.

[0092] For example, when selecting hybridoma cells that produce antibodies with high affinity for a desired protein, the protein is added to a plate and allowed to remain there, followed by the addition of the hybridoma cell culture supernatant, followed by the removal of antibodies not bound to the protein from the plate, and the measurement of the amount of antibody retained on the plate. According to this method, the higher the affinity of the antibodies contained in the hybridoma cell culture supernatant added to the plate for the protein, the greater the amount of antibody retained on the plate. Therefore, the amount of antibody retained on the plate can be measured, and hybridoma cells corresponding to plates retaining a greater number of antibodies can be selected as cell lines that produce antibodies with relatively high affinity for the protein. From the cell lines selected in this way, mRNA can be extracted to synthesize cDNA, and the cDNA can be used as a template to amplify a DNA fragment containing the gene encoding the antibody against the protein using PCR, thereby isolating the gene encoding the high-affinity antibody.

[0093] When selecting a gene encoding an antibody against a target protein with high affinity from the above-mentioned artificially reconstructed antibody genes, the artificially reconstructed antibody gene is first incorporated into an expression vector, and this expression vector is then introduced into a host cell. The host cells used in this case are not particularly limited, regardless of whether they are prokaryotic or eukaryotic, as long as they can express the antibody gene by introducing an expression vector incorporating the artificially reconstructed antibody gene. However, mammalian cells such as human, mouse, or Chinese hamster are preferred, with Chinese hamster ovary-derived CHO cells or mouse myeloma-derived NS / 0 cells being particularly preferred. Furthermore, the expression vector used to incorporate and express a gene encoding an antibody gene can be used without particular limitation, as long as it expresses the gene when introduced into mammalian cells. The gene incorporated into the expression vector is located downstream of a DNA sequence (gene expression control site) that can regulate the frequency of gene transcription in mammalian cells. Examples of gene expression control sites that can be used in the present invention include a cytomegalovirus-derived promoter, the SV40 early promoter, the human elongation factor-1 alpha (EF-1α) promoter, and the human ubiquitin C promoter.

[0094] Mammalian cells into which such an expression vector has been introduced express the artificially reconstructed antibody incorporated into the expression vector. To select cells that produce antibodies with high affinity for antibodies against a desired protein from the cells expressing the artificially reconstructed antibody obtained in this manner, the protein is added to a plate and allowed to remain there, then the cell culture supernatant is contacted with the protein, and antibodies not bound to the protein are removed from the plate, and the amount of antibody retained on the plate is measured. According to this method, the higher the affinity of the antibody contained in the cell culture supernatant for the protein, the greater the amount of antibody retained on the plate. Therefore, by measuring the amount of antibody retained on the plate, cells corresponding to plates retaining a greater amount of antibody can be selected as cell lines that produce antibodies with a relatively high affinity for the protein, and thus a gene encoding an antibody with high affinity for the protein can be selected. From the cell lines selected in this manner, a DNA fragment containing the gene encoding the antibody against the protein can be amplified using PCR, thereby isolating the gene encoding the high-affinity antibody.

[0095] Methods for producing a conjugate of an hNAGLU variant and an antibody according to the present invention include linking them via a non-peptide linker or a peptide linker. Examples of non-peptide linkers that can be used include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethers, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, as well as derivatives thereof, or combinations thereof. A peptide linker is a peptide chain or derivative thereof consisting of 1 to 50 peptide-bonded amino acids, whose N-terminus and C-terminus form covalent bonds with either the hNAGLU variant or the antibody, respectively, thereby linking the hNAGLU variant and the antibody.

[0096] An antibody and an hNAGLU variant can also be linked by a peptide bond at the N- or C-terminus of the antibody heavy or light chain, respectively, via a linker sequence or directly. A conjugate formed by linking an antibody and an hNAGLU variant in this manner can be obtained as a fusion protein by inserting a DNA fragment, in which a cDNA encoding an hNAGLU variant is placed in frame at the 3'- or 5'-end of the cDNA encoding the antibody heavy or light chain, directly or via a DNA fragment encoding a linker sequence, into an expression vector for mammalian cells and culturing mammalian cells transfected with this expression vector. When a DNA fragment encoding an hNAGLU variant is linked to the heavy chain, an expression vector for mammalian cells incorporating a cDNA fragment encoding the antibody light chain can also be introduced into the same host cell. When a DNA fragment encoding an hNAGLU variant is linked to the light chain, an expression vector for mammalian cells incorporating a cDNA fragment encoding the antibody heavy chain can also be introduced into the same host cell. When the antibody is a single-chain antibody, a fusion protein combining the antibody and an hNAGLU mutant can be obtained by incorporating a DNA fragment in which a cDNA encoding the single-chain antibody is linked to the 5'- or 3'-end of the cDNA encoding the hNAGLU mutant, either directly or via a DNA fragment encoding a linker sequence, into an expression vector (for mammalian cells, eukaryotic cells such as yeast, or prokaryotic cells such as E. coli), and expressing the DNA fragment in these cells into which this expression vector has been introduced. The fusion protein of the antibody and the hNAGLU mutant can be produced as a recombinant protein by the above-mentioned method.

[0097] Any medium can be used for culturing mammalian cells into which an expression vector has been introduced, as long as it is capable of culturing and growing mammalian cells, but preferably a serum-free medium is used. In the present invention, a serum-free medium used as a medium for producing recombinant proteins is preferably one containing, for example, 3 to 700 mg / L of amino acids, 0.001 to 50 mg / L of vitamins, 0.3 to 10 g / L of monosaccharides, 0.1 to 10,000 mg / L of inorganic salts, 0.001 to 0.1 mg / L of trace elements, 0.1 to 50 mg / L of nucleosides, 0.001 to 10 mg / L of fatty acids, 0.01 to 1 mg / L of biotin, 0.1 to 20 μg / L of hydrocortisone, 0.1 to 20 mg / L of insulin, 0.1 to 10 mg / L of vitamin B12, 0.01 to 1 mg / L of putrescine, 10 to 500 mg / L of sodium pyruvate, and a water-soluble iron compound. If desired, thymidine, hypoxanthine, a conventional pH indicator, an antibiotic, etc. may be added to the medium.

