Methods and compositions for increasing enzyme activity in CNS

Abstract

To provide methods and compositions for treating a subject suffering from an enzyme deficiency in the central nervous system (CNS).SOLUTION: Bifunctional fusion antibodies provided herein comprise an antibody to an endogenous blood brain barrier (BBB) receptor and an enzyme deficient in mucopolysaccharidosis III (MPS-III). Fusion antibodies provided herein comprise N-sulfoglucosamine sulfohydrolase (SGSH), α-N-acetylglucosaminidase (NAGLU), heparin-α-glucosaminide N-acetyltransferase (HGSNAT), or N-acetylglucosamine-6-sulfatase (GNS).SELECTED DRAWING: Figure 12

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 61 / 857,140, ​​filed July 22, 2013, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web, the entirety of which is incorporated herein by reference. The ASCII copy was created on May 14, 2014, is named 28570_713_601_SL.txt, and is 84 kilobytes in size. [Background technology]

[0003] Mucopolysaccharidosis (MPS) type III, also called MPS-III or Sanfilippo syndrome, is an inherited metabolic disorder that primarily affects the central nervous system (CNS). MPS III is caused by a deficiency of enzymes required to break down long-chain sugar molecules called glycosaminoglycans. There are four major types of MPS-III. Type A (MPS-IIIA) is caused by a deficiency of the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), also called sulfamidase or N-heparan sulfatase, which acts to break down heparan sulfate glycosaminoglycans (GAGs). SGSH is responsible for the hydrolysis of N-linked sulfate groups from the non-reducing terminal glucosaminide residues of heparan sulfate. Type B (MPS-IIIB) is caused by a deficiency of α-N-acetylglucosaminidase (NAGLU). Type C (MPS-IIIC) is caused by a deficiency of heparin-α-glucosaminide N-acetyltransferase (HGSNAT). Type D (MPS-IIID) is caused by a deficiency of N-acetylglucosamine-6-sulfatase (GNS). Deficiencies in the levels of these enzymes result in pathological accumulation of glycosaminoglycans, for example in peripheral tissues and the CNS. However, the clinical features of MPS-III are almost exclusively neurological. Symptoms include behavioral disturbances beginning at an early age and progressing to dementia, and developmental regression, followed by death in the teens or twenties. In general, treatment of lysosomal storage disorders such as MPS-III involves intravenous enzyme replacement therapy with the missing recombinant enzyme. However, systemically administered recombinant enzymes do not cross the blood-brain barrier (BBB) ​​and therefore have little effect on disease effects in the CNS. Summary of the Invention [Problem to be solved by the invention]

[0004] The present specification describes methods and compositions for treating subjects suffering from sulfamidase, i.e., N-sulfoglucosamine sulfohydrolase ("SGSH") deficiency. In some embodiments, the methods described herein feature delivering SGSH to the CNS by systemically administering a therapeutically effective amount of a bifunctional fusion antibody or protein. In some embodiments, the bifunctional fusion antibody comprises an antibody against an endogenous blood-brain barrier (BBB) ​​receptor and an amino acid sequence of SGSH. In some embodiments, the bifunctional fusion antibody is a human insulin antibody (HIR Ab) genetically fused with SGSH ("HIR Ab-SGSH fusion antibody"). In some embodiments, the HIR Ab-SGSH fusion antibody binds to the extracellular domain of the insulin receptor and is transported across the blood-brain barrier ("BBB") into the CNS while retaining SGSH enzymatic activity. In some embodiments, the HIR Ab acts as a molecular Trojan horse that binds to the endogenous insulin receptor on the BBB and transports SGSH into the brain. In some embodiments, the therapeutically effective systemic dose of the HIR Ab-SGSH fusion antibody used for systemic administration is based, in part, on the specific CNS uptake properties of the fusion antibody from peripheral blood, as described herein. [Means for solving the problem]

[0005] In some embodiments, the present disclosure provides a method for treating SGSH deficiency in the central nervous system of a subject in need of treatment, comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody having SGSH activity. In some embodiments, the fusion antibody comprises an amino acid sequence of an immunoglobulin heavy chain, an amino acid sequence of SGSH, and an amino acid sequence of an immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor (e.g., human insulin receptor) and catalyzes the hydrolysis of N-linked sulfate groups from non-reducing terminal glucosaminide residues of heparan sulfate. In some embodiments, the amino acid sequence of SGSH is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin heavy chain.

[0006] In some embodiments, the fusion antibody is post-translationally modified by sulfatase modifier type 1 (SUMF1). In some embodiments, the post-translational modification comprises a cysteine ​​to formylglycine conversion. In some embodiments, the fusion antibody comprises formylglycine.

