Mutants of α-N-acetylglucosaminidase

Abstract

We provide a novel hNAGLU mutant that can increase the expression level of human α-N-acetylglucosaminidase (hNAGLU) in a host cell into which a gene encoding hNAGLU has been introduced, compared to when a gene encoding wild-type hNAGLU is introduced. [Solution] An hNAGLU mutant obtained by modifying the amino acid sequence of hNAGLU, the mutant having a specific amino acid sequence or an amino acid sequence obtained by adding a mutation to this amino acid sequence.

Description

[Technical Field]

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

[0002] Mucopolysaccharidosis type IIIB (MPS-IIIB), also known as Sanfilippo syndrome type B, is a genetic disorder caused by a gene abnormality in α-N-acetylglucosaminidase (NAGLU), an enzyme necessary for the breakdown of heparan sulfate (HS), a type of glycosaminoglycan (GAG), within lysosomes. This abnormality leads to decreased or deficient enzyme activity. In severe cases, the accumulation of HS in various organs, including the brain, results in serious neurological and tissue damage, such as cognitive decline and behavioral disorders, which manifest between the ages of 2 and 6. Behavioral problems such as hyperactivity and intellectual disability are observed. Severe intellectual disability and motor disorders develop with the rapid progression of central nervous system degeneration, and language ability is lost by the age of 7 or 8. In the teens, sleep disorders, hepatosplenomegaly, and seizures occur, leading to immobility and bedriddenness. Many die in their 20s from respiratory infections or other causes.

[0003] Enzyme replacement therapy is used to replenish enzymes that are deficient or lacking in patients with Sanfilippo syndrome type B. The enzyme α-N-acetylglucosaminidase (NAGLU) catalyzes the hydrolysis of the non-reducing α-N-acetylglucosamine residue of heparan sulfate, and when administered to patients, it can break down heparin (HS) accumulated in lysosomes within 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 produced using cells transformed with an expression vector incorporating the gene encoding this enzyme.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a novel hNAGLU variant capable of increasing the expression level of hNAGLU in host cells into which the gene encoding hNAGLU has been introduced, compared to the case where the gene encoding wild-type hNAGLU is introduced, by introducing mutations into the amino acid sequence of hNAGLU.

Means for Solving the Problems

[0007] In research directed towards the above object, as a result of intensive studies, the present inventors have found that host cells into which the gene encoding an hNAGLU variant obtained by modifying the amino acid sequence of hNAGLU, which is described in detail herein, is introduced express more hNAGLU than host cells into which the gene encoding wild-type hNAGLU is introduced, and have completed the present invention. That is, the present invention includes the following. 1. A variant of human α-N-acetylglucosaminidase (hNAGLU) selected from the group consisting of the following (1) to (7): (1) Those having the amino acid sequence shown in SEQ ID NO: 3, in which lysine at position 36 and proline at position 37 in the amino acid sequence of wild-type hNAGLU shown in SEQ ID NO: 1 are each replaced with glutamic acid and serine, respectively; (2) Those having the amino acid sequence represented by SEQ ID NO: 5, in which serine is 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) Those having the 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) Those having the amino acid sequence represented by SEQ ID NO: 11, in which glutamate at position 228 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with lysine; (5) Those having the amino acid sequence represented by SEQ ID NO: 15, in which threonine at position 320 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with proline, and glutamate at position 321 is substituted with aspartic acid; (6) Those having the amino acid sequence represented by SEQ ID NO: 17, in which serine at position 505 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with alanine, and isoleucine at position 506 is substituted with valine; and (7) Those having the amino acid sequence represented by SEQ ID NO: 19, in which serine at position 526 in the amino acid sequence of wild-type hNAGLU represented by SEQ ID NO: 1 is substituted with asparagine, and alanine at position 528 is substituted with threonine. 2. An hNAGLU variant of the above 1 having the amino acid sequence represented by SEQ ID NO: 3, which is an hNAGLU variant with mutations added while conserving glutamate at position 36 and serine at position 37 of the amino acid sequence, and is selected from the group consisting of the following (1'-a) to (1'-h): (1'-a) Those in which the amino acid residues constituting the amino acid sequence are substituted with other amino acid residues, 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) Those in which the amino acid residues constituting the amino acid sequence are 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 of 1'-a and the deletion of 1'-b above; (1'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (1'-f) A combination of the deletion of 1'-b and the addition of 1'-d; (1'-g) A combination of the above substitution of 1'-a, deletion of 1'-b, and addition of 1'-d; and (1'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 3. hNAGLU mutants obtained by adding a mutation to the above hNAGLU mutant (1) having the amino acid sequence shown in Sequence ID No. 5, while preserving the serine at position 45 of the amino acid sequence, and selected from the group consisting of (2'-a) to (2'-h) below: (2'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence is characterized by the deletion of amino acid residues, the number of deleted amino acid residues being 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (2'-c) A combination of the substitution 2'-a and the deletion 2'-b above; (2'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (2'-f) A combination of the deletion of 2'-b and the addition of 2'-d; (2'-g) A combination of the above substitution of 2'-a, deletion of 2'-b, and addition of 2'-d; and (2'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 4. hNAGLU mutants obtained by introducing a mutation to the hNAGLU mutant of 1 above, having the amino acid sequence shown in Sequence ID No. 9, while preserving the arginine at position 209 of the amino acid sequence, and selected from the group consisting of (3'-a) to (3'-h) below: (3'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence is characterized by the deletion of amino acid residues, the number of deleted amino acid residues being 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (3'-c) A combination of the substitution 3'-a and the deletion 3'-b above; (3'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 substitution of 3'-a and the addition of 3'-d above; (3'-f) A combination of the deletion of 3'-b and the addition of 3'-d; (3'-g) A combination of the above substitution of 3'-a, deletion of 3'-b, and addition of 3'-d; and (3'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 5. hNAGLU mutants obtained by adding mutations to the hNAGLU mutant of 1 above, having the amino acid sequence shown in Sequence ID No. 11, while preserving the lysine at position 228 of the amino acid sequence, and selected from the group consisting of (4'-a) to (4'-h) below: (4'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence is characterized by the deletion of amino acid residues, the number of deleted amino acid residues being 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (4'-c) A combination of the substitution 4'-a and the deletion 4'-b above; (4'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (4'-f) A combination of the deletion of 4'-b and the addition of 4'-d; (4'-g) A combination of the above substitution of 4'-a, deletion of 4'-b, and addition of 4'-d; and (4'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 6. hNAGLU mutants obtained by introducing mutations to the above 1 hNAGLU mutant 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, selected from the following group consisting of (5'-a) to (5'-h): (5'-a) The amino acid sequence in which the amino acid residues constituting the amino acid sequence are replaced by other amino acid residues, 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) The amino acid sequence is characterized by the deletion of amino acid residues, the number of deleted amino acid residues being 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (5'-c) A combination of the substitution of 5'-a and the deletion of 5'-b; (5'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the substitution of 5'-a and the addition of 5'-d above; (5'-f) A combination of the deletion of 5'-b and the addition of 5'-d; (5'-g) A combination of the above substitution of 5'-a, deletion of 5'-b, and addition of 5'-d; and (5'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 7. hNAGLU mutants obtained by introducing mutations to the above 1 hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 17, while preserving alanine at position 505 and valine at position 506 of the amino acid sequence, selected from the following group consisting of (6'-a) to (6'-h): (6'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence is characterized by the deletion of amino acid residues, the number of deleted amino acid residues being 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (6'-c) A combination of the substitution 6'-a and the deletion 6'-b above; (6'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 substitution of 6'-a and the addition of 6'-d above; (6'-f) A combination of the deletion of 6'-b and the addition of 6'-d; (6'-g) A combination of the above substitution of 6'-a, deletion of 6'-b, and addition of 6'-d; and (6'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 8. hNAGLU mutants obtained by introducing mutations to the above 1 hNAGLU mutant 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, selected from the following group consisting of (7'-a) to (7'-h): (7'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence having been deleted, wherein 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) A combination of the substitution of 7'-a and the deletion of 7'-b above; (7'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (7'-f) A combination of the deletion of 7'-b and the addition of 7'-d; (7'-g) A combination of the above substitution of 7'-a, deletion of 7'-b, and addition of 7'-d; and (7'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 9. hNAGLU variants of the above 4, selected from the group consisting of (8) to (15) below: (8) An hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, in which a mutation is introduced while preserving the arginine at position 209 of the amino acid sequence, and having the amino acid sequence shown in SEQ ID NO: 25, in which lysine at position 36 is replaced with glutamic acid and proline at position 37 is replaced with serine; (9) An hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, in which a mutation is introduced 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 is added between leucine at position 44 and glycine at position 45; (10) An hNAGLU mutant having the amino acid sequence shown in SEQ ID NO: 9, in which a mutation is introduced while preserving the arginine at position 209 of the amino acid sequence, and which 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, in which a mutation is introduced while preserving the arginine at position 209 of the amino acid sequence, and having the amino acid sequence shown in SEQ ID NO: 31, in which lysine at position 36 is replaced with glutamic acid, proline at position 37 is replaced with serine, and serine is added between leucine at position 44 and glycine at position 45; (12) An hNAGLU mutant having the amino acid sequence shown in Sequence ID No. 9, in which a mutation is introduced while preserving the arginine at position 209 of the amino acid sequence, and having the amino acid sequence shown in Sequence ID No. 33, in which valine at position 54 is replaced with isoleucine and arginine at position 620 is replaced with lysine; (13) An hNAGLU mutant having the amino acid sequence shown in Sequence ID No. 9, in which a mutation is introduced while preserving the arginine at position 209 of the amino acid sequence, and having the amino acid sequence shown in Sequence ID No. 35, in which valine at position 54 is replaced with isoleucine, and serine is added between leucine at position 44 and glycine at position 45; (14) hNAGLU mutants having the amino acid sequence shown in SEQ ID NO: 9, in which mutations are introduced while preserving the arginine at position 209 of the amino acid sequence, the arginine at position 620 is replaced with lysine, and a serine molecule is added between leucine at position 44 and glycine at position 45, having the amino acid sequence shown in SEQ ID NO: 37; and (15) A variant of hNAGLU having the amino acid sequence shown in Sequence ID No. 9, wherein the arginine at position 209 of the amino acid sequence is preserved, the valine at position 54 is replaced with isoleucine, the arginine at position 620 is replaced with lysine, and a serine is added between the leucine at position 44 and the glycine at position 45, thereby having the amino acid sequence shown in Sequence ID No. 39. 10. hNAGLU mutants obtained by adding mutations to the hNAGLU mutant described in 9 above, having the amino acid sequence shown in SEQ ID NO: 25, while preserving arginine at position 209, glutamic acid at position 36, and serine at position 37 of the amino acid sequence, selected from the following group consisting of (8'-a) to (8'-h): (8'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence is characterized by the deletion of amino acid residues, the number of deleted amino acid residues being 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (8'-c) A combination of the substitution of 8'-a above and the deletion of 8'-b; (8'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the deletion of 8'-b and the addition of 8'-d; (8'-g) A combination of the above substitution of 8'-a, deletion of 8'-b, and addition of 8'-d; (8'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 11. hNAGLU mutants obtained by adding mutations to the hNAGLU mutant described in 9 above, having the amino acid sequence shown in Sequence ID No. 27, while preserving the arginine at position 210 and the serine at position 45 of the amino acid sequence, selected from the following group consisting of (9'-a) to (9'-h): (9'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence is characterized by the deletion of amino acid residues, the number of deleted amino acid residues being 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (9'-c) A combination of the substitution 9'-a and the deletion 9'-b above; (9'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (9'-f) A combination of the deletion of 9'-b and the addition of 9'-d; (9'-g) A combination of the above substitution of 9'-a, deletion of 9'-b, and addition of 9'-d; (9'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 12. hNAGLU mutants obtained by adding mutations to the hNAGLU mutant described in 9 above, having the amino acid sequence shown in Sequence ID No. 29, while preserving arginine at position 209, proline at position 320, and aspartic acid at position 321 of the amino acid sequence, selected from the following group consisting of (10'-a) to (10'-h): (10'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence is characterized by the deletion of amino acid residues, the number of deleted amino acid residues being 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2; (10'-c) A combination of the substitution of 10'-a and the deletion of 10'-b; (10'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the deletion of 10'-b and the addition of 10'-d; (10'-g) A combination of the above substitution of 10'-a, deletion of 10'-b, and addition of 10'-d; (10'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 13. hNAGLU mutants described in 9 above, having the amino acid sequence shown in Sequence ID No. 31, to which mutations are introduced 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, selected from the following group consisting of (11'-a) to (11'-h): (11'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence having been deleted, wherein 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) A combination of the substitution of 11'-a and the deletion of 11'-b above; (11'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (11'-f) A combination of the deletion of 11'-b and the addition of 11'-d; (11'-g) A combination of the substitution of 11'-a, the deletion of 11'-b, and the addition of 11'-d; (11'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 14. hNAGLU mutants obtained by adding mutations to the hNAGLU mutant described in 9 above, having the amino acid sequence shown in SEQ ID NO: 33, while preserving arginine at position 209, isoleucine at position 54, and lysine at position 620 of the amino acid sequence, selected from the group consisting of (12'-a) to (12'-h) below: (12'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence having been deleted, wherein 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) A combination of the substitution of 12'-a and the deletion of 12'-b; (12'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the deletion of 12'-b and the addition of 12'-d; (12'-g) A combination of the substitution of 12'-a, the deletion of 12'-b, and the addition of 12'-d; (12'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 15. hNAGLU mutants obtained by adding mutations to the hNAGLU mutant described in 9 above, having the amino acid sequence shown in SEQ ID NO: 35, while preserving arginine at position 210, isoleucine at position 55, and serine at position 45 of the amino acid sequence, selected from the group consisting of (13'-a) to (13'-h) below: (13'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence is substituted with another amino acid residue, wherein 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) The amino acid sequence having been deleted, wherein 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) A combination of the substitution of 13'-a and the deletion of 13'-b; (13'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the deletion of 13'-b and the addition of 13'-d; (13'-g) A combination of the substitution of 13'-a, the deletion of 13'-b, and the addition of 13'-d; (13'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 16. hNAGLU mutants obtained by adding mutations to the hNAGLU mutant described in 9 above, having the amino acid sequence shown in SEQ ID NO: 37, while preserving the arginine at position 210, the lysine at position 621, and the serine at position 45 of the amino acid sequence, selected from the group consisting of (14'-a) to (14'-h) below: (14'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence having been deleted, wherein 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) A combination of the substitution of 14'-a and the deletion of 14'-b; (14'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (14'-f) A combination of the deletion of 14'-b and the addition of 14'-d; (14'-g) A combination of the substitution of 14'-a, the deletion of 14'-b, and the addition of 14'-d; (14'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 17. hNAGLU mutants obtained by adding mutations to the hNAGLU mutant described in 9 above, having the amino acid sequence shown in SEQ ID NO: 39, while preserving arginine at position 210, isoleucine at position 55, lysine at position 621, and serine at position 45, selected from the following group consisting of (15'-a) to (15'-h): (15'-a) an amino acid sequence in which an amino acid residue is substituted with another amino acid residue, wherein 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) The amino acid sequence having been deleted, wherein 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) A combination of the substitution of 15'-a and the deletion of 15'-b; (15'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (15'-f) A combination of the deletion of 15'-b and the addition of 15'-d; (15'-g) A combination of the substitution of 15'-a, the deletion of 15'-b, and the addition of 15'-d; (15'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence. 18. DNA comprising a gene encoding any of the hNAGLU variants described in 1 to 17 above. 19. An expression vector comprising the DNA described in item 18 above. 20. Mammalian cells transformed with the expression vector described in item 19 above. 21. A method for producing hNAGLU mutants, comprising the step of culturing the mammalian cells described in 20 above in serum-free medium. 22. A fusion protein comprising an hNAGLU variant described in any of items 1 to 17 above and an antibody, wherein the antibody binds to receptors on cerebral vascular endothelial cells, thereby enabling the fusion protein to cross the blood-brain barrier (BBB). 23. The fusion protein according to 22 above, wherein the receptor on the cerebral vascular endothelial cell 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 the cerebral vascular endothelial cell is a transferrin receptor. 25. A fusion protein according to any one of items 22 to 24 above, wherein the antibody is one of a Fab antibody, an F(ab')2 antibody, an F(ab') antibody, a single-domain antibody, a single-chain antibody, or an Fc antibody. 26. The fusion protein according to any one of items 22 to 25 above, wherein the hNAGLU variant is bound to either the C-terminal or N-terminal side of the light chain of the antibody. 27. The fusion protein according to any one of items 22 to 25 above, wherein the hNAGLU variant is bound to either the C-terminal or N-terminal side of the heavy chain of the antibody. 28. The fusion protein according to any one of items 22 to 27 above, wherein the hNAGLU variant is bound to either the C-terminal or N-terminal side of the light chain, or to either the C-terminal or N-terminal side of the heavy chain of the antibody, via a linker sequence. 29. The fusion protein according to 28 above, wherein the linker sequence consists of 1 to 50 amino acid residues. 30. The fusion protein according to 29 above, wherein the linker sequence comprises one glycine molecule, one serine molecule, 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 an amino acid sequence consisting of 1 to 10 consecutive such amino acid sequences. 31. DNA comprising a gene encoding a fusion protein as described in any of items 22-30 above. 32. An expression vector comprising the DNA described in 31 above. 33. Mammalian cells transformed with the expression vector described in 32 above. 34. A method for producing a fusion protein of an hNAGLU mutant and an antibody, comprising the step of culturing the mammalian cells described in 33 above in 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 drawing]