[0098] As a serum-free medium used as a medium for producing a fusion protein of an antibody and a hNAGLU mutant, DMEM / F12 medium (a mixed medium of DMEM and F12) may be used as a basal medium, and these media are well known to those skilled in the art. Furthermore, as a serum-free medium, DMEM (HG) HAM modified (R5) medium, which contains sodium bicarbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, ferrous sulfate (II), asparagine, aspartic acid, serine, and polyvinyl alcohol, may also be used. Furthermore, commercially available serum-free media, such as CD OptiCHO TM Culture medium, CHO-S-SFM II medium or CD CHO medium (Thermo Fisher Scientific, formerly Life Technologies), IS cho-V TM Culture medium (Irvine Scientific), EX-CELL TM 302 medium or EX-CELL TM 325-PF medium (SAFC Biosciences) or the like can also be used as the basal medium.

[0099] Preferred embodiments of the fusion protein of an antibody and a hNAGLU mutant include the following (1) to (7): (1) A conjugate comprising an hNAGLU variant bound directly or via a linker to the C-terminus of an antibody heavy chain and an antibody light chain; (2) A conjugate comprising an hNAGLU variant bound directly or via a linker to the N-terminus of an antibody heavy chain and an antibody light chain; (3) A conjugate comprising an hNAGLU variant bound directly or via a linker to the C-terminus of an antibody light chain and an antibody heavy chain; (4) A conjugate comprising an hNAGLU mutant bound directly or via a linker to the N-terminus of an antibody light chain and an antibody heavy chain.

[0100] When an antibody and an hNAGLU mutant are linked via a linker sequence, the linker sequence placed between the antibody and the hNAGLU mutant is a peptide chain preferably consisting of 1 to 60 or 1 to 50, more preferably 1 to 17, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids, but the number of amino acids constituting the linker sequence can be appropriately adjusted to 1, 2, 3, 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, 27, etc. Such a linker sequence is not limited in its amino acid sequence, as long as the antibody linked thereto retains its affinity for the receptor on cerebrovascular endothelial cells and the hNAGLU mutant linked via the linker sequence can exert the physiological activity of the hNAGLU mutant under physiological conditions, but preferably consists of glycine and serine. Examples include those consisting of a single amino acid, either glycine or serine, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 58), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 59), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 60), or a sequence consisting of 1 to 10 or 2 to 5 consecutive amino acids of these amino acid sequences. Also included are sequences consisting of 1 to 50 amino acids, or sequences consisting of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, or 27 amino acids. For example, those containing the amino acid sequence Gly-Ser can be suitably used as a linker sequence. In addition, a linker sequence containing a total of 27 amino acids consisting of the amino acid sequence Gly-Ser followed by five consecutive amino acids of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 58) can be suitably used.Furthermore, a linker sequence containing a total of 25 amino acids consisting of five consecutive amino acids of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 58) can also be suitably used.

[0101] In the present invention, when one peptide chain contains multiple linker sequences, for convenience, the linker sequences are named in order from the N-terminus side as the first linker sequence, the second linker sequence, and so on.

[0102] Specific examples of antibodies to be bound to the hNAGLU mutants of the present invention include the following anti-human transferrin receptor antibodies (anti-hTfR antibodies): (1) an anti-hTfR antibody, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 61 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 62; and (2) An anti-hTfR antibody, which is a Fab antibody, the light chain of which comprises the amino acid sequence of SEQ ID NO: 61 and the heavy chain of which comprises the amino acid sequence of SEQ ID NO: 63; However, the present invention is not limited to these, and the above amino acid sequences can be appropriately mutated by substitution, deletion, addition, etc. The anti-hTfR antibodies in (1) and (2) above are humanized anti-hTfR antibodies.

[0103] When amino acids in the amino acid sequence of the light chain of the anti-human transferrin receptor antibody are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2. When amino acids in the light chain amino acid sequence are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2. Mutations that combine these amino acid substitutions and deletions can also be added.

[0104] When amino acids are added to the amino acid sequence of the light chain of the above-mentioned anti-human transferrin receptor antibody, preferably 1 to 10 amino acids are added to the amino acid sequence of the light chain or to the N-terminus or C-terminus, more preferably 1 to 5 amino acids, even more preferably 1 to 3 amino acids, and even more preferably 1 or 2 amino acids. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated light chain preferably exhibits 80% or more identity, more preferably 90% or more identity, and even more preferably 95% or more identity with the amino acid sequence of the original light chain.

[0105] When amino acids in the amino acid sequence of the heavy chain of the anti-human transferrin receptor antibody are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2. When amino acids in the heavy chain amino acid sequence are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2. Mutations that combine these amino acid substitutions and deletions can also be added.

[0106] When amino acids are added to the amino acid sequence of the heavy chain of the above-mentioned anti-human transferrin receptor antibody, preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2 amino acids are added within the amino acid sequence of the heavy chain or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated heavy chain preferably exhibits 80% or more identity, more preferably 90% or more identity, and even more preferably 95% or more identity with the amino acid sequence of the original heavy chain.

[0107] The hNAGLU variants of the present invention can be bound to antibodies against receptors on cerebrovascular endothelial cells, allowing them to cross the blood-brain barrier and function in the brain. Therefore, they can be used to manufacture drugs for administration into the bloodstream to treat central nervous system disease states caused by NAGLU deficiency. Furthermore, hNAGLU variants bound to antibodies can be used in treatment methods that involve administering a therapeutically effective amount of a central nervous system disease state caused by NAGLU deficiency into the bloodstream (including intravenous injection such as intravenous drip infusion) to a patient. The hNAGLU variants bound to antibodies administered into the bloodstream can reach not only the brain but also other organs and tissues that express NAGLU. Furthermore, these drugs can also be used to prevent the onset of these disease states.

[0108] The hNAGLU variants of the present invention can be used as drugs that exert their pharmacological effects in the central nervous system (CNS) by binding to antibodies against receptors on cerebrovascular endothelial cells and administering them into the bloodstream. Such drugs are generally administered to patients by intravenous injection, such as intravenous drip infusion, subcutaneous injection, or intramuscular injection, but the administration route is not particularly limited.

[0109] In one embodiment of the present invention, the hNAGLU mutant is not limited to an antibody, and can also be conjugated with other proteins. The other proteins are not particularly limited, but may be, for example, proteins of human origin. A conjugate of an hNAGLU mutant with the other protein can be obtained by the above-mentioned method for producing a conjugate of an hNAGLU mutant with an antibody. Furthermore, a fusion protein of an hNAGLU mutant with the other protein can be obtained as a recombinant protein by the above-mentioned method for producing a fusion protein with an antibody. One embodiment of the present invention includes a gene encoding a fusion protein of an hNAGLU mutant with the other protein, an expression vector incorporating the gene, and a host cell into which the expression vector has been introduced.