[0007] In some embodiments, SGSH retains at least 20% of its activity compared to its activity as a separate entity, hi some embodiments, SGSH and the immunoglobulin each retain at least 20% of their activity compared to their activity as a separate entity.

[0008] In some embodiments, at least about 10 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 20 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 30 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 40 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 50 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 100 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 200 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 300 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 400 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 500 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 1000 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 5 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 1 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 0.5 μg of SGSH enzyme is delivered to the brain. In some embodiments, at least about 0.1 μg of SGSH enzyme is delivered to the brain.

[0009] In some embodiments, normalized per 50 kg body weight, at least about 200 μg of SGSH enzyme is delivered to the brain. In some embodiments, normalized per 50 kg body weight, at least about 250 μg of SGSH enzyme is delivered to the brain. In some embodiments, normalized per 50 kg body weight, at least about 300 μg of SGSH enzyme is delivered to the brain. In some embodiments, normalized per 50 kg body weight, at least about 400 μg of SGSH enzyme is delivered to the brain. In some embodiments, normalized per 50 kg body weight, at least about 500 μg of SGSH enzyme is delivered to the brain. In some embodiments, normalized per 50 kg body weight, at least about 1000 μg of SGSH enzyme is delivered to the brain. In some embodiments, normalized per 50 kg body weight, at least about 2000 μg of SGSH enzyme is delivered to the brain. In some embodiments, normalized per 50 kg body weight, at least about 150 μg of SGSH enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 100 μg of SGSH enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 50 μg of SGSH enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 10 μg of SGSH enzyme is delivered to the brain.

[0010] In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.5 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.6 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.7 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.8 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.9 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 1 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 2 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 5 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.4 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.3 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.2 mg / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 0.1 mg / Kg body weight.

[0011] In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 1000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 1500 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 2000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 3000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 4000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 5000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 10,000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 15,000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 20,000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 25,000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 900 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 800 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 700 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 600 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 500 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 400 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 300 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 200 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 100 units / Kg body weight.

[0012] In some embodiments, the fusion antibody has a SGSH specific activity of at least 1000 units / mg protein. In some embodiments, the fusion antibody has a SGSH specific activity of at least 1500 units / mg. In some embodiments, the fusion antibody has a SGSH specific activity of at least 2000 units / mg. In some embodiments, the fusion antibody has a SGSH specific activity of at least 3000 units / mg. In some embodiments, the fusion antibody has a SGSH specific activity of at least 4000 units / mg. In some embodiments, the fusion antibody has a SGSH specific activity of at least 5000 units / mg. In some embodiments, the fusion antibody has a SGSH specific activity of at least 10,000 units / mg. In some embodiments, the fusion antibody has a SGSH specific activity of at least 12,000 units / mg. In some embodiments, the fusion antibody has a SGSH specific activity of at least It is 15,000 units / mg.

[0013] In some embodiments, systemic administration is parenteral, intravenous, subcutaneous, intramuscular, intranasal, intraarterial, transdermal, or by inhalation.

[0014] In some embodiments, the fusion antibody is a chimeric antibody. In some embodiments, the fusion antibody is a humanized antibody.

[0015] In some embodiments, the immunoglobulin heavy chain is an IgG immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is an IgG1 class immunoglobulin heavy chain.

[0016] In some embodiments, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1 containing no more than four single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2 containing no more than six single amino acid mutations, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3 containing no more than three single amino acid mutations, where the single amino acid mutation is a substitution, deletion, or insertion.

[0017] In another embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1 containing not more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2 containing not more than 6 single amino acid mutations, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3 containing not more than 3 single amino acid mutations.

[0018] In another embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1 containing not more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2 containing not more than 6 single amino acid mutations, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3 containing one single amino acid mutation.

[0019] In another embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1 containing one single amino acid mutation, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2 containing one single amino acid mutation, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3 containing one single amino acid mutation.

[0020] In another embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3.

[0021] In a further embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3.

[0022] In some embodiments, the immunoglobulin light chain is an immunoglobulin light chain of the kappa or lambda class.

[0023] In some embodiments, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4 containing no more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5 containing no more than 5 single amino acid mutations, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6 containing no more than 5 single amino acid mutations, where the single amino acid mutation is a substitution, deletion, or insertion.

[0024] In another embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4 containing not more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5 containing not more than 5 single amino acid mutations, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6 containing not more than 5 single amino acid mutations.

[0025] In another embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4 containing not more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5 containing not more than 3 single amino acid mutations, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6 containing not more than 3 single amino acid mutations.

[0026] In another embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4 containing one single amino acid mutation, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5 containing one single amino acid mutation, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6 containing one single amino acid mutation.