[0009] [Figure 1]Figure (Example 6) shows the results of an experiment to confirm the expression level of the hNAGLU mutant by transient expression. The vertical axis of the bar graph represents fluorescence intensity. (a) shows the SDS-page pattern of the band corresponding to wild-type hNAGLU or the hNAGLU mutant, and (b) shows the Western blotting pattern of the band corresponding to wild-type hNAGLU or the hNAGLU mutant. The following data shows the expression levels of (1) the K36E / P37S hNAGLU mutant, (2) the L44_G45insS hNAGLU mutant, (3) the R129Q hNAGLU mutant, (4) the Q209R hNAGLU mutant, (5) the E228K hNAGLU mutant, (6) the T240V hNAGLU mutant, (7) the T320P / E321D hNAGLU mutant, (8) the S505A / I506V hNAGLU mutant, (9) the S526N / A528T hNAGLU mutant, (10) the D613Q hNAGLU mutant, (11) the H204K hNAGLU mutant, and (12) the wild-type hNAGLU. [Figure 2] Figure (Example 10) shows the results of an experiment to confirm the expression level of hNAGLU mutants in bulk cells. The vertical axis of the bar graph shows the enzyme activity level of hNAGLU expressed by 1 × 10⁶ cells (nmol / h / 10⁶ cells) for each hNAGLU mutant. (1) shows the expression levels of the K36E / P37S hNAGLU mutant, (2) shows the L44_G45insS hNAGLU mutant, (3) shows the R129Q hNAGLU mutant, (4) shows the Q209R hNAGLU mutant, (5) shows the E228K hNAGLU mutant, (6) shows the T240V hNAGLU mutant, (7) shows the T320P / E321D hNAGLU mutant, (8) shows the S505A / I506V hNAGLU mutant, (9) shows the S526N / A528T hNAGLU mutant, (10) shows the D613Q hNAGLU mutant, (11) shows the H204K hNAGLU mutant, and (12) shows the wild-type hNAGLU. The measurements for each of (1) to (4) were repeated four times, and the measured values ​​are shown in the figure. [Figure 3]Figure (Example 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 shows the fluorescence intensity. (a) shows the SDS-page pattern of the band corresponding to wild-type hNAGLU or the hNAGLU mutant, and (b) shows the Western blotting pattern of the band corresponding to wild-type hNAGLU or the hNAGLU mutant. (1) shows the expression level data for wild-type hNAGLU, (2) for the Q209R hNAGLU mutant, (3) for the K36E / P37S / Q209R hNAGLU mutant, (4) for the L44_G45insS / Q209R hNAGLU mutant, (5) for the Q209R / T320P / E321D hNAGLU mutant, and (6) for the K36E / P37S / L44_G45insS / Q209R hNAGLU mutant. [Figure 4] Figure (Example 16) shows the results of an experiment to confirm the expression level of the hNAGLU mutant by transient expression. The vertical axis of the bar graph shows the fluorescence intensity. (a) shows the pattern of the band corresponding to wild-type hNAGLU or the hNAGLU mutant in SDS-page. (1) shows the expression level data for wild-type hNAGLU, (2) for the Q209R hNAGLU mutant, (3) for the L44_G45insS / Q209R hNAGLU mutant, (4) for the V54I / Q209R / R620K hNAGLU mutant, (5) for the L44_G45insS / V54I / Q209R hNAGLU mutant, (6) for the L44_G45insS / Q209R / R620K hNAGLU mutant, (7) for the L44_G45insS / V54I / Q209R / R620K hNAGLU mutant, and (8) for the negative control. [Modes for carrying out the invention]

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

[0011] When amino acid residues are added to the amino acid sequence shown in Sequence ID No. 1, one or more amino acid residues are added to the amino acid sequence of hNAGLU or to the N-terminus or C-terminus of the amino acid sequence. The number of amino acid residues added is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. Addition and substitution of amino acid residues may be combined, as may addition and deletion of amino acid residues, or addition, substitution, and deletion of amino acid residues. Normally, wild-type hNAGLU is biosynthesized as a precursor consisting of 743 amino acid residues, and hNAGLU is obtained by removing the leader peptide consisting of 23 amino acid residues from the N-terminus, as shown in Sequence ID No. 67. When amino acids are added to the N-terminus of hNAGLU, these amino acids may originate from the leader peptide. In this case, the amino acid added to the N-terminus is Gly if there is one amino acid, Ala-Gly if there are two amino acids, and Ala-Ala-Gly if there are two amino acids.

[0012] The following are examples of hNAGLU variants introduced by combining at least two of these three types of mutations—substitution, deletion, and addition of amino acids—but are not limited to these: (i) to (iv): (i) Having an amino acid sequence obtained by deleting 0 to 10 amino acid residues, substituting 0 to 10 amino acid residues with other amino acid residues, and adding 0 to 10 amino acid residues to the amino acid sequence shown in Sequence ID No. 1; (ii) Having 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 amino acid residues to the amino acid sequence shown in Sequence ID No. 1; (iii) Having 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 amino acid residues to the amino acid sequence shown in Sequence ID No. 1; (iv) Having an amino acid sequence obtained by deleting 0 to 2 amino acid residues, substituting 0 to 2 amino acid residues with other amino acid residues, and adding 0 to 2 amino acid residues to the amino acid sequence shown in Sequence ID No. 1.

[0013] The above-mentioned wild-type or mutant hNAGLUs are also considered hNAGLUs if their constituent amino acids are modified by sugar chains. Furthermore, the above-mentioned wild-type or mutant hNAGLUs are also considered hNAGLUs if their constituent amino acids are modified by phosphate. In addition, hNAGLUs are also considered hNAGLUs if they are modified by substances other than sugar chains and phosphate. Furthermore, the above-mentioned wild-type or mutant hNAGLUs are also considered hNAGLUs if the side chains of their constituent amino acids are modified by substitution reactions or the like. Such modifications include, but are not limited to, the conversion of cysteine ​​residues to formylglycine.