[0110] In one embodiment of the present invention, the hNAGLU variant can be conjugated with not only an anti-hTfR antibody but also a substance capable of binding to human hTfR. The substance is not particularly limited, and an example of the protein is human transferrin. Human transferrin is not limited to wild-type, and may be a partial fragment or mutant thereof as long as it has affinity for hTfR. Such a conjugate can pass through the blood-brain barrier and exert its function in the brain. A fusion protein of an hNAGLU variant and human transferrin can be obtained as a recombinant protein by the method for producing a fusion protein with an antibody described above. One embodiment of the present invention includes a gene encoding the fusion protein of an hNAGLU variant and human transferrin, an expression vector incorporating the gene, and a host cell into which the expression vector has been introduced. [Example]

[0111] 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.

[0112] Example 1: Construction of expression vectors for hNAGLU mutants A DNA fragment containing the wild-type hNAGLU gene and having the nucleotide sequence shown in SEQ ID NO: 41 was synthesized. Using this as a template, PCRs Nos. 1 to 7 and 11 shown in Table 1 were performed using an MluI-tagged 5' primer having the nucleotide sequence shown in SEQ ID NO: 42 as the forward primer and a primer shown in Table 1 as the reverse primer. Table 1 shows the SEQ ID NOs corresponding to the nucleotide sequences of each reverse primer.

[0113] [Table 1]

[0114] Next, PCR was performed using a DNA fragment having the base sequence shown in SEQ ID NO: 41 as a template, the PCR products obtained by PCR Nos. 1 to 7 and 11 shown in Table 1 as forward primers, and a His-tag-NotI-added 3' primer having the base sequence shown in SEQ ID NO: 43 as a reverse primer, to obtain PCR products containing genes encoding the hNAGLU mutants shown in Table 2. Table 2 shows the sequence numbers corresponding to the amino acid sequences of each hNAGLU mutant, the sequence numbers corresponding to the base sequences encoding them, and the number of each hNAGLU mutant (mutant number).

[0115] [Table 2]

[0116] Using a DNA fragment containing the wild-type hNAGLU gene and having the nucleotide sequence shown in SEQ ID NO: 41 as a template, PCRs Nos. 8 to 10 shown in Table 3 were performed using the primers shown in Table 3 as forward primers and the His-tag-NotI-added 3' primer having the nucleotide sequence shown in SEQ ID NO: 43 as a reverse primer. Table 3 shows the SEQ ID NOs corresponding to the nucleotide sequences of each forward primer.

[0117] [Table 3]

[0118] Next, PCR was performed using a DNA fragment having the base sequence shown in SEQ ID NO: 41 as a template, an MluI-added 5' primer having the base sequence shown in SEQ ID NO: 42 as a forward primer, and the PCR products obtained by PCR Nos. 8 to 10 shown in Table 3 as reverse primers, to obtain PCR products containing genes encoding the hNAGLU mutants shown in Table 4. Table 4 shows the sequence numbers corresponding to the amino acid sequences of each hNAGLU mutant, the sequence numbers corresponding to the base sequences encoding them, and the number of each hNAGLU mutant (mutant number).

[0119] [Table 4]

[0120] Next, the PCR products obtained from PCR Nos. 1 to 11 and the DNA fragment encoding wild-type hNAGLU having the nucleotide sequence shown in SEQ ID NO: 41 were digested with restriction enzymes MluI and NotI (Takara Bio Inc.) and separated by agarose gel electrophoresis. After EtBr staining, the band containing the target DNA fragment was excised under UV irradiation, and the DNA was extracted from the gel using a QIAEX II Gel Extraction Kit (QIAGEN). Similarly, the pCI-neo vector (Promega Corp.) and pEI-puro vector were digested with restriction enzymes MluI and NotI, followed by gel extraction and purification. Each restriction enzyme-digested PCR product was mixed with each restriction enzyme-digested vector, and ligation reactions were carried out at 16°C for 30 to 60 minutes using Ligation Mix (Takara Bio Inc.). A similar ligation reaction was also carried out for the DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 41, which contains the wild-type hNAGLU gene.

[0121] The pEI-puro vector was constructed as follows: pEF / myc / nuc vector (Invitrogen) was digested with restriction enzymes (KpnI and NcoI) to excise a DNA fragment containing the EF-1a promoter and its first intron, and this DNA fragment was blunt-ended with T4 DNA polymerase. Separately, pCI-neo (Invitrogen) was digested with restriction enzymes (BglII and EcoRI) to excise the region containing the CMV enhancer / promoter and intron, and then the region was blunt-ended with T4 DNA polymerase. The region containing the EF-1a promoter and its first intron (after blunt-ending) was inserted into this vector. This vector was named pE-neo vector. The pCAGIpuro vector (Miyahara M. et al., J. Biol. Chem. 275, 613-618 (2000)) was digested with restriction enzymes (NotI and BamHI) to introduce an internal ribosome binding site (IRES) derived from murine encephalomyocarditis virus (EMCV), a puromycin resistance gene (Puro), and a nucleotide sequence encoding the puromycin-resistant gene (Puro). r A DNA fragment containing the polyadenylation signal (polyA) derived from bovine growth hormone (bGH) was excised. Separately, the pE-neo vector was digested with restriction enzymes (NotI and BamHI) to clone the neomycin resistance gene (Neomycin-1). r A region of approximately 2 kbp containing the IRES, PuroR, and polyA derived from bGH was excised. A DNA fragment containing the IRES, PuroR, and polyA derived from bGH was inserted into this region. This vector was named pEI-puro vector.

[0122] Next, each ligation reaction mixture was used to infect Escherichia coli (ECOS TMXCompetent E. coli DH5α (Nippon Gene) was transformed with each transformant. To confirm whether the resulting transformants contained the desired plasmid DNA, single colonies were cultured overnight in LB liquid medium (LB Broth, Sigma-Aldrich), and a small amount of plasmid DNA was purified using the FastGene Plasmid Mini Kit (Nippon Genetics). The purified plasmid DNA was digested with restriction enzymes MluI and NotI and separated by agarose gel electrophoresis to confirm that the desired insert DNA had been inserted. In addition, Sanger sequencing analysis confirmed that the desired modifications had been introduced into each hNAGLU gene. Plasmids that were confirmed to contain the desired hNAGLU mutant or wild-type hNAGLU were purified using standard methods.

[0123] [Example 2] Transient expression of hNAGLU mutants Transient expression of hNAGLU mutants was carried out using a plasmid in which a gene encoding each hNAGLU mutant was inserted into the purified pCI-neo vector obtained in Example 1. As a control, a plasmid in which a gene encoding wild-type NAGLU was inserted into the pCI-neo vector was used.