[0014] In other words, hNAGLU modified by a sugar chain is included in hNAGLU having the original amino acid sequence before modification. Similarly, hNAGLU modified by a phosphate group is included in hNAGLU having the original amino acid sequence before modification. Furthermore, hNAGLU modified by something other than sugar chains and phosphates is also included in hNAGLU having the original amino acid sequence before modification. Additionally, hNAGLU in which the side chains of the amino acids constituting the hNAGLU have been altered by substitution reactions or other means is also included in hNAGLU having the original amino acid sequence before the alteration. Such alterations include, but are not limited to, the alteration of cysteine ​​residues 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 are substituted, deleted, and / or added (in this specification, "addition" of an amino acid residue means adding a residue to the end or middle of the sequence) to the amino acid sequence of normal wild-type hNAGLU (the amino acid sequence shown in SEQ ID NO: 1), and which has the same function as normal wild-type hNAGLU, such as having the enzymatic activity to degrade heparan sulfate. Preferred hNAGLU variants in the present invention include those in which one or more amino acid residues are substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO: 1. When an amino acid residue in the amino acid sequence is substituted with another amino acid residue, the number of amino acid residues to be substituted 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 deleted can be 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. Furthermore, hNAGLU mutants may also be a combination of these amino acid residue substitutions and deletions.

[0016] When amino acid residues are added to the amino acid sequence shown in Sequence ID No. 1, one or more amino acid residues are added to the amino acid sequence of hNAGLU or to the N-terminus or C-terminus of said amino acid sequence. The number of amino acid residues added is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. In addition, the addition and substitution of amino acid residues may be combined, the addition and deletion of amino acid residues may be combined, or the addition, substitution, and deletion of amino acid residues may be combined. In other words, the hNAGLU mutant is introduced into the amino acid sequence shown in Sequence ID No. 1 by combining at least two of the three types of mutations: amino acid substitution, deletion, and addition.

[0017] The following are examples of hNAGLU variants introduced by combining at least two of these three types of mutations—substitution, deletion, and addition of amino acids—but are not limited to these: (i) to (iv): (i) Those having an amino acid sequence obtained by deleting 0 to 10 amino acid residues, substituting 0 to 10 amino acid residues with other amino acid residues, and adding 0 to 10 amino acid residues to the amino acid sequence shown in Sequence ID No. 1 (excluding wild-type hNAGLU); (ii) Having an amino acid sequence that is 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 amino acid residues to the amino acid sequence shown in Sequence ID No. 1 (excluding wild-type hNAGLU); (iii) Having an amino acid sequence that is 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 amino acid residues to the amino acid sequence shown in Sequence ID No. 1 (excluding wild-type hNAGLU); (iv) Having an amino acid sequence obtained by deleting 0 to 2 amino acid residues, substituting 0 to 2 amino acid residues with other amino acid residues, and adding 0 to 2 amino acid residues to the amino acid sequence shown in Sequence ID No. 1 (excluding wild-type hNAGLU).

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

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

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

[0021] Substitutions of amino acids in the amino acid sequence of wild-type hNAGLU or hNAGLU mutants are For example, substitutions occur within amino acid families that are related by their side chains and chemical properties. Such substitutions within amino acid families are expected 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) Aspartic acid and glutamic acid, which are acidic amino acids, (2) Basic amino acids histidine, lysine, and arginine (3) Aromatic amine acids such as phenylalanine, tyrosine, and tryptophan, (4) Serine and threonine, which are amino acids having a hydroxyl group (hydroxy amino acids) (5) Hydrophobic amino acids methionine, alanine, valine, leucine, and isoleucine, (6) Neutral hydrophilic amino acids such as cysteine, serine, threonine, asparagine, and glutamine, (7) Glycine and proline, amino acids that affect the orientation of peptide chains, (8) Asparagine and glutamine, which are amide-type amino acids (polar amino acids) (9) Aliphatic amino acids, alanine, leucine, isoleucine, and valine, (10) Alanine, glycine, serine, and threonine, which are amino acids with small side chains. (11) Alanine and glycine, which are amino acids with particularly small side chains, (12) Valine, leucine, and isoleucine, which are branched amino acids.

[0022] In one embodiment of the present invention, a high-expression hNAGLU mutant is characterized in that, when expressed as a recombinant protein in host cells, it achieves an expression level 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 higher, compared to when wild-type hNAGLU is expressed as a recombinant protein in host cells under the same conditions. Here, "under the same conditions" means that the expression vector, host cells, culture conditions, etc., are identical.

[0023] The following are some preferred embodiments of such high-expression hNAGLU mutants: (1) to (7): (1) Having the amino acid sequence shown in SEQ ID NO: 3, in which the lysine at position 36 of the amino acid sequence of the wild-type hNAGLU shown in SEQ ID NO: 1 is replaced with glutamic acid, and the proline at position 37 is replaced with serine. (2) Having the amino acid sequence shown in SEQ ID NO: 5, in which serine is added between leucine at position 44 and glycine at position 45 in the amino acid sequence of wild-type hNAGLU shown in SEQ ID NO: 1, (3) Having the amino acid sequence shown in SEQ ID NO: 9, in which the glutamine at position 209 of the amino acid sequence of the wild-type hNAGLU shown in SEQ ID NO: 1 is replaced with arginine, (4) Having the amino acid sequence shown in SEQ ID NO: 11, in which the glutamic acid at position 228 of the amino acid sequence of the wild-type hNAGLU shown in SEQ ID NO: 1 is replaced with lysine, (5) Having the amino acid sequence shown in SEQ ID NO: 15, in which the threonine at position 320 of the wild-type hNAGLU shown in SEQ ID NO: 1 is replaced with proline, and the glutamic acid at position 321 is replaced with aspartic acid, (6) Having the amino acid sequence shown in SEQ ID NO: 17, in which the serine at position 505 of the wild-type hNAGLU shown in SEQ ID NO: 1 is replaced with alanine and the isoleucine at position 506 is replaced with valine, (7) Having the amino acid sequence shown in SEQ ID NO: 19, in which the serine at position 526 of the 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, the following (1') to (7') are preferred embodiments of such high-expression hNAGLU mutants. (1') Without making any mutations in the glutamic acid at position 36 and the serine at position 37 of the amino acid sequence shown in SEQ ID NO: (1'-a) The amino acid sequence in which the amino acid residues constituting the amino acid sequence are replaced with other amino acid residues, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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 of 1'-a and the deletion of 1'-b above; (1'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (1'-f) A combination of the deletion of 1'-b and the addition of 1'-d; (1'-g) A combination of the substitution of 1'-a, the deletion of 1'-b, and the addition of 1'-d; (1'-h) Showing 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 introducing a mutation to the serine at position 45 of the amino acid sequence shown in SEQ ID NO: (2'-a) The amino acid residues constituting the amino acid sequence are replaced with other amino acid residues, 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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 2'-a and the deletion 2'-b above; (2'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (2'-f) A combination of the deletion of 2'-b and the addition of 2'-d; (2'-g) A combination of the above substitution of 2'-a, deletion of 2'-b, and addition of 2'-d; (2'-h) Showing 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 introducing a mutation to the arginine at position 209 of the amino acid sequence shown in SEQ ID NO: (3'-a) The amino acid sequence in which the amino acid residues constituting the amino acid sequence are replaced with other amino acid residues, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution 3'-a and the deletion 3'-b above; (3'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 substitution of 3'-a and the addition of 3'-d above; (3'-f) A combination of the deletion of 3'-b and the addition of 3'-d; (3'-g) A combination of the substitution of 3'-a, the deletion of 3'-b, and the addition of 3'-d; (3'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0027] (4') Without introducing a mutation to the lysine at position 228 of the amino acid sequence shown in SEQ ID NO: 11: (4'-a) The amino acid sequence in which the amino acid residues constituting the amino acid sequence are replaced with other amino acid residues, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution 4'-a and the deletion 4'-b above; (4'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (4'-f) A combination of the deletion of 4'-b and the addition of 4'-d; (4'-g) A combination of the above substitution of 4'-a, deletion of 4'-b, and addition of 4'-d; (4'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0028] (5') Without making any mutations in the proline at position 320 and the aspartic acid at position 321 of the amino acid sequence shown in SEQ ID NO: (5'-a) The amino acid residues constituting the amino acid sequence are replaced with other amino acid residues, 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 are deleted, 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) A combination of the substitution of 5'-a and the deletion of 5'-b; (5'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the substitution of 5'-a and the addition of 5'-d above; (5'-f) A combination of the deletion of 5'-b and the addition of 5'-d; (5'-g) A combination of the above substitution of 5'-a, deletion of 5'-b, and addition of 5'-d; (5'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0029] (6') Without making any mutations in the alanine at position 505 and the valine at position 506 of the amino acid sequence shown in SEQ ID NO: (6'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence is replaced by another amino acid residue, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution 6'-a and the deletion 6'-b above; (6'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 substitution of 6'-a and the addition of 6'-d above; (6'-f) A combination of the deletion of 6'-b and the addition of 6'-d; (6'-g) A combination of the above substitution of 6'-a, deletion of 6'-b, and addition of 6'-d; (6'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0030] (7') Without making any mutations in the asparagine at position 526 and the threonine at position 528 of the amino acid sequence shown in SEQ ID NO: (7'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence is replaced by another amino acid residue, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution of 7'-a and the deletion of 7'-b above; (7'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (7'-f) A combination of the deletion of 7'-b and the addition of 7'-d; (7'-g) A combination of the above substitution of 7'-a, deletion of 7'-b, and addition of 7'-d; (7'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

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

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

[0033] (8') Without making any mutations in the arginine at position 209, glutamic acid at position 36, and serine at position 37 of the amino acid sequence shown in SEQ ID NO: (8'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence is replaced by another amino acid residue, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution of 8'-a and the deletion of 8'-b; (8'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the deletion of 8'-b and the addition of 8'-d; (8'-g) A combination of the above substitution of 8'-a, deletion of 8'-b, and addition of 8'-d; (8'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0034] (9') Without making any mutations in the arginine at position 210 and the serine at position 45 of the amino acid sequence shown in SEQ ID NO: (9'-a) The amino acid sequence in which the amino acid residues constituting the amino acid sequence are replaced with other amino acid residues, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution 9'-a and the deletion 9'-b above; (9'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (9'-f) A combination of the deletion of 9'-b and the addition of 9'-d; (9'-g) A combination of the above substitution of 9'-a, deletion of 9'-b, and addition of 9'-d; (9'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0035] (10') Without making any mutations in the amino acid sequence shown in SEQ ID NO: 29, specifically in the arginine at position 209, the proline at position 320, and the aspartic acid at position 321: (10'-a) The amino acid sequence in which the amino acid residues constituting the amino acid sequence are replaced with other amino acid residues, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution of 10'-a and the deletion of 10'-b; (10'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the deletion of 10'-b and the addition of 10'-d; (10'-g) A combination of the above substitution of 10'-a, deletion of 10'-b, and addition of 10'-d; (10'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0036] (11') Without making any mutations to the 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 is replaced by another amino acid residue, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution of 11'-a and the deletion of 11'-b above; (11'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (11'-f) A combination of the deletion of 11'-b and the addition of 11'-d; (11'-g) A combination of the substitution of 11'-a, the deletion of 11'-b, and the addition of 11'-d; (11'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0037] (12') Without making any mutations in the amino acid sequence shown in SEQ ID NO: 33, specifically in the arginine at position 209, isoleucine at position 54, and lysine at position 620: (12'-a) The amino acid sequence in which the amino acid residues constituting the amino acid sequence are replaced with other amino acid residues, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution of 12'-a and the deletion of 12'-b; (12'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the deletion of 12'-b and the addition of 12'-d; (12'-g) A combination of the substitution of 12'-a, the deletion of 12'-b, and the addition of 12'-d; (12'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0038] (13') Without making any mutations in the arginine at position 210, isoleucine at position 55, and serine at position 45 of the amino acid sequence shown in SEQ ID NO: (13'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence is replaced by another amino acid residue, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution of 13'-a and the deletion of 13'-b; (13'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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) A combination of the deletion of 13'-b and the addition of 13'-d; (13'-g) A combination of the substitution of 13'-a, the deletion of 13'-b, and the addition of 13'-d; (13'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0039] (14') Without making any mutations in the arginine at position 210, lysine at position 621, and serine at position 45 of the amino acid sequence shown in SEQ ID NO: (14'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence is replaced by another amino acid residue, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution of 14'-a and the deletion of 14'-b; (14'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (14'-f) A combination of the deletion of 14'-b and the addition of 14'-d; (14'-g) A combination of the substitution of 14'-a, the deletion of 14'-b, and the addition of 14'-d; (14'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

[0040] (15') Without making any mutations in the amino acid sequence shown in SEQ ID NO: 39, the arginine at position 210, the isoleucine at position 55, the lysine at position 621, and the serine at position 45: (15'-a) an amino acid sequence in which an amino acid residue constituting the amino acid sequence is replaced by another amino acid residue, wherein 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 sequence in which amino acid residues constituting the amino acid sequence are deleted, wherein 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) A combination of the substitution of 15'-a and the deletion of 15'-b; (15'-d) The amino acid sequence having one or more amino acid residues added to it or to the N-terminus or C-terminus, wherein 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 above; (15'-f) A combination of the deletion of 15'-b and the addition of 15'-d; (15'-g) A combination of the substitution of 15'-a, the deletion of 15'-b, and the addition of 15'-d; (15'-h) Showing 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% identity with the amino acid sequence.

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

[0042] Genes encoding hNAGLU mutants that show increased expression levels compared to wild-type hNAGLU when expressed as recombinant proteins in host cells can be obtained, for example, by the following method: The genes encoding the mutated hNAGLU and the wild-type hNAGLU are each incorporated into the same type of expression vector, and the same type of host cells are transformed using 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 the cells showing a larger quantification value compared to the quantification value of the cells into which the wild-type hNAGLU gene has been introduced is identified as the gene encoding the target high-expression hNAGLU mutant. Note that, in addition to the quantification value of hNAGLU as a protein, the enzyme activity value of hNAGLU can also be used as the quantification value. For example, the total amount of hNAGLU enzyme activity contained in the culture supernatant can be used as the quantification value.

[0043] Mutations in wild-type hNAGLU can be introduced using a method that randomly introduces mutations into the gene encoding wild-type hNAGLU. For example, by incorporating the gene encoding wild-type hNAGLU into a vector and then applying a mutagen (radiation, mutagenic substance, etc.) to host cells transformed using this vector, mutations can be introduced into the gene encoding wild-type hNAGLU.

[0044] The introduction of mutations into wild-type hNAGLU can also be carried out by introducing mutations at a predetermined location in the gene encoding wild-type hNAGLU. For example, such gene mutations can be introduced by chemically synthesizing a gene with a mutation at a predetermined location.

[0045] High-expression hNAGLU mutants can be produced as recombinant proteins by culturing host cells transformed using an expression vector incorporating the gene encoding the mutant.

[0046] The host cells used in this process are not particularly limited as long as they can express a high-expression hNAGLU mutant by introducing such an expression vector. They may be any eukaryotic cells such as mammalian cells, yeast, plant cells, or insect cells, or prokaryotic cells such as Escherichia coli or Bacillus subtilis, but 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 human, mouse, and Chinese hamster-derived cells are preferred, and CHO cells derived from Chinese hamster ovary cells or NS / 0 cells derived from mouse myeloma are particularly preferred. In this case, the expression vector used to incorporate and express the DNA fragment encoding the high-expression hNAGLU mutant can be used without particular limitations as long as it brings about the 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 regulatory site) that can regulate the frequency of gene transcription in mammalian cells. Examples of gene expression regulatory sites that can be used in the present invention include cytomegalovirus-derived promoters, SV40 initial promoters, human elongation factor-1α (EF-1α) promoters, and human ubiquitin C promoters.

[0048] Expression vectors are known in which glutamine synthase (GS) is positioned downstream of the gene encoding the target protein via an internal ribosome entry site (IRES) as a selection marker (International Patent Publications WO2012 / 063799, WO2013 / 161958). The expression vectors described in these publications can be used particularly suitably for the production of high-expression hNAGLU mutants.

[0049] For example, an expression vector for expressing a target protein, comprising a first gene expression regulatory site, a gene encoding the protein downstream thereof, an internal ribosome binding site further downstream, and a gene encoding glutamine synthase further downstream, and further comprising a dihydrofolate reductase gene or a drug resistance gene downstream of the first gene expression regulatory site or a different second gene expression regulatory site, can be suitably used to produce high-expression hNAGLU mutants. In this expression vector, a cytomegalovirus-derived promoter, an SV40 initial promoter, a human elongation factor-1α promoter (hEF-1α promoter), and a human ubiquitin C promoter are suitably used as the first gene expression regulatory site or the second gene expression regulatory site, but the hEF-1α promoter is particularly suitable.

[0050] Furthermore, as the internal ribosome binding site, it is preferable to use one derived from 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 enterovirus, Syler's murine encephalomyelitis virus, and coxsackie B virus, or from the 5' untranslated region of a gene selected from the group consisting of human immunoglobulin heavy chain binding protein gene, Drosophila Antennapedia gene, and Drosophila Ultravitrachus gene, but an internal ribosome binding site derived from the 5' untranslated region of the mouse encephalomyocarditis virus genome is particularly preferable. When using an internal ribosome binding site derived from the 5' untranslated region of the mouse encephalomyocarditis virus genome, in addition to the wild type, a wild-type internal ribosome binding site in which some of the multiple start codons contained in the internal ribosome binding site have been disrupted can also be suitably used. Furthermore, the drug resistance gene suitably 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, comprising 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 mouse encephalomyocarditis virus genome further downstream, and a gene encoding glutamine synthase further downstream, and further comprising another gene expression regulatory site and a dihydrofolate reductase gene downstream thereof, wherein the internal ribosome binding site has some of the multiple start codons contained in the wild-type internal ribosome binding site disrupted, can be suitably used to produce high-expression hNAGLU mutants. An example of such an expression vector is the expression vector described in WO2013 / 161958.

[0052] Furthermore, for example, an expression vector for expressing a target protein, comprising 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 mouse encephalomyocarditis virus genome further downstream, and a gene encoding glutamine synthase further downstream, and further comprising another gene expression regulatory site and a drug resistance gene downstream thereof, wherein the internal ribosome binding site has some of the multiple start codons contained in the wild-type internal ribosome binding site disrupted, can be suitably used to produce high-expression hNAGLU mutants. 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 3' end of the internal ribosome binding site, derived from the 5' untranslated region of the wild-type mouse encephalomyocarditis virus genome, contains three start codons (ATGs). The above-mentioned pE-mIRES-GS-puro and pE-mIRES-GS-mNeo are expression vectors that contain IRESs in which some of the start codons are disrupted.

[0054] hNAGLU variants (including high-expression hNAGLU variants) can be expressed in cells or culture media by culturing host cells into which an expression vector containing the gene encoding the variant has been introduced. The method for expressing hNAGLU variants when mammalian cells are the host cells is described in detail below.

[0055] As a culture medium for mammalian cells, any medium capable of culturing and growing mammalian cells can be used without particular limitations, but serum-free medium is preferably used. In the present invention, a serum-free medium used as a culture medium for recombinant protein production 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 10000 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, conventional pH indicators, and antibiotics may be added to the culture medium.

[0056] As a serum-free medium used for recombinant protein production, DMEM / F12 medium (a mixed medium of DMEM and F12) may be used as a basic 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 containing sodium bicarbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, ferrous sulfate, asparagine, aspartic acid, serine, and polyvinyl alcohol may be used. Furthermore, commercially available serum-free media, such as CDOptiCHO, may also be used. 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 Medium 302 or EX-CELL TM 325-PF medium (SAFC Biosciences) and other similar media can also be used as the basic culture medium.

[0057] The high-expression hNAGLU mutant is characterized in that, when mammalian cells into which an expression vector containing the gene encoding it has been introduced are cultured in the serum-free medium described above and expressed as 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, or 2 to 8 times higher, compared to when wild-type hNAGLU is expressed as recombinant protein under the same conditions. The mammalian cells used in this case are CHO cells, NS / 0 cells, etc., but CHO cells are particularly preferred.

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

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

[0060] In one embodiment of the present invention, the hNAGLU variant can be conjugated with an antibody. For example, the hNAGLU variant in one embodiment of the present invention can be conjugated with an antibody that can specifically bind to a receptor on cerebral vascular endothelial cells. By conjugating the hNAGLU variant with an antibody that can specifically bind to a receptor on cerebral vascular endothelial cells, the hNAGLU variant can be allowed to cross the blood-brain barrier (BBB) ​​and exert its function in the central nervous system (CNS). Examples of such receptors on cerebral vascular endothelial cells include, but are not limited to, insulin receptors, transferrin receptors, leptin receptors, lipoprotein receptors, and IGF receptors. Furthermore, the receptor is preferably a human-derived receptor.

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

[0062] In this invention, the term "human antibody" refers to an antibody whose entire protein is encoded by a human-derived gene. However, antibodies encoded by genes that have been mutated without altering the original amino acid sequence, for purposes such as increasing gene expression efficiency, are also included in "human antibodies." Furthermore, antibodies produced by combining two or more genes encoding human antibodies and replacing a part of one human antibody with a part of another human antibody are also considered "human antibodies." 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 called CDR1, CDR2, and CDR3, in order from the N-terminus. The three CDRs in the heavy chain are also called CDR1, CDR2, and CDR3, in order from the N-terminus. Antibodies whose antigen specificity, affinity, etc., has been modified by replacing the CDR of one human antibody with the CDR of another human antibody are also included in "human antibodies."

[0063] In this invention, antibodies that have undergone mutations such as substitution, deletion, or addition to the amino acid sequence of the original antibody by modifying the gene of the original human antibody are also included in the definition of "human antibody." When substituting amino acids in the amino acid sequence of the original antibody with other amino acids, the number of amino acids to be substituted is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. When deleting amino acids in the amino acid sequence of the original antibody, the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, antibodies that have undergone mutations combining these amino acid substitutions and deletions are also considered human antibodies. When adding amino acids, preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence of the original antibody or to the N-terminus or C-terminus. Antibodies that have undergone mutations combining these amino acid additions, substitutions, and deletions are also considered 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 mutation is added to a human antibody, the mutation can be added to the variable region of the antibody. When the above mutation is added to the variable region of the antibody, the mutation may be added to either the CDR of the variable region or the framework region, but is particularly likely to be added to the framework region. That is, in this invention, when we refer to "human-derived genes," we include not only the original human-derived genes but also genes obtained by modifying them.

[0064] In the present invention, the term "humanized antibody" refers to an antibody in which the amino acid sequence of a part of the variable region (for example, all or part of the CDR) is derived from a mammal other than a human, and the remaining region is derived from a human. For example, a humanized antibody may be one which is produced by replacing three complementarity-determining regions (CDRs) in the light chain and three complementarity-determining regions (CDRs) in the heavy chain of a human antibody with CDRs from another mammal. The species of the other mammal from which the CDRs transplanted to the appropriate positions in the human antibody are derived is not particularly limited as long as it is a mammal other than a human, but is preferably a mouse, rat, rabbit, horse, or a primate other than a human, more preferably a mouse and a rat, and even more preferably a mouse. Furthermore, an antibody in which the amino acid sequence of the original humanized antibody has been modified in the same way as the mutations that can be added to the human antibody described above is also included in the term "humanized antibody".

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

[0066] A chimeric antibody is an antibody in which a portion of a human antibody is replaced by a portion of an antibody from a non-human mammal. The antibody consists of an Fc region, a Fab region, and a hinge region, as described below. A specific example of such a chimeric antibody is one in which the Fc region originates from a human antibody while the Fab region originates from an antibody from another mammal. The hinge region originates from either the human antibody or the antibody from the other mammal. Conversely, an example of a chimeric antibody is one in which the Fc region originates from another mammal while the Fab region originates from a human antibody. The hinge region originates from either the human antibody or the antibody from the other mammal.