[0124] ExpiCHO cells were transformed with a plasmid carrying the gene encoding the hNAGLU mutants and a plasmid carrying the gene encoding wild-type hNAGLU according to the High Titer protocol of the ExpiCHO Expression System (Thermo Fisher Scientific). After transformation, the cells were cultured for 8 days, and each hNAGLU mutant and wild-type hNAGLU were expressed in the culture supernatant. After cultivation, the culture medium was centrifuged to collect the culture supernatant.

[0125] [Example 3] Confirmation of hNAGLU mutant expression level by transient expression (SDS page electrophoresis) Ten microliters of the culture supernatant obtained in Example 2 was mixed with 8 μL of 2× Sample Buffer (Bio-Rad) and 2 μL of 2-mercaptoethanol and heat-denatured under reducing conditions by incubating at 100°C for 3 minutes. Five microliters of the heat-denatured sample was applied to each well of a 5-20% polyacrylamide gel placed in 50 mM Tris buffer / 380 mM glycine buffer (pH 8.3) containing 0.1% SDS, and electrophoresis was performed at a constant current of 25 mA. After electrophoresis, the gel was immersed in Oriole Fluorescent Gel Stain (Bio-Rad) and shaken at room temperature for 90 minutes. After washing the gel with pure water, protein bands were detected using a luminometer image analyzer (Amersham Imager 600RGB, Cytiva).

[0126] [Example 4] Confirmation of hNAGLU mutant expression level by transient expression (Western blotting method) Electrophoresis was performed in the same manner as in Example 3. The nitrocellulose membrane and the gel after electrophoresis were sandwiched between blotting papers soaked in 25 mM Tris buffer / 192 mM glycine buffer containing 20% ​​methanol, and the proteins were transferred to the nitrocellulose membrane by applying a current of 1.0 A and 25 V for 10 minutes in a blotting apparatus. After transfer, the nitrocellulose membrane was immersed in PBST containing 5% skim milk and shaken for 1 hour, then immersed in a solution of mouse anti-His tag mAb (Medical and Biological Laboratories) diluted to 0.4 μg / mL and shaken for 1 hour. After washing the membrane with PBST, it was immersed in a solution of anti-mouse IgG (H+L), HRP Conjugate (Promega) diluted to 0.4 μg / mL and shaken for 30 minutes, and then washed again with PBST. An HRP detection reagent (Bio-Rad) was dropped onto the transfer surface of the membrane and reacted for 5 minutes, and bands corresponding to each hNAGLU mutant and wild-type hNAGLU were detected using a luminometer image analyzer.

[0127] [Example 5] Confirmation of hNAGLU mutant expression level by transient expression (enzyme activity measurement) The sample solution was prepared by diluting the culture supernatant obtained in Example 2 10-fold with 100 mM citrate buffer (pH 4.2) containing 0.1% BSA. The standard solution was prepared by serially diluting 4-MU (4-methylumbelliferone, Sigma-Aldrich) from 400 to 35.12 μM with 100 mM citrate buffer (pH 4.2) containing 0.1% BSA. The substrate solution was prepared by diluting 4-methylumbelliferyl-N-acetyl-α-D-glucosaminide (Sigma-Aldrich), an artificial substrate for NAGLU, to 1 mmol / L with 100 mM citrate buffer (pH 4.2) containing 0.1% BSA. 25 μL of sample solution or standard solution was added to each well of a microplate, followed by 25 μL / well of substrate solution, which was then mixed using a plate shaker. After incubating the plate at 37°C for 1 hour, 150 μL of 200 mmol / L glycine-NaOH buffer (pH 10.7) was added to each well to stop the reaction. The fluorescence intensity of the released 4-MU (4-methylumbelliferone) was measured using a fluorescence plate reader (Gemini XPS, Molecular Devices) (excitation wavelength: 355 nm, emission wavelength: 460 nm). A calibration curve was created based on the measurement results of the standard solution, and the measured values ​​for each sample solution were interpolated to determine the enzyme activity.

[0128] [Example 6] Confirmation of hNAGLU mutant expression level by transient expression (Results) Figure 1 shows the results of measuring the expression levels of hNAGLU mutants by transient expression measured in Examples 2 to 4. Table 5 shows the expression levels of each hNAGLU mutant based on the enzyme activity measurement results shown in the bar graph in Figure 1, expressed as relative values ​​when the expression level of the wild type is set to 1.

[0129] [Table 5]

[0130] Seven hNAGLU mutants (mutant numbers 1 to 7), including the K36E / P37S hNAGLU mutant, L44_G45insS hNAGLU mutant, Q209R hNAGLU mutant, E228K hNAGLU mutant, T320P / E321 D hNAGLU mutant, S505A / I506V hNAGLU mutant, and S526N / A528 T hNAGLU mutant, showed higher transient expression than the wild-type hNAGLU mutant. In particular, the Q209R hNAGLU mutant (mutant number 3) showed 4.4-fold higher expression than the wild-type hNAGLU mutant. On the other hand, the R129Q hNAGLU mutant (mutant number 16), S526N / A528T hNAGLU mutant (mutant number 17), D613Q hNAGLU mutant (mutant number 18), and H204K hNAGLU mutant (mutant number 19) showed lower transient expression than the wild-type hNAGLU mutant.

[0131] Furthermore, a positive correlation was observed between the enzyme activity and the expression level of the hNAGLU mutants confirmed by SDS-PAGE electrophoresis (Fig. 1(a)).Furthermore, a positive correlation was observed between the enzyme activity and the expression level of the hNAGLU mutants confirmed by Western blotting (Fig. 1(b)).

[0132] [Example 7] Preparation of bulk cells expressing hNAGLU mutants Using a gene transfer device (Superelectroporator NEPA21, Nepa Gene), expression plasmids encoding the hNAGLU mutants or wild-type hNAGLU obtained in Example 1 were introduced into serum-free adapted CHO-K1 cells, and selective culture was performed in CD OptiCHO medium (Thermo Fisher Scientific) containing 10 μg / mL Puromycin (Thermo Fisher Scientific). The culture volume was gradually increased with repeated medium changes every 3 to 4 days, and when the cell viability during culture exceeded 90%, the cells were harvested and used as hNAGLU mutant-expressing bulk cells and wild-type hNAGLU-expressing bulk cells.

[0133] Example 8: Cultivation of bulk cells expressing hNAGLU mutants Each of the hNAGLU mutant-expressing bulk cells obtained in Example 7 and the wild-type hNAGLU-expressing bulk cells were cultured at 2 × 10 in a CD OptiCHO medium containing 10 μg / mL furomycin. 5 The cells were seeded at a cell density of 1000 cells / mL and cultured statically at 37°C in the presence of 5% CO2. After 9 days of culture, the culture supernatant was collected by centrifugation.