[0067] Furthermore, antibodies can be said to consist of a variable region and a constant region. Another specific example of a chimeric antibody is the constant region of the heavy chain (C H ) and the steady region of the light chain (C L ) is derived from human antibodies, while the variable region of the heavy chain (V H ) and the variable region of the light chain (V L) is derived from an antibody of another mammal. Conversely, while the constant region (C H ) of the heavy chain and the constant region (C L ) of the light chain are derived from an antibody of another mammal, the variable region (V H ) of the heavy chain and the variable region (V L ) of the light chain are derived from a human antibody. Here, the species of other mammals are not particularly limited as long as they are mammals other than humans, but are preferably mice, rats, rabbits, horses, or non-human primates, for example, mice.

[0068] The antibody in one embodiment of the present invention has a basic structure consisting of a total of four polypeptide chains, namely two immunoglobulin light chains (or simply "light chains") and two immunoglobulin heavy chains (or simply "heavy chains"). However, in the present invention, when referring to an "antibody", in addition to those having this basic structure, (1) Those consisting of a total of two polypeptide chains, namely one light chain and one heavy chain, (2) Those consisting of a Fab region that lacks the Fc region from the basic structure of an antibody in the original sense and those consisting of a Fab region and all or part of the hinge region (including Fab, F(ab’) and F(ab’)2), (3) A single-chain antibody formed by binding a linker sequence to the C-terminal side of the light chain and further binding a heavy chain to the C-terminal side thereof, (4) A single-chain antibody formed by binding a linker sequence to the C-terminal side of the heavy chain and further binding a light chain to the C-terminal side thereof, (5) Those consisting of an Fc region that lacks the Fab region from the basic structure of an antibody in the original sense and are modified such that the amino acid sequence of the Fc region has the property of specifically binding to a specific antigen (Fc antibody), (6) The single-domain antibodies described below are also included in the "antibody" in the present invention.

[0069] The antibody in one embodiment of the present invention is an antibody derived from a camelid (including alpaca). Some antibodies from camelids consist of two heavy chains linked by disulfide bonds. Antibodies consisting of these two heavy chains are called heavy chain antibodies. VHH is an antibody consisting of one heavy chain that includes the variable region of the heavy chain constituting the heavy chain antibody, or an antibody consisting of one heavy chain that lacks the constant region (CH) constituting the heavy chain antibody. VHH is also one of the antibodies in the embodiments of the present invention. In addition, an antibody consisting of two light chains linked by disulfide bonds is also one of the antibodies in the embodiments of the present invention. Antibodies consisting of these two light chains are called light chain antibodies. To reduce the antigenicity when antibodies derived from camelids (including VHH) are administered to humans, antibodies in which mutations have been added to the amino acid sequence of camelid antibodies are also antibodies in one embodiment of the present invention. When mutations are added to the amino acids of camelid antibodies, the same mutations that can be added to the antibodies described herein can be added.

[0070] The antibody in one embodiment of the present invention is a shark-derived antibody. A shark antibody consists of two heavy chains linked by a disulfide bond. An antibody consisting of these two heavy chains is called a heavy chain antibody. A VNAR is an antibody consisting of one heavy chain that includes the variable region of the heavy chain constituting the heavy chain antibody, or an antibody consisting of one heavy chain that lacks the constant region (CH) constituting the heavy chain antibody. A VNAR is also one of the antibodies in the embodiments of the present invention. An antibody in one embodiment of the present invention is a shark antibody in which a mutation has been made in the amino acid sequence of the shark antibody in order to reduce its antigenicity when administered to a human. When a mutation is made in the amino acid sequence of the shark antibody, the same mutations as those that can be made in the antibodies described herein can be made. A humanized shark antibody is also one of the antibodies in the embodiments of the present invention.

[0071] Antibodies, which have a basic structure consisting of a total of four polypeptide chains, two light chains and two heavy chains, have a variable region (V) in the light chain. L ) has three complementarity determination regions (CDRs) and a variable region of the heavy chain (V HThe light chain has three complementarity-determining regions (CDRs). The three CDRs on the light chain are called CDR1, CDR2, and CDR3, in order from the N-terminus. The three CDRs on the heavy chain are also called CDR1, CDR2, and CDR3, in order from the N-terminus. However, even if some or all of these CDRs are incomplete or absent, the antibody is still included as long as it has the property of specifically binding to a particular antigen. Variable regions (V) of the light and heavy chains L and V H The region of the computer other than the CDR is called the framework region (FR). The FRs are numbered FR1, FR2, FR3, and FR4, starting from the N-terminus. Typically, the CDR and FRs exist in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, starting from the N-terminus.

[0072] In one embodiment of the present invention, an antibody is also included in the definition of an antibody, in which mutations such as substitution, deletion, or addition have been added to the amino acid sequence of the original antibody. When an amino acid in the amino acid sequence of the original antibody is substituted with another amino acid, the number of amino acids to be substituted is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. When an amino acid is deleted from the amino acid sequence of the original antibody, the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, an antibody is also included in which mutations combining these amino acid substitutions and deletions have been added. When amino acids are added, preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence of the original antibody or to the N-terminus or C-terminus. An antibody is also included in which mutations combining these amino acid additions, substitutions, and deletions have been added. The amino acid sequence of the mutated antibody preferably shows 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.

[0073] In one embodiment of the present invention, an antibody is also included in the definition of an antibody in which mutations such as substitution, deletion, or addition have been added to the amino acid sequence of the variable region of the original antibody. When an amino acid in the amino acid sequence of the original antibody is substituted with another amino acid, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When an amino acid is deleted from the amino acid sequence of the original antibody, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, an antibody is also included in which mutations combining these amino acid substitutions and deletions have been added. 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 of the original antibody or to the N-terminus or C-terminus. An antibody is also included in which mutations combining these amino acid additions, substitutions, and deletions have been added. 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 a mutation is added to the variable region of the antibody, the mutation may be added to either the CDR of the variable region or the framework region, but is particularly likely to be added to the framework region.

[0074] In one embodiment of the present invention, an antibody is also included in which mutations such as substitution, deletion, or addition have been added to the amino acid sequence in the framework region of the variable region of the original antibody. When an amino acid in the amino acid sequence of the original antibody is substituted with another amino acid, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When an amino acid is deleted from the amino acid sequence of the original antibody, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, an antibody is also included in which mutations combining these amino acid substitutions and deletions have been added. 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 of the original antibody or to the N-terminus or C-terminus. An antibody is also included in which mutations combining these amino acid additions, substitutions, and deletions have been added. The amino acid sequence of the mutated antibody preferably shows 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.

[0075] In one embodiment of the present invention, an antibody is also included in which a mutation such as substitution, deletion, or addition of amino acids has been added to the amino acid sequence in the CDR region of the variable region of the original antibody. When an amino acid in the amino acid sequence of the original antibody is substituted with another amino acid, the number of amino acids to be substituted is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2. When an amino acid is deleted from the amino acid sequence of the original antibody, the number of amino acids to be deleted is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2. Furthermore, an antibody is also included in which a mutation combining these amino acid substitutions and deletions has been added. 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 of the original antibody or to the N-terminus or C-terminus. An antibody is also included in which a mutation combining these amino acid additions, substitutions, and deletions has been added. The amino acid sequence of the mutated antibody preferably shows 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.

[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. Examples of such algorithms include BLAST (Altschul SF. J Mol . Biol. 215. 403-10, (1990)), the Pearson and Lipman similarity search method (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), and the Smith and Waterman local homology algorithm (Adv. Appl. Math. 2. 482-9 (1981)).

[0077] In one embodiment of the present invention, Fab is a variable region and C L A single light chain containing a region (the steady region of the light chain), and a variable region and C HThis refers to a molecule in which one heavy chain, containing one region (part 1 of the constant region of the heavy chain), is linked to the cysteine ​​residues present in each region by disulfide bonds. In Fab, the heavy chain consists of a variable region and a C H In addition to one region (part 1 of the constant region of the heavy chain), 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 the antibody together. In Fab, the light chain and the heavy chain are defined as the constant region of the light chain (C L Cysteine ​​residues located in the region and the constant region of the heavy chain (C H The bonds are formed by disulfide bonds between cysteine ​​residues located in the 1st region or the hinge region. The heavy chain that forms the Fab is called the Fab heavy chain. Since the Fab lacks cysteine ​​residues located in the hinge region that bind antibody heavy chains together, it consists of one light chain and one heavy chain. The light chain that makes up the Fab consists of a variable region and C L It includes a region. The heavy chains that make up Fab are variable regions and C H It may consist of one region, a variable region, C H It may also include a portion of the hinge region in addition to one region. However, in this case, the hinge region is selected so as not to contain cysteine ​​residues that connect the heavy chains, so that a disulfide bond is not formed between the two heavy chains in the hinge region. In F(ab'), the heavy chain consists of a variable region and C HIn addition to the 1st region, it includes all or part of the hinge region containing cysteine ​​residues that link the heavy chains together. F(ab')2 refers to a molecule in which two F(ab') molecules are linked by disulfide bonds between cysteine ​​residues present in their hinge regions. The heavy chain that forms F(ab') or F(ab')2 is called a Fab' heavy chain. Furthermore, polymers such as dimers and trimers formed by the direct or linker linkage of multiple antibodies are also antibodies. Moreover, not limited to these, anything that includes a part of an antibody molecule and has the property of specifically binding to an antigen is included in the definition of "antibody" in this invention. That is, in this invention, when we refer to a light chain, it includes those derived from a light chain and having all or part of the amino acid sequence of its variable region. Similarly, when we refer to a heavy chain, it 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 its variable region, for example, even if the Fc region is deleted, it is still a heavy chain.

[0078] Furthermore, here, Fc or the Fc region refers to the C region in the antibody molecule. H 2 regions (part 2 of the steady-state region of the heavy chain), and C H This refers to a region containing a fragment consisting of three regions (part 3 of the steady region of the heavy chain).

[0079] Furthermore, the antibody in the present invention is (7) The present invention also includes scFab, scF(ab'), and scF(ab')2, which are single-chain antibodies obtained by linking the light chain and heavy chain that constitute Fab, F(ab'), or F(ab')2 as shown in (2) above via a linker sequence. Here, in the case of scFab, scF(ab'), and scF(ab')2, the linker sequence may be attached to the C-terminus of the light chain, and the heavy chain may be attached to the C-terminus of the light chain, or the linker sequence may be attached to the C-terminus of the heavy chain, and the light chain may be attached to the C-terminus of the heavy chain. Furthermore, scFv, which is a single-chain antibody obtained by linking the variable region of the light chain and the variable region of the heavy chain via a linker sequence, is also included in the antibodies of the present invention. In the case of scFv, a linker array may be attached to the C-terminal side of the variable region of the light chain, and the variable region of the heavy chain may be attached to the C-terminal side of that linker array, or a linker array may be attached to the C-terminal side of the variable region of the heavy chain, and the variable region of the light chain may be attached to the C-terminal side of that linker array.

[0080] Furthermore, the term "antibody" as used herein includes not only full-length antibodies and those described in (1) to (7) above, but also a broader concept encompassing (1) to (7), which includes antigen-binding fragments (antibody fragments) in which a portion of a full-length antibody is missing. Antigen-binding fragments include heavy-chain antibodies, light-chain antibodies, VHH, VNAR, and those in which a portion of these is missing.

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

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

[0083] In a single-chain antibody, the linker sequence positioned between the light and heavy chains of the antibody is preferably a peptide chain consisting of 2 to 50 amino acid residues, more preferably 8 to 50, even more preferably 10 to 30, and even more preferably 12 to 18 or 15 to 25, for example, 15 or 25 amino acid residues. Such a linker sequence is not limited to its amino acid sequence as long as the anti-hTfR antibody formed by linking the two chains maintains affinity for hTfR, but is preferably composed of glycine alone or glycine and serine, for example, the amino acid sequence Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly, Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or sequences in which these amino acid sequences are repeated 2 to 10 times or 2 to 5 times. For example, when linking the variable region of the light chain to the C-terminal side of the amino acid sequence comprising the entire variable region of the heavy chain via a linker sequence, a linker sequence containing a total of 15 amino acids, corresponding to three consecutive Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), is preferred.

[0084] In one embodiment of the present invention, a single-domain antibody is an antibody that has the property of specifically binding to an antigen with a single variable region. Single-domain antibodies include antibodies in which the variable region consists only of the variable region of the heavy chain (heavy chain single-domain antibodies) and antibodies in which the variable region consists only of the variable region of the 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 (the extracellular region of the transferrin receptor), but is not limited thereto.

[0086] A common method for producing antibodies against a desired protein involves creating recombinant proteins using cells into which an expression vector containing the gene encoding the protein has been introduced, and then immunizing animals such as mice with these recombinant proteins. By extracting antibody-producing cells against the recombinant protein from the immunized animals and fusing them with myeloma cells, hybridoma cells capable of producing antibodies against the recombinant protein can be created.

[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 originate, but preferably they are primates including mice, rats, rabbits, guinea pigs, dogs, cats, horses, and humans, more preferably mice, rats, and humans, and even more preferably mice and humans. As mouse immune system cells, for example, splenocytes prepared from the spleen of a mouse can be used. As human immune system cells, cells prepared from human peripheral blood, bone marrow, spleen, etc. can be used. When human immune system cells are immunized using in vitro immunization, human antibodies against recombinant proteins can be obtained.

[0088] By immunizing immune system cells using in vitro immunization and then fusing these cells with myeloma cells, hybridoma cells capable of producing antibodies can be created. Furthermore, mRNA can be extracted from immunized cells to synthesize cDNA, and this cDNA can be used as a template to amplify DNA fragments containing genes encoding the light and heavy chains of immunoglobulins via PCR. These fragments can then be used to artificially reconstruct antibody genes.

[0089] Hybridoma cells obtained by the above method may include cells that produce antibodies that recognize proteins other than the target protein as antigens. Furthermore, not all hybridoma cells that produce antibodies against the desired protein will necessarily produce antibodies that exhibit the desired characteristics, such as high affinity for that protein.

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

[0091] Therefore, it is necessary to select hybridoma cells that produce antibodies with the desired characteristics from the hybridoma cells obtained as described above. Furthermore, in the case of artificially reconstructed antibody genes, it is necessary to select the gene encoding an antibody with the desired characteristics from the antibody gene. For example, the method detailed below is effective for selecting hybridoma cells that produce antibodies that show high affinity for a desired protein (high-affinity antibodies), or genes encoding high-affinity antibodies.

[0092] For example, when selecting hybridoma cells that produce antibodies with high affinity to a desired protein, a method is used in which the protein is added to a plate and retained thereon, the culture supernatant of the hybridoma cells is added, then antibodies that are not bound to the protein are removed from the plate, and the amount of antibodies retained on the plate is measured. According to this method, the higher the affinity of the antibodies contained in the culture supernatant of the hybridoma cells added to the plate to the protein, the greater the amount of antibodies retained on the plate. Therefore, by measuring the amount of antibodies retained on the plate, hybridoma cells corresponding to the plate with the most antibodies retained can be selected as cell lines that produce antibodies with relatively high affinity to the protein. From the cell line selected in this way, mRNA can be extracted and cDNA synthesized, and using this cDNA as a template, a DNA fragment containing the gene encoding the antibody against the protein can be amplified using PCR to isolate the gene encoding the high-affinity antibody.

[0093] When selecting a gene encoding an antibody against a target protein with high affinity from the artificially reconstructed antibody genes described above, the artificially reconstructed antibody gene is first incorporated into an expression vector, and this expression vector is then introduced into host cells. The host cells used are not particularly limited, regardless of whether they are prokaryotic or eukaryotic, as long as they are capable of expressing the antibody gene by introducing the expression vector containing the artificially reconstructed antibody gene. However, cells derived from mammals such as humans, mice, and Chinese hamsters are preferred, and CHO cells derived from Chinese hamster ovaries or NS / 0 cells derived from mouse myeloma are particularly preferred. Furthermore, the expression vector used to incorporate and express the gene encoding the antibody gene is not particularly limited, as long as it expresses the gene when introduced into mammalian cells. The gene incorporated into the expression vector is positioned downstream of a DNA sequence (gene expression regulatory site) that can regulate the frequency of gene transcription within mammalian cells. Examples of gene expression regulatory sites that can be used in this invention include cytomegalovirus-derived promoters, SV40 initial promoters, human elongation factor-1 alpha (EF-1α) promoters, and human ubiquitin C promoters.

[0094] Mammalian cells into which such expression vectors have been introduced begin to express the artificially reconstituted antibodies incorporated into the expression vectors. When selecting cells that produce antibodies with high affinity for a desired protein from among the cells expressing these artificially reconstituted antibodies, a method is used in which the protein is added to a plate and retained thereon, the cell culture supernatant is brought into contact with the protein, and then antibodies that are not bound to the protein are removed from the plate, and the amount of antibodies retained on the plate is measured. According to this method, the higher the affinity of the antibodies contained in the cell culture supernatant to the protein, the greater the amount of antibodies retained on the plate. Therefore, by measuring the amount of antibodies retained on the plate, cells corresponding to plates with a larger amount of retained antibodies can be selected as cell lines that produce antibodies with relatively high affinity for the protein, and consequently, the gene encoding the antibody with high affinity for the protein can be selected. From the cell lines selected in this way, the gene encoding the high-affinity antibody can also be isolated by amplifying the DNA fragment containing the gene encoding the antibody against the protein using PCR.

[0095] The present invention provides a method for producing a conjugate of the hNAGLU variant and an antibody, which can be done via a non-peptide linker or a peptide linker. Non-peptide linkers include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, or derivatives thereof, or combinations thereof. The peptide linker is a peptide chain or derivative thereof consisting of 1 to 50 amino acids linked by peptide bonds, where the N-terminus and C-terminus of the peptide chain form covalent bonds with either the hNAGLU variant or the antibody, respectively, thereby conjugating the hNAGLU variant and the antibody.

[0096] The antibody and the hNAGLU mutant can also be linked to the N-terminus or C-terminus of the antibody's heavy or light chain via a peptide bond, either directly or via a linker sequence. The conjugate formed by linking the antibody and the hNAGLU mutant in this way can be obtained as a fusion protein by incorporating a DNA fragment in which the cDNA encoding the hNAGLU mutant is positioned in-frame, either directly or with a DNA fragment encoding the linker sequence flanked, at the 3' or 5' end of the cDNA encoding the antibody's heavy or light chain, into a mammalian cell expression vector, and culturing mammalian cells into which this expression vector has been introduced. When the DNA fragment encoding the hNAGLU mutant is linked to the heavy chain, a mammalian cell expression vector incorporating the cDNA fragment encoding the antibody's light chain can also be introduced into the same host cells. Similarly, when the DNA fragment encoding the hNAGLU mutant is linked to the light chain, a mammalian cell expression vector incorporating the cDNA fragment encoding the antibody's heavy chain can also be introduced into the same host cells. If the antibody is a single-chain antibody, the fusion protein of the antibody and the hNAGLU mutant can be obtained by incorporating a DNA fragment into an expression vector (for mammalian cells, eukaryotes such as yeast, or prokaryotic cells such as E. coli) to which the cDNA encoding the single-chain antibody is linked, either directly to the 5' or 3' end of the cDNA encoding the hNAGLU mutant, or with a DNA fragment encoding a linker sequence in between. The expression vector is then introduced into these cells and the resulting fusion protein can be produced as a recombinant protein using the method described above.

[0097] The culture medium for mammalian cells into which the expression vector has been introduced can be any medium capable of culturing and growing mammalian cells, but serum-free medium is preferred. In the present invention, a serum-free medium used as a culture medium for recombinant protein production 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 10000 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, conventional pH indicators, and antibiotics may be added to the culture medium.

[0098] As a serum-free medium used for the production of antibody-hNAGLU mutant fusion proteins, DMEM / F12 medium (a mixed medium of DMEM and F12) may be used as the basic 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 containing sodium bicarbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, ferrous sulfate, asparagine, aspartic acid, serine, and polyvinyl alcohol may be used. Furthermore, commercially available serum-free media, such as CDOptiCHO, may also be used. 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 Medium 302 or EX-CELL TM 325-PF medium (SAFC Biosciences) and other similar media can also be used as the basic culture medium.

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

[0100] When an antibody and an hNAGLU variant are linked via a linker sequence, the linker sequence positioned between the antibody and the hNAGLU variant is preferably a peptide chain consisting of 1 to 60 or 1 to 50 amino acids, more preferably 1 to 17, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids. However, 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 linked antibody maintains affinity to receptors on cerebral vascular endothelial cells and the linked hNAGLU variant exhibits its physiological activity under physiological conditions. However, it is preferably composed of glycine and serine. Examples include sequences consisting of one amino acid, either glycine or serine; amino acid sequences Gly-Ser, Ser-Ser, Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 58), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 59), Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 60); or sequences containing 1 to 10 or 2 to 5 consecutive such amino acid sequences. These include sequences consisting of 1 to 50 amino acids, 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, or 27 amino acids. For example, sequences containing the amino acid sequence Gly-Ser can be suitably used as linker sequences. Furthermore, a sequence containing a total of 27 amino acids, consisting of five consecutive Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 58) amino acid sequences following the Gly-Ser sequence, can be suitably used as a linker sequence. Moreover, a sequence containing a total of 25 amino acids, consisting of five consecutive Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 58) amino acid sequences, can also be suitably used as a linker sequence.

[0101] In this invention, if a single peptide chain contains multiple linker sequences, for convenience, each linker sequence will be named sequentially from the N-terminus as the first linker sequence, the second linker sequence, and so on.

[0102] Specific examples of antibodies to be conjugated to the hNAGLU variant of the present invention include the following anti-human transferrin receptor antibodies (anti-hTfR antibodies). That is, (1) An anti-hTfR antibody in which 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 that is also a Fab antibody, wherein the light chain of the antibody contains the amino acid sequence of SEQ ID NO: 61 and the heavy chain contains the amino acid sequence of SEQ ID NO: 63, However, these are not the only possible mutations; substitutions, deletions, additions, and other modifications can be made to the above amino acid sequences as appropriate. Note that the anti-hTfR antibodies in (1) and (2) above are humanized anti-hTfR antibodies.

[0103] When substituting amino acids in the amino acid sequence of the light chain of the above-mentioned anti-human transferrin receptor antibody 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 deleting amino acids in the amino acid sequence of the light chain, 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. Furthermore, mutations combining these amino acid substitutions and deletions can also be introduced.

[0104] When adding amino acids to the amino acid sequence of the light chain of the above-mentioned anti-human transferrin receptor antibody, preferably 1 to 10 amino acids, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2 amino acids are added to the amino acid sequence of the light chain, either at the N-terminus or C-terminus. Mutations combining these amino acid additions, substitutions, and deletions can also be introduced. The amino acid sequence of the mutated light chain preferably has 80% or more identity with the original light chain amino acid sequence, more preferably 90% or more identity, and even more preferably 95% or more identity.

[0105] When substituting amino acids in the heavy chain amino acid sequence of the above-mentioned anti-human transferrin receptor antibody 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 deleting amino acids in the heavy chain amino acid sequence, 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. Furthermore, mutations combining these amino acid substitutions and deletions can also be introduced.

[0106] When adding amino acids to the amino acid sequence of the heavy chain of the above-mentioned anti-human transferrin receptor antibody, preferably 1 to 10 amino acids, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2 amino acids are added to the amino acid sequence of the heavy chain, either at the N-terminus or C-terminus. Mutations combining these amino acid additions, substitutions, and deletions can also be introduced. The amino acid sequence of the mutated heavy chain preferably has 80% or more identity with the original heavy chain's amino acid sequence, more preferably 90% or more identity, and even more preferably 95% or more identity.