[0134] [Example 9] Confirmation of hNAGLU mutant expression level in bulk cells (enzyme activity measurement) The sample solution was prepared by diluting the culture supernatant obtained in Example 8 10-fold with citrate buffer (pH 4.2) containing 0.1% BSA. The substrate solution was prepared by diluting 4-methylumbelliferyl-N-acetyl-α-D-glucosaminide, an artificial substrate for hNAGLU, to 1 mmol / L with citrate buffer (pH 4.2). 25 μL of the sample solution or standard solution was added to each well of a microplate, followed by 25 μL of substrate solution per well, which was then shaken and mixed using a plate shaker. The plate was incubated at 37°C for 1 hour, and then 150 μL of 200 mmol / L glycine-NaOH buffer (pH 10.7) was added to each well to terminate the reaction. The fluorescence intensity of the released 4-MU (4-methylumbelliferone) was measured using a fluorescence plate reader (excitation wavelength: 355 nm, emission wavelength: 460 nm). A calibration curve was prepared based on the measurement results of the standard solution, and the measured values ​​of each sample solution were interpolated to determine the enzyme activity. The enzyme activity was calculated for each hNAGLU mutant at 1 × 10 6 The enzyme activity of hNAGLU expressed from 1 x 10 cells (nmol / h / 1 x 10 6 The number of cells was calculated as individual cells.

[0135] [Example 10] Confirmation of hNAGLU mutant expression level in bulk cells (Results) Figure 2 shows the results of measuring the expression levels of hNAGLU mutants in bulk cells measured in Example 9. Table 6 shows the expression levels of each hNAGLU mutant based on the enzyme activity measurement results shown in the bar graph in Figure 2, expressed as relative values ​​when the expression level of the wild type was set to 1. Bulk cell culture was repeated four times, and enzyme activity measurements were performed for each.

[0136] [Table 6]

[0137] As with transient expression, seven hNAGLU mutants (mutant numbers 1 to 7), namely, K36E / P37S hNAGLU mutant, L44_G45insS hNAGLU mutant, Q209R hNAGLU mutant, E228K hNAGLU mutant, T320P / E321D hNAGLU mutant, S505A / I506V hNAGLU mutant, and S526N / A528T hNAGLU mutant, were expressed at higher levels than the wild-type hNAGLU in bulk cells. In particular, the Q209R hNAGLU mutant (mutant number 3) showed 2.2-fold higher expression levels than the wild-type hNAGLU. Furthermore, as in the case of transient expression, the R129Q hNAGLU mutant (mutant number 16), S526N / A528T hNAGLU mutant (mutant number 17), D613Q hNAGLU mutant (mutant number 18), and H204K hNAGLU mutant (mutant number 19) had lower expression levels compared to the wild type.

[0138] Example 11: Mutation introduction into Q209R hNAGLU mutant and construction of expression vector The experimental results of Examples 1 to 10 revealed that, among the hNAGLU mutants, the Q209R hNAGLU mutant exhibited the highest expression level. Therefore, to obtain an even more highly expressing hNAGLU mutant, further mutations were introduced into the Q209R hNAGLU mutant. Using the plasmid containing the gene encoding the Q209R hNAGLU mutant obtained in Example 1 as a template, PCRs Nos. 12 to 15 shown in Table 7 were performed using an MluI-tagged 5' primer having the nucleotide sequence shown in SEQ ID NO: 42 as the forward primer and the primers shown in Table 7 as the reverse primers. Table 7 shows the sequence numbers corresponding to the nucleotide sequences of each reverse primer.

[0139] [Table 7]

[0140] Furthermore, PCR No. 16 shown in Table 8 was performed using the plasmid containing the gene encoding the Q209R hNAGLU mutant obtained in Example 1 as a template, the primer shown in Table 8 as the forward primer, and the His-tag-NotI-added 3' primer having the nucleotide sequence shown in SEQ ID NO: 43 as the reverse primer. Table 8 shows the SEQ ID NOs corresponding to the nucleotide sequences of the forward primers.

[0141] [Table 8]

[0142] Next, PCR Nos. 17 to 26 shown in Table 9 were performed using the forward and reverse primers shown in Table 9 and the plasmid containing the gene encoding the Q209R hNAGLU mutant obtained in Example 1 as a template.

[0143] [Table 9]

[0144] By PCR of the above Nos. 17 to 19, 21, and 23 to 26, DNA fragments encoding the hNAGLU mutants shown in Table 10, in which 1 to 3 additional mutations were introduced into the Q209R hNAGLU mutant, were amplified. Table 10 shows the sequence number corresponding to the amino acid sequence of each hNAGLU mutant, the sequence number corresponding to the nucleotide sequence encoding it, and the number of each hNAGLU mutant (mutant number).

[0145] [Table 10]

[0146] Next, the PCR products obtained from PCR Nos. 17-19, 21, and 23-26 were digested with restriction enzymes MluI and NotI (Takara Bio) and separated by agarose gel electrophoresis. After EtBr staining, the band containing the target DNA fragment was excised under UV irradiation, and the DNA was extracted from the gel using a QIAEX II Gel Extraction Kit (QIAGEN). Similarly, the pCI-neo vector (Promega) and pEI-puro vector were digested with restriction enzymes MluI and NotI, then gel extracted and purified. Each restriction enzyme-digested PCR product was mixed with each restriction enzyme-digested vector, and ligation reactions were carried out using Ligation Mix (Takara Bio) at 16°C for 30-60 minutes.

[0147] Next, each ligation reaction mixture was used to infect Escherichia coli (ECOS TMXCompetent E. coli DH5α (Nippon Gene) was transformed with each transformant. To confirm whether the resulting transformants contained the desired plasmid DNA, single colonies were cultured overnight in LB liquid medium (LB Broth, Sigma-Aldrich), and a small amount of plasmid DNA was purified using the FastGene Plasmid Mini Kit (Nippon Genetics). The purified plasmid DNA was digested with restriction enzymes MluI and NotI and separated by agarose gel electrophoresis to confirm that the desired insert DNA had been inserted. In addition, Sanger sequencing analysis confirmed that the desired modifications had been introduced into each hNAGLU gene. Plasmids that were confirmed to contain the desired hNAGLU mutants were purified using standard methods.