[0107] The hNAGLU variant of the present invention can be made to cross the blood-brain barrier and exert its function in the brain by binding it to an antibody against a receptor on cerebral vascular endothelial cells. Therefore, it can be used to manufacture drugs for intravenous administration for the treatment of central nervous system disorders caused by NAGLU deficiency. Furthermore, the hNAGLU variant bound to the antibody can be used in a treatment method that includes administering an effective amount of the hNAGLU variant to a patient in question via the bloodstream (including intravenous injection such as intravenous infusion). The hNAGLU variant bound to the antibody administered via the bloodstream can reach the brain as well as other organs and tissues that express NAGLU. In addition, this drug can also be used to prevent the onset of the disease.

[0108] The hNAGLU variant of the present invention can be used as a drug to exert its therapeutic effect in the central nervous system (CNS) by conjugating it with an antibody against a receptor on cerebral vascular endothelial cells and administering it into the bloodstream. Such drugs are generally administered to patients by intravenous injection, subcutaneous injection, or intramuscular injection, such as by intravenous infusion, but there are no particular limitations on the route of administration.

[0109] In one embodiment of the present invention, the hNAGLU mutant is not limited to an antibody, but can also be a conjugate with other proteins. There are no particular limitations on the other proteins, but such proteins are, for example, human-derived proteins. The conjugate of the hNAGLU mutant with the other protein can be obtained by the method described above for producing the conjugate of the hNAGLU mutant with an antibody. Furthermore, the fusion protein of the hNAGLU mutant with the other protein can be obtained as a recombinant protein by the method described above for producing the fusion protein with an antibody. One embodiment of the present invention includes a gene encoding the fusion protein of the hNAGLU mutant with the other protein, an expression vector incorporating the gene, and host cells into which the expression vector has been introduced.

[0110] In one embodiment of the present invention, the hNAGLU variant is not limited to an anti-hTfR antibody, but can also be a conjugate with a substance capable of binding to human hTfR. While there are no particular limitations on the substance, the protein is, for example, human transferrin. The human transferrin is not limited to the wild type; it may be a partial fragment or a variant thereof, as long as it has affinity for hTfR. Such a conjugate can cross the blood-brain barrier and exert its function in the brain. The fusion protein of the hNAGLU variant and human transferrin can be obtained as a recombinant protein by the method described above for producing a fusion protein with an antibody. One embodiment of the present invention includes a gene encoding the fusion protein of the hNAGLU variant and human transferrin, an expression vector incorporating the gene, and host cells into which the expression vector has been introduced. [Examples]

[0111] The present invention will be described in more detail below with reference to examples, but the present invention is not intended to be limited to these examples.

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

[0113] [Table 1]

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

[0115] [Table 2]

[0116] Using a DNA fragment containing the wild-type hNAGLU gene and the sequence shown in SEQ ID NO. 41 as a template, PCR Nos. 8-10 shown in Table 3 were performed using the primers shown in Table 3 as forward primers and the His-tagged-NotI-added 3' primer with the 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 nucleotide sequence shown in SEQ ID NO. 41 as a template, with a MluI-added 5' primer having the nucleotide sequence shown in SEQ ID NO. 42 as a forward primer, and PCR products obtained from PCR No. 8-10 shown in Table 3 as reverse primers, to obtain PCR products containing the genes encoding the hNAGLU variants shown in Table 4. Table 4 shows the SEQ ID NO corresponding to the amino acid sequence of each hNAGLU variant, the SEQ ID NO corresponding to the encoding nucleotide sequence, and the number (mutant number) of each hNAGLU variant.

[0119] [Table 4]

[0120] Next, the PCR products obtained from PCRs No. 1-11, and the DNA fragments containing the nucleotide sequence shown in SEQ ID NO. 41, which encodes wild-type hNAGLU, were restricted enzyme-treated with MluI and NotI (Takara Bio), and separated by agarose gel electrophoresis. After EtBr staining, the bands containing the target DNA fragments were excised under UV irradiation, and the DNA was extracted from the gel using the QIAEX II Gel Extraction Kit (QIAGEN). Similarly, the pCI-neo vector (Promega) and pEI-puro vectors were restricted enzyme-treated with MluI and NotI, and then gel extracted and purified. Each restriction enzyme-treated vector was mixed with each restriction enzyme-treated PCR product, and a ligation reaction was performed using Ligation Mix (Takara Bio) at 16°C for 30-60 minutes. The DNA fragments containing the nucleotide sequence shown in SEQ ID NO. 41, which contains the wild-type hNAGLU gene, were similarly subjected to the ligation reaction.

[0121] The pEI-puro vector was prepared using the following procedure: The 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 blunt-ended with T4 DNA polymerase. The region containing the EF-1a promoter and its first intron (after blunt-ending) was then inserted into this excised region. This was named the 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 obtain the internal ribosome binding site (IRES) and puromycin resistance gene (Puro) derived from mouse encephalomyocarditis virus (EMCV). r DNA fragments containing the polyadenylation signal (polyA) derived from ) and bovine growth hormone (bGH) were excised. Separately, the pE-neo vector was digested with restriction enzymes (NotI and BamHI) to extract the neomycin resistance gene (Neo r A region of approximately 2 kbp containing the above-mentioned IRES, PuroR, and bGH-derived polyA was excised. A DNA fragment containing these was then inserted. This was named the pEI-puro vector.

[0122] Next, using each ligation reaction solution, E. coli (ECOS) TMXCompetent E. coli DH5 α (Nippon Gene Co., Ltd.) was transformed into each of the transformed cells. To confirm that the resulting transformants held the target 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 Co., Ltd.). The purified plasmid DNA was restricted with MluI and NotI enzymes, and the insertion of the target insert DNA was confirmed by agarose gel electrophoresis. Furthermore, the introduction of the target modification into each hNAGLU gene was confirmed by Sanger sequencing analysis. Each plasmid in which the target hNAGLU mutant or wild-type hNAGLU was confirmed to be incorporated was purified by conventional methods.

[0123] [Example 2] Transient expression of hNAGLU mutant Transient expression of hNAGLU mutants was performed using plasmids containing the genes encoding each hNAGLU mutant in the purified pCI-neo vector obtained in Example 1. As a control, a plasmid containing the gene encoding wild-type NAGLU in the pCI-neo vector was used.

[0124] ExpiCHO cells were transformed using plasmids containing the gene encoding the hNAGLU mutant and the gene encoding the 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 to express each hNAGLU mutant and the wild-type hNAGLU in the culture supernatant. After culturing, the culture medium was centrifuged and the culture supernatant was collected.

[0125] [Example 3] Confirmation of hNAGLU mutant expression level by transient expression (SDS-Page electrophoresis) Ten μL of the culture supernatant obtained in Example 2 was mixed with eight μL of 2× Sample Buffer (Bio-Rad) and two μL of 2-Mercaptoethanol, and the mixture was thermally denatured under reducing conditions by incubation at 100°C for three minutes. After thermal denaturation, five μL of the sample was added 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. The gel after electrophoresis 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 luminography 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 described in Example 3. The nitrocellulose membrane and the gel after electrophoresis were sandwiched between blotting paper 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, 25 V for 10 minutes using a blotting apparatus. The transferred nitrocellulose membrane was immersed in PBST containing 5% skim milk and shaken for 1 hour, then immersed in a 0.4 μg / mL diluted Mouse anti-His tag mAb (Laboratory Institute of Medicine) solution and shaken for 1 hour. After washing the membrane with PBST, it was immersed in a 0.4 μg / mL diluted Anti-mouse IgG (H+L), HRP Conjugate (Promega) solution and shaken for 30 minutes, and then washed again with PBST. HRP detection reagent (Bio-Rad) was dropped onto the transfer surface of the membrane and allowed to react for 5 minutes. Bands corresponding to each hNAGLU variant and wild-type hNAGLU were detected using a luminography analyzer.

[0127] [Example 5] Confirmation of hNAGLU mutant expression level by transient expression (enzyme activity measurement) As a sample solution, the culture supernatant obtained in Example 2 was prepared by diluting it 10-fold with 100 mM citrate buffer (pH 4.2) containing 0.1% BSA. As a standard solution, 4-MU (4-Methylumbelliferone, Sigma-Aldrich) was prepared by serially diluting it to 400-35.12 μM with 100 mM citrate buffer (pH 4.2) containing 0.1% BSA. As a substrate solution, 4-Methylumbelliferyl-N-acetyl-α-D-glucosaminide (Sigma-Aldrich), an artificial substrate of NAGLU, was prepared by diluting it to 1 mmol / L with 100 mM citrate buffer (pH 4.2) containing 0.1% BSA. 25 μL of either the sample solution or the standard solution was added to each well of a microplate, and then 25 μL of the substrate solution was added per well. The mixture was then stirred using a plate shaker. After incubating the plate at 37°C for 1 hour, the reaction was stopped by adding 150 μL of 200 mmol / L glycine-NaOH buffer (pH 10.7) to each well. 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, fluorescence wavelength 460 nm). A calibration curve was created based on the measurement results of the standard solution, and the measured values ​​of 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 transient expression levels of hNAGLU mutants in Examples 2-4. Table 5 shows the expression levels of each hNAGLU mutant, based on the enzyme activity measurement results shown as bar graphs in Figure 1, as relative values ​​with the wild-type expression level set to 1.

[0129] [Table 5]

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

[0131] Furthermore, a positive correlation was observed between enzyme activity and the expression level of the hNAGLU mutant as confirmed by SDS-Page electrophoresis, as shown in Figure 1(a). Additionally, a positive correlation was observed between enzyme activity and the expression level of the hNAGLU mutant as confirmed by Western blotting, as shown in Figure 1(b).

[0132] [Example 7] Production of hNAGLU mutant-expressing bulk cells Using a gene transfer device (Super Electroporator NEPA21, Neppageen), expression plasmids containing the genes encoding the respective hNAGLU mutants or wild-type hNAGLU obtained in Example 1 were introduced into serum-adapted CHO-K1 cell lines. Selective culture was then performed in CD OptiCHO medium (Thermo Fisher Scientific) containing 10 μg / mLPuromycin (Thermo Fisher Scientific). The culture volume was gradually increased while changing the culture medium every 3-4 days. When the viability of the cultured cells exceeded 90%, the cells were harvested and designated as hNAGLU mutant-expressing bulk cells and wild-type hNAGLU-expressing bulk cells.

[0133] [Example 8] Culture of hNAGLU mutant-expressing bulk cells In CD OptiCHO medium containing 10 μg / mLPuromycin, 2 × 10⁶ hNAGLU mutant-expressing bulk cells and wild-type hNAGLU-expressing bulk cells obtained in Example 7 were added. 5 Cells were seeded at a cell density of cells / mL and cultured statically at 37°C in the presence of 5% CO2. Nine days after the start of culture, the culture supernatant was collected by centrifugation.

[0134] [Example 9] Confirmation of hNAGLU mutant expression level in bulk cells (enzyme activity measurement) As the sample solution, the culture supernatant obtained in Example 8 was diluted 10-fold with citrate buffer (pH 4.2) containing 0.1% BSA. As the substrate solution, 4-Methylumbelliferyl-N-acetyl-α-D-glucosaminide, an artificial substrate for hNAGLU, was diluted 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 the substrate solution per well. The mixture was then stirred with 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 (excitation wavelength 355 nm, fluorescence wavelength 460 nm). A calibration curve was created based on the measurement results of the standard solution, and the measured values ​​of each sample solution were interpolated into it to determine the enzyme activity. The enzyme activity for each hNAGLU mutant was 1 × 10⁻⁶ during culture. 6 Enzyme activity of hNAGLU expressed from individual cells (nmol / h / 1×10) 6 It was determined as an individual cell.

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

[0136] [Table 6]

[0137] Similar to transient expression, seven variants (mutant numbers 1-7)—K36E / P37S hNAGLU, L44_G45insS hNAGLU, Q209R hNAGLU, E228K hNAGLU, T320P / E321D hNAGLU, S505A / I506V hNAGLU, and S526N / A528T hNAGLU—showed higher expression levels compared to the wild type in bulk cells. In particular, the Q209R hNAGLU variant (mutant number 3) showed 2.2 times higher expression levels compared to wild-type hNAGLU. Furthermore, similar to 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) showed lower expression levels compared to the wild type.