[0148] Example 12: Transient expression of hNAGLU mutants Transient expression of the hNAGLU mutant was carried out using a plasmid in which the hNAGLU mutant was integrated into the pCI-neo vector purified in Example 11. As a control, a plasmid in which wild-type hNAGLU was integrated into the pCI-neo vector was used.

[0149] According to the High Titer protocol of the ExpiCHO Expression System (Thermo Fisher Scientific), ExpiCHO cells were transformed with a plasmid carrying the gene encoding the hNAGLU mutants and a plasmid carrying the gene encoding wild-type hNAGLU. After transformation, the cells were cultured for 8 days, and each hNAGLU mutant and wild-type hNAGLU were expressed in the culture supernatant. After cultivation, the culture medium was centrifuged to collect the culture supernatant. As a negative control, the culture supernatant of untransformed cells was also collected in the same manner.

[0150] Example 13: Confirmation of hNAGLU mutant expression level by transient expression (SDS page electrophoresis) Ten microliters of the culture supernatant obtained in Example 12 was mixed with 8 μL of 2× Sample Buffer and 2 μL of 2-mercaptoethanol and heat-denatured under reducing conditions by incubating at 100°C for 3 minutes. Five microliters of the heat-denatured sample was applied to wells of a 5-20% polyacrylamide gel placed in 50 mM Tris buffer / 380 mM glycine buffer (pH 8.3) containing 0.1% SDS, and electrophoresis was performed at a constant current of 25 mA. After electrophoresis, the gel was immersed in Oriole Fluorescent Gel Stain (Bio-Rad) and shaken at room temperature for 90 minutes. After washing the gel with pure water, protein bands were detected using a luminometer image analyzer.

[0151] Example 14: Confirmation of hNAGLU mutant expression level by transient expression (Western blotting) Electrophoresis was performed as described in Example 13. The nitrocellulose membrane and the electrophoresed gel were sandwiched between blotting papers soaked in 25 mM Tris buffer / 192 mM glycine buffer containing 20% ​​methanol, and the proteins were transferred to the nitrocellulose membrane by applying a current of 1.0 A and 25 V for 10 minutes in a blotting apparatus. After transfer, the nitrocellulose membrane was immersed in PBST containing 5% skim milk and shaken for 1 hour, then immersed in a mouse anti-His tag mAb solution diluted to 0.4 μg / mL and shaken for 1 hour. After washing the membrane with PBST, it was immersed in an anti-mouse IgG (H+L), HRP conjugate solution diluted to 0.4 μg / mL and shaken for 30 minutes, then washed again with PBST. HRP detection reagent was added dropwise to the transfer surface of the membrane, allowed to react for 5 minutes, and detected using a luminometer image analyzer.

[0152] Example 15: Confirmation of hNAGLU mutant expression level by transient expression (enzyme activity measurement) The sample solution was prepared by diluting the culture supernatant obtained in Example 12 10-fold with citrate buffer (pH 4.2) containing 0.1% BSA. The substrate solution was prepared by diluting 4-methylumbelliferyl-N-acetyl-α-D-glucosaminide, an artificial substrate for hNAGLU, to 1 mmol / L with citrate buffer (pH 4.2). The sample solution or standard solution was added to a microplate at 25 μL / well, and 25 μL of substrate solution was added to each well and mixed using a plate shaker. The plate was incubated at 37°C for 1 hour, after which the reaction was stopped by adding 150 μL / well of 200 mmol / L glycine-NaOH buffer (pH 10.7). The fluorescence intensity of the released 4-MU (4-methylumbelliferone) was measured using a fluorescence plate reader (excitation wavelength: 355 nm, emission wavelength: 460 nm). A calibration curve was prepared based on the measurement results of the standard solution, and the enzyme activity was calculated by interpolating the measured values ​​of each sample solution.

[0153] [Example 16] Confirmation of hNAGLU mutant expression level by transient expression (Results) Figures 3 and 4 show the results of measuring the expression levels of hNAGLU mutants by transient expression measured in Examples 12 to 15. Table 11 shows the expression levels of each hNAGLU mutant based on the enzyme activity measurement results shown in the bar graphs in Figures 3 and 4, as relative values ​​when the expression level of the wild type is set to 1.

[0154] [Table 11]

[0155] Further mutations in the Q209R hNAGLU mutant, K36E / P37S / Q209R hNAGLU mutant, L44_G45insS / Q209R hNAGLU mutant, Q209R / T320P / E321D hNAGLU mutant, K36E / P37S / L44_G45insS / Q209R hNAGLU mutant, V54I / Q209R / R620K hNAGLU mutant, L44_G45insS / V54I / Q209Rh NAGLU mutant, L44_G45insS / Q209R / R620K hNAGLU mutant, and L44_G45insS / V54I / Q209R / R620K hNAGLU mutant (mutants 8 to 15), all of which are Q209R hNAGLU mutants. The V54I / Q209R / R620K hNAGLU mutant showed higher transient expression than the wild-type hNAGLU mutant. In particular, the V54I / Q209R / R620K hNAGLU mutant showed approximately 1.8-fold higher transient expression (5.9-fold higher than the wild-type hNAGLU mutant) than the Q209R hNAGLU mutant. Furthermore, a positive correlation was observed between the enzyme activity and the expression levels of the hNAGLU mutants confirmed by SDS-PAGE electrophoresis (Figure 3(b) and Figure 4(a)). Furthermore, a positive correlation was observed between the enzyme activity and the expression levels of the hNAGLU mutants confirmed by Western blotting (Figure 4(b)).

[0156] Example 17 The above results indicate that when producing recombinant hNAGLU, production yields can be increased by 2 to 5 times by producing the recombinant Q209R hNAGLU mutant instead of recombinant wild-type hNAGLU. Further mutations were added to the Q209R hNAGLU mutant to produce recombinant K36E / P37S / Q209R hNAGLU mutant, recombinant L44_G45insS / Q209R hNAGLU mutant, recombinant Q209R / T320P / E321D hNAGLU mutant, recombinant K36E / P37S / L44_G45insS / Q209R hNAGLU mutant, recombinant V54I / Q209R / R620K hNAGLU mutant, recombinant L44_G45insS / V54I / Q209R hNAGLU mutant, recombinant L44_G45insS / Q209R / R620K hNAGLU mutant, and recombinant L44_G45insS / V54I / Q209R / R620K It is shown that the production of recombinant hNAGLU can be further increased by producing it as an hNAGLU mutant.

[0157] Example 18: Construction of cells for expressing a fusion protein of an anti-human transferrin receptor antibody (anti-hTfR antibody) and an hNAGLU mutant The fusion protein of anti-human transferrin receptor antibody (anti-hTfR antibody) and hNAGLU mutant can be produced by the method described in detail below.