[0138] [Example 11] Mutation introduction into Q209R hNAGLU mutant and construction of expression vector From the experimental results of Examples 1 to 10, it was found that among the hNAGLU mutants, the Q209R hNAGLU mutant exhibited the highest expression level. Therefore, in order to obtain a further highly expressed hNAGLU mutant, mutations were further introduced into the Q209R hNAGLU mutant. Using the plasmid incorporating the gene encoding the Q209R hNAGLU mutant obtained in Example 1 as a template, the MluI-added 5' primer having the nucleotide sequence shown in SEQ ID NO: 42 as a forward primer and the primers shown in Table 7 as reverse primers, PCRs of Nos. 12 to 15 shown in Table 7 were performed. Table 7 shows the SEQ ID NOs corresponding to the nucleotide sequences of each reverse primer.

[0139]

Table 7

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

[0141]

Table 8

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

[0143]

Table 9

[0144] By the above-described PCR of Nos. 17 to 19, 21, and 23 to 26, a DNA fragment encoding the hNAGLU variant shown in Table 10, in which one to three additional mutations were introduced into the Q209R hNAGLU variant, was amplified. Table 10 shows the SEQ ID NOs corresponding to the amino acid sequences of each hNAGLU variant, the SEQ ID NOs corresponding to the nucleotide sequences encoding them, and the number of each hNAGLU variant (variant number).

[0145] [Table 10]

[0146] Next, each PCR product obtained by the PCR of Nos. 17 to 19, 21, and 23 to 26 was treated 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 cut out under UV irradiation, and DNA was extracted from the gel using the QIAEX II Gel Extraction Kit (QIAGEN). Similarly, the pCI-neo vector (Promega) and the pEI-puro vector were also treated with restriction enzymes MluI and NotI, and gel extraction and purification were performed. Each PCR product treated with restriction enzymes was mixed with each vector treated with restriction enzymes, and a ligation reaction was carried out at 16°C for 30 to 60 minutes using Ligation Mix (Takara Bio Inc.).

[0147] Next, using each ligation reaction solution, Escherichia coli (ECOS TMXCompetent E. coli DH5 α (Nippon Gene Co., Ltd.) was transformed into each of the transformed cells. To confirm that the resulting transformants held the target 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 Co., Ltd.). The purified plasmid DNA was restricted with MluI and NotI enzymes, and the insertion of the target insert DNA was confirmed by agarose gel electrophoresis. Furthermore, the introduction of the target modification into each hNAGLU gene was confirmed by Sanger sequencing analysis. Each plasmid in which the target hNAGLU mutant was confirmed to be incorporated was purified by conventional methods.

[0148] [Example 12] Transient expression of hNAGLU mutant Transient expression of the hNAGLU mutant was performed using a plasmid containing the hNAGLU mutant incorporated into the pCI-neo vector purified in Example 11. A plasmid containing wild-type hNAGLU incorporated into the pCI-neo vector was used as a control.

[0149] ExpiCHO cells were transformed using plasmids containing the gene encoding the hNAGLU mutant and the gene encoding the 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 to express each hNAGLU mutant and the wild-type hNAGLU in the culture supernatant. After culturing, the culture medium was centrifuged and the culture supernatant was collected. In addition, the culture supernatant of untransformed cells was collected in the same manner as a negative control.

[0150] [Example 13] Confirmation of hNAGLU mutant expression level by transient expression (SDS-Page electrophoresis) Ten μL of the culture supernatant obtained in Example 12 was mixed with eight μL of 2×Sample Buffer and two μL of 2-Mercaptoethanol, and the mixture was thermally denatured under reducing conditions by incubation at 100°C for three minutes. After thermal denaturation, five μL of the 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. The gel after electrophoresis 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 luminography image analyzer.

[0151] [Example 14] Confirmation of hNAGLU mutant expression level by transient expression (Western blotting method) Electrophoresis was performed in the same manner as described in Example 13. The nitrocellulose membrane and the gel after electrophoresis were sandwiched between blotting paper 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, 25 V for 10 minutes using a blotting apparatus. The transferred 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, and washed again with PBST. HRP detection reagent was dropped onto the transfer surface of the membrane and reacted for 5 minutes, and detected using a luminography image analyzer.

[0152] [Example 15] Confirmation of hNAGLU mutant expression level by transient expression (enzyme activity measurement) As the sample solution, the culture supernatant obtained in Example 12 was diluted 10-fold with citrate buffer (pH 4.2) containing 0.1% BSA. As the substrate solution, 4-Methylumbelliferyl-N-acetyl-α-D-glucosaminide, an artificial substrate for hNAGLU, was diluted to 1 mmol / L with citrate buffer (pH 4.2). 25 μL / well of the sample solution or standard solution was added to a microplate, and then 25 μL of the substrate solution was added to each well. The mixture was then stirred with a plate shaker. After incubating the plate at 37°C for 1 hour, 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, fluorescence wavelength 460 nm). A calibration curve was created based on the measurement results of the standard solution, and the measured values ​​of each sample solution were interpolated into it to determine the enzyme activity level.

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

[0154] [Table 11]

[0155] The following variants, which are further mutations of the Q209R hNAGLU mutant, are all Q209R hNAGLU: 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 8-15). The mutants showed higher transient expression compared to the hNAGLU mutant. In particular, the V54I / Q209R / R620K hNAGLU mutant showed approximately 1.8 times more transient expression than the Q209R hNAGLU mutant (5.9 times more than the wild type). Furthermore, a positive correlation was observed between enzyme activity and the expression level of the hNAGLU mutant as confirmed by SDS-Page electrophoresis, as shown in Figures 3(b) and 4(a). In addition, a positive correlation was observed between enzyme activity and the expression level of the hNAGLU mutant as confirmed by Western blotting, as shown in Figure 4(b).

[0156] [Example 17] The above results indicate that when producing recombinant hNAGLU, a 2 to 5 times increase in production volume can be achieved by using the recombinant Q209R hNAGLU mutant instead of the recombinant wild-type hNAGLU. Furthermore, by adding further mutations to the Q209R hNAGLU mutant, 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 This study demonstrates that the production volume 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. A fusion protein of an anti-human transferrin receptor antibody (anti-hTfR antibody) and an hNAGLU variant can be produced by the method detailed below.

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

[0159] At the C-terminus of the Fab heavy chain of an anti-hTfR antibody comprising the amino acid sequence of SEQ ID NO: 63, a DNA fragment encoding a protein of SEQ ID NO: 64 to which the L44_G45insS / Q209R hNAGLU variant (variant number 9) binds via a linker sequence consisting of a total of 15 amino acids in which the amino acid sequence of SEQ ID NO: 3 is continuous three times is synthesized. On the 5'-side of this DNA fragment, an MluI sequence and a sequence encoding a leader peptide that functions as a secretion signal are arranged in order from the 5'-end, and a NotI sequence is arranged on the 3'-side. This DNA fragment is digested with MluI and NotI and incorporated between MluI and NotI of the pE-neo vector to construct pE-neo(HC-mhNAGLU).

[0160] Also, at the C-terminus of the L44_G45insS / Q209R hNAGLU variant (variant number 9), a DNA fragment encoding a protein of SEQ ID NO: 65 to which the Fab heavy chain of an anti-hTfR antibody comprising the amino acid sequence of SEQ ID NO: 63 binds via a linker sequence consisting of a total of 15 amino acids in which the amino acid sequence of SEQ ID NO: 3 is continuous three times is synthesized. On the 5'-side of this DNA fragment, an MluI sequence and a sequence encoding a leader peptide that functions as a secretion signal are arranged in order from the 5'-end, and a NotI sequence is arranged on the 3'-side. This DNA fragment is digested with MluI and NotI and incorporated between MluI and NotI of the pE-neo vector to construct pE-neo(mhNAGLU-HC).

[0161] A DNA fragment encoding a DNA fragment (SEQ ID NO: 66) encoding the light chain of an anti-hTfR comprising the amino acid sequence of SEQ ID NO: 61 is synthesized. This DNA fragment is digested with MluI and NotI and incorporated between MluI and NotI of the pE-hygr vector 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 GenePulser (Bio-Rad) according to the following method. Cell transformation is generally carried out using the following method. 5X10 5 Individual CHO-K1 cells are CD OptiCHO TM Seeds are seeded in a 3.5 cm culture dish with culture medium (Life Technology Co., Ltd.) added, and cultured overnight at 37°C and 5% CO2. The culture medium is then converted to Opti-MEM. TM Replace with culture medium I (Life Technology Co., Ltd.) and divide the cells into 5x10 cells. 6 Suspend the cells to a density of cells / mL. Take 100 μL of the cell suspension and add Opti-MEM to it. TM Add 5 μL each of pE-neo(HC-mhNAGLU) and pE-neo(HC-mhNAGLU) plasmid DNA solution diluted to 100 μg / mL in Medium I. Electroporation is performed using GenePulser (Bio-Rad) to introduce the plasmid into the cells. The cells are cultured overnight at 37°C and 5% CO2, and then treated with CD OptiCHO by adding 0.5 mg / mL hygromycin and 0.8 mg / mL G418. TM Selective culture is performed in culture medium.

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

[0164] The ELISA method used in this case is generally carried out as follows: 100 μL of 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). The plate is left to stand at room temperature for at least 1 hour to allow the antibody to adsorb to the plate. Next, each well is washed three times with PBS-T, and 200 μL of StartingBlock (PBS) Blocking Buffer (Thermo Fisher Scientific) is added to each well. The plate is left 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 an appropriate concentration in PBS with 0.5% BSA and 0.05% Tween20 (PBS-BT), is added to each well. The plate is left 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 with 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 to stand at room temperature for 8–20 minutes. Then, add 100 μL of 1 mol / L sulfuric acid to each well to stop the reaction, and measure the absorbance of each well at 490 nm using a 96-well plate reader. Cells corresponding to wells showing high values ​​can be used as high-expression cell lines for the production of fusion proteins.

[0165] [Example 19] Production of a fusion protein of anti-hTfR antibody and hNAGLU variant The fusion protein of the anti-hTfR antibody and the hNAGLU mutant can be produced by the following method: Using the high-expression cell line described in Example 18, the cell concentration is approximately 2 x 10⁻⁶. 5 CD OptiCHO TMDilute with culture medium, add 200 mL of cell suspension to a 1 L Erlenmeyer flask, and culture at 37°C in a humid environment of 5% CO2 and 95% air with a stirring speed of approximately 70 rpm for 6-7 days. Collect the culture supernatant by centrifugation and filter through a 0.22 μm filter (Millipore). Add 150 mL of 20 mM Tris buffer (pH 8.0) containing 5 times the column volume of NaCl to the culture supernatant and load it onto a Protein A column (column volume: 1 mL, Bio-Rad) that has been pre-equilibrated with 20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, 3 times the column volume. Then, wash the column with 5 times the column volume of the same buffer, and elute the adsorbed fusion protein with 50 mM glycine buffer (pH 2.8) containing 150 mM NaCl, 4 times the column volume. The pH of the eluate containing this fusion protein is adjusted to pH 7.0 by adding 1 M Tris buffer (pH 8.0). The resulting solution is then stored as a purified fusion protein at 4°C or frozen. [Industrial applicability]

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

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

Claims

1. A therapeutic agent for mucopolysaccharidosis type IIIB, containing as an active ingredient a fusion protein of a human α-N-acetylglucosaminidase (hNAGLU) variant having the amino acid sequence shown in SEQ ID NO: 27 and an anti-transferrin receptor antibody.

2. The therapeutic agent according to claim 1, wherein the anti-transferrin receptor antibody is Fab.

3. The therapeutic agent according to claim 1 or 2, wherein the fusion protein comprises a conjugate in which the hNAGLU variant is bound to the N-terminus of the heavy chain of the antibody via a linker, and the light chain of the antibody.

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