[0158] The expression vectors pE-neo and pE-hygr were constructed using the method described in patent document WO2018 / 124121. The pE-neo and pE-hygr vectors were digested with MluI and NotI, respectively.

[0159] A DNA fragment encoding the protein shown in SEQ ID NO: 64 is synthesized in which the L44_G45insS / Q209R hNAGLU mutant (mutant no. 9) is linked to the C-terminus of the Fab heavy chain of an anti-hTfR antibody comprising the amino acid sequence of SEQ ID NO: 63 via a linker sequence consisting of three consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 3, for a total of 15 amino acids. An MluI sequence and a sequence encoding a leader peptide that functions as a secretion signal are placed, in order from the 5' end, on the 5' side of this DNA fragment, and a NotI sequence is placed on the 3' side. This DNA fragment is digested with MluI and NotI and inserted into the MluI-NotI region of the pE-neo vector to construct pE-neo(HC-mhNAGLU).

[0160] Furthermore, a DNA fragment encoding the protein shown in SEQ ID NO: 65, to which the Fab heavy chain of an anti-hTfR antibody comprising the amino acid sequence shown in SEQ ID NO: 63 is linked via a linker sequence consisting of a total of 15 amino acids, consisting of three consecutive amino acid sequences shown in SEQ ID NO: 3, is synthesized at the C-terminus of the L44_G45insS / Q209R hNAGLU mutant (mutant no. 9). At the 5' side of this DNA fragment, an MluI sequence and a sequence encoding a leader peptide that functions as a secretion signal are placed, in this order from the 5' end, and at the 3' side, a NotI sequence. This DNA fragment is digested with MluI and NotI and inserted into the pE-neo vector between the MluI and NotI sites to construct pE-neo(mhNAGLU-HC).

[0161] A DNA fragment encoding the light chain of anti-hTfR (SEQ ID NO: 66) comprising the amino acid sequence of SEQ ID NO: 61 is synthesized. This DNA fragment is digested with MluI and NotI and inserted into the pE-hygr vector between MluI and NotI to construct pE-hygr (LC).

[0162] CHO cells (CHO-K1: obtained from the American Type Culture Collection) are transformed with pE-neo(HC-mhNAGLU) and pE-neo(HC-mhNAGLU), or pE-neo(HC-mhNAGLU) and pE-neo(mhNAGLU-HC), respectively, using a GenePulser (Bio-Rad) according to the following method. Cell transformation is generally carried out as follows. 5x10 5 CHO-K1 cells were cultured in CD OptiCHO TM The cells are seeded in a 3.5 cm culture dish containing medium (Life Technologies) and cultured overnight at 37°C in 5% CO2. TM The medium was replaced with I medium (Life Technologies), and the cells were cultured at 5x10 6 Suspend the cells to a density of 100 cells / mL. Take 100 μL of the cell suspension and add Opti-MEM TM Five microliters of pE-neo(HC-mhNAGLU) and pE-neo(HC-mhNAGLU) plasmid DNA solutions diluted to 100 μg / mL in I medium were added. Electroporation was performed using a GenePulser (Bio-Rad) to introduce the plasmids into the cells. The cells were cultured overnight at 37°C in 5% CO2, and then incubated in CD OptiCHO medium supplemented with 0.5 mg / mL hygromycin and 0.8 mg / mL G418. TM Selective culture on medium.

[0163] Next, the cells selected by selective culture are seeded onto a 96-well plate by limiting dilution so that no more than one cell is seeded per well, and cultured for approximately 10 days so that each cell forms a monoclonal colony. The culture supernatant from the well in which a monoclonal colony has formed is collected, and the humanized antibody content in the culture supernatant is examined by ELISA to select cell lines with high humanized antibody expression.

[0164] The ELISA procedure is generally as follows: 100 μL of a goat anti-human IgG polyclonal antibody solution diluted to 4 μg / mL in 0.05 M bicarbonate buffer (pH 9.6) is added to each well of a 96-well microtiter plate (Nunc) and allowed to stand at room temperature for at least 1 hour to allow the antibody to adsorb to the plate. Next, after washing each well three times with PBS-T, 200 μL of StartingBlock (PBS) Blocking Buffer (Thermo Fisher Scientific) is added to each well and the plate is allowed to stand at room temperature for 30 minutes. After washing each well three times with PBS-T, 100 μL of culture supernatant or human IgG standard diluted to the appropriate concentration in PBS supplemented with 0.5% BSA and 0.05% Tween 20 (PBS-BT) is added to each well and the plate is allowed to stand at room temperature for at least 1 hour. After washing the plate three times with PBS-T, add 100 μL of HRP-labeled anti-human IgG polyclonal antibody solution diluted in PBS-BT to each well and allow the plate to stand at room temperature for at least 1 hour. After washing each well three times with PBS-T, add 100 μL of 0.4 mg / mL o-phenylenediamine in phosphate-citrate buffer (pH 5.0) to each well and allow the plate to stand at room temperature for 8 to 20 minutes. Next, add 100 μL of 1 mol / L sulfuric acid to each well to stop the reaction, and measure the absorbance at 490 nm in each well using a 96-well plate reader. Cells corresponding to wells that showed high values ​​can be used as high-expressing cell lines for fusion protein production.

[0165] Example 19: Preparation of fusion protein of anti-hTfR antibody and hNAGLU mutant The fusion protein of the anti-hTfR antibody and the hNAGLU mutant can be produced by the following method. The highly expressing cell line described in Example 18 was cultured at a cell concentration of about 2×10 5 CD OptiCHO TMThe cell suspension was diluted with medium, and 200 mL of the cell suspension was added to a 1 L Erlenmeyer flask. The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air at an agitation speed of approximately 70 rpm for 6–7 days. The culture supernatant was collected by centrifugation and filtered through a 0.22 μm filter (Millipore). The culture supernatant was added to 20 mM Tris buffer (pH 8.0) containing 150 mL of NaCl (5 column volumes). The supernatant was then loaded onto a Protein A column (column volume: 1 mL, Bio-Rad) pre-equilibrated with 20 mM Tris buffer (pH 8.0) containing 150 mM NaCl (3 column volumes). The column was then washed with 5 column volumes of the same buffer, and the adsorbed fusion protein was eluted with 4 column volumes of 50 mM glycine buffer (pH 2.8) containing 150 mM NaCl. The pH of the eluate containing the fusion protein is adjusted to pH 7.0 by adding 1 M Tris buffer (pH 8.0). The resulting solution is stored at 4°C or frozen as a purified fusion protein. [Industrial Applicability]

[0166] According to the present invention, it is possible to provide an hNAGLU mutant that can be administered as enzyme replacement therapy for the treatment of patients with mucopolysaccharidosis type IIIB, and that can be produced more efficiently as a recombinant protein compared to wild-type hNAGLU. [Sequence List Free Text]

[0167] SEQ ID NO: 1: Amino acid sequence of wild-type hNAGLU SEQ ID NO: 2: Nucleotide sequence of a DNA fragment encoding wild-type hNAGLU, synthetic sequence SEQ ID NO: 3: Amino acid sequence of hNAGLU variant no. 1 SEQ ID NO: 4: Nucleotide sequence of the DNA fragment encoding hNAGLU mutant No. 1, synthetic sequence SEQ ID NO: 5: Amino acid sequence of hNAGLU variant no. 2 SEQ ID NO: 6: Nucleotide sequence of the DNA fragment encoding hNAGLU mutant No. 2, synthetic sequence SEQ ID NO: 7: Amino acid sequence of hNAGLU variant no. 16 SEQ ID NO: 8: Nucleotide sequence of the DNA fragment encoding hNAGLU mutant No. 16, synthetic sequence SEQ ID NO: 9: Amino acid sequence of hNAGLU variant no. 3 SEQ ID NO: 10: Nucleotide sequence of the DNA fragment encoding hNAGLU mutant No. 3, synthetic sequence SEQ ID NO: 11: Amino acid sequence of hNAGLU variant no. 4 SEQ ID NO: 12: Nucleotide sequence of the DNA fragment encoding hNAGLU mutant No. 4, synthetic sequence SEQ ID NO: 13: Amino acid sequence of hNAGLU variant no. 17 SEQ ID NO: 14: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 17, synthetic sequence SEQ ID NO: 15: Amino acid sequence of hNAGLU variant no. 5 SEQ ID NO: 16: Nucleotide sequence of the DNA fragment encoding hNAGLU mutant No. 5, synthetic sequence SEQ ID NO: 17: Amino acid sequence of hNAGLU variant no. 6 SEQ ID NO: 18: Nucleotide sequence of the DNA fragment encoding hNAGLU mutant No. 6, synthetic sequence SEQ ID NO: 19: Amino acid sequence of hNAGLU variant no. 7 SEQ ID NO: 20: Nucleotide sequence of the DNA fragment encoding hNAGLU mutant No. 7, synthetic sequence SEQ ID NO: 21: Amino acid sequence of hNAGLU variant no. 18 SEQ ID NO: 22: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 18, synthetic sequence SEQ ID NO: 23: Amino acid sequence of hNAGLU variant no. 19 SEQ ID NO: 24: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 19, synthetic sequence SEQ ID NO: 25: Amino acid sequence of hNAGLU variant no. 8 SEQ ID NO: 26: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 8, synthetic sequence SEQ ID NO: 27: Amino acid sequence of hNAGLU variant no. 9 SEQ ID NO: 28: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 9, synthetic sequence SEQ ID NO: 29: Amino acid sequence of hNAGLU variant no. 10 SEQ ID NO: 30: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 10, synthetic sequence SEQ ID NO: 31: Amino acid sequence of hNAGLU variant no. 11 SEQ ID NO: 32: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 11, synthetic sequence SEQ ID NO: 33: Amino acid sequence of hNAGLU variant no. 12 SEQ ID NO: 34: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 12, synthetic sequence SEQ ID NO: 35: Amino acid sequence of hNAGLU variant no. 13 SEQ ID NO: 36: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 13, synthetic sequence SEQ ID NO: 37: Amino acid sequence of hNAGLU variant no. 14 SEQ ID NO: 38: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 14, synthetic sequence SEQ ID NO: 39: Amino acid sequence of hNAGLU variant no. 15 SEQ ID NO: 40: Nucleotide sequence of a DNA fragment encoding hNAGLU mutant No. 15, synthetic sequence SEQ ID NO: 41: Nucleotide sequence containing the gene encoding hNAGLU, synthetic sequence SEQ ID NO: 42: MluI-added 5' primer, synthetic sequence SEQ ID NO: 43: His tag-NotI-added 3' primer, synthetic sequence SEQ ID NO: 44: K36E / P37S mutation-introducing 3' primer, synthetic sequence SEQ ID NO: 45: L44_G45insS mutation-introduced 3' primer, synthetic sequence SEQ ID NO: 46: R129Q mutation-introducing 3' primer, synthetic sequence SEQ ID NO: 47: Q209R mutation-introducing 3' primer, synthetic sequence SEQ ID NO: 48: E228K mutation-introducing 3' primer, synthetic sequence SEQ ID NO: 49: T240V mutation-introducing 3' primer, synthetic sequence SEQ ID NO: 50: T320P / E321 D mutation-introducing 3' primer, synthetic sequence SEQ ID NO: 51: S505A / I506V mutation-introducing 5' primer, synthetic sequence SEQ ID NO: 52: S526N / A528 T mutation-introducing 5' primer, synthetic sequence SEQ ID NO: 53: D613Q mutation-introducing 5' primer, synthetic sequence SEQ ID NO: 54: H204K mutation-introducing 3' primer, synthetic sequence SEQ ID NO: 55: V54I mutation-introduced 3' primer, synthetic sequence SEQ ID NO: 56: R620K mutation-introduced 5' primer, synthetic sequence SEQ ID NO: 57: Amino acid sequence of human transferrin receptor SEQ ID NO: 58: Example 1 of the amino acid sequence of the linker SEQ ID NO: 59: Example 2 of the amino acid sequence of the linker SEQ ID NO: 60: Example 3 of the amino acid sequence of the linker SEQ ID NO: 61: Amino acid sequence of the light chain of the anti-hTfR antibody SEQ ID NO: 62: Amino acid sequence of the heavy chain of the anti-hTfR antibody SEQ ID NO: 63: Amino acid sequence of the Fab heavy chain of the anti-hTfR antibody SEQ ID NO: 64: Amino acid sequence 1 of the fusion protein of hNAGLU variant No. 9 and the Fab heavy chain of an anti-hTfR antibody SEQ ID NO: 65: Amino acid sequence 2 of the fusion protein of hNAGLU variant No. 9 and the Fab heavy chain of an anti-hTfR antibody SEQ ID NO: 66: Nucleotide sequence encoding the amino acid sequence of the light chain of anti-hTfR antibody, synthetic sequence SEQ ID NO: 67: Amino acid sequence of the signal peptide of wild-type hNAGLU precursor

Claims

[Claim 1] The invention described herein.

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