[0027] In another embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6.

[0028] In a further embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6.

[0029] In some embodiments, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3, and the immunoglobulin light chain comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6.

[0030] In some embodiments, the immunoglobulin heavy chain of the fusion antibody is at least 90% identical to SEQ ID NO:7 and the amino acid sequence of the light chain immunoglobulin is at least 90% identical to SEQ ID NO:8.

[0031] In some embodiments, the immunoglobulin heavy chain of the fusion antibody is at least 95% identical to SEQ ID NO:7 and the amino acid sequence of the light chain immunoglobulin is at least 95% identical to SEQ ID NO:8.

[0032] In some embodiments, the immunoglobulin heavy chain of the fusion antibody comprises SEQ ID NO:7 and the amino acid sequence of the light chain immunoglobulin comprises SEQ ID NO:8.

[0033] In some embodiments, SGSH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, SGSH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, SGSH comprises the amino acid sequence of SEQ ID NO: 9.

[0034] In another embodiment, the amino acid sequence of the immunoglobulin heavy chain of the fusion antibody is at least 90% identical to SEQ ID NO:7, the amino acid sequence of the light chain immunoglobulin is at least 90% identical to SEQ ID NO:8, and the amino acid sequence of SGSH is at least 95% identical to or includes SEQ ID NO:9.

[0035] In another embodiment, the amino acid sequence of the immunoglobulin heavy chain of the fusion antibody comprises SEQ ID NO:8, the amino acid sequence of the immunoglobulin light chain comprises SEQ ID NO:8, and the amino acid sequence of SGSH comprises SEQ ID NO:9.

[0036] In some embodiments, the fusion antibody herein crosses the BBB by binding an endogenous BBB receptor-mediated transport system. In some embodiments, the fusion antibody crosses the BBB via an endogenous BBB receptor selected from the group consisting of insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and insulin-like growth factor (IGF) receptor. In some embodiments, the fusion antibody crosses the BBB by binding the insulin receptor.

[0037] In some embodiments, systemic administration is parenteral, intravenous, subcutaneous, intramuscular, intranasal, intraarterial, transdermal, or by inhalation.

[0038] In some embodiments, the SGSH deficiency in the central nervous system is Mucopolysaccharidosis type IIIA (MPS-IIIA) or Sanfilippo syndrome type A.

[0039] In some embodiments, the present disclosure provides a method for treating N-sulfoglucosamine sulfohydrolase (SGSH) deficiency in the central nervous system of a subject in need of such treatment, the method comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody having SGSH activity, the fusion antibody comprising (a) an immunoglobulin light chain and an amino acid sequence of SGSH, and (b) an immunoglobulin heavy chain, the fusion antibody crossing the blood-brain barrier (BBB). In some embodiments, the amino acid sequence of SGSH is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin light chain.

[0040] In some embodiments, the present disclosure provides a method for treating SGSH deficiency in the central nervous system of a subject in need of treatment, the method comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody having SGSH activity, the fusion antibody comprising (a) a fusion protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 10, and (b) an immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor. In some embodiments, the endogenous BBB receptor is the human insulin receptor. In some embodiments, the fusion antibody catalyzes the hydrolysis of N-linked sulfate esters from heparan sulfate. In some embodiments, the fusion protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 10.

[0041] In some embodiments, the present disclosure provides a fusion antibody having SGSH activity, the fusion antibody comprising (a) a fusion protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 10, and (b) an immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor. In some embodiments, the endogenous BBB receptor is a human insulin receptor. In some embodiments, the fusion antibody is an antibody that binds to an endogenous BBB receptor. In some embodiments, the fusion antibody is an antibody that binds to a human insulin receptor. In some embodiments, the fusion antibody catalyzes the hydrolysis of N-linked sulfate esters from heparan sulfate. In some embodiments, the fusion protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 10.

[0042] In some embodiments, the present disclosure provides a fusion antibody having SGSH activity, the fusion antibody comprising (a) an immunoglobulin heavy chain and an amino acid sequence of SGSH, and (b) an immunoglobulin light chain. In some embodiments, the amino acid sequence of SGSH is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin heavy chain. In some embodiments, the present disclosure provides a fusion antibody having SGSH activity, the fusion antibody comprising (a) an immunoglobulin light chain and an amino acid sequence of SGSH, and (b) an immunoglobulin heavy chain. In some embodiments, the amino acid sequence of SGSH is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor. In some embodiments, the endogenous BBB receptor is the human insulin receptor. In some embodiments, the fusion antibody is an antibody that binds to an endogenous BBB receptor. In some embodiments, the fusion antibody is an antibody that binds to the human insulin receptor. In some embodiments, the fusion antibody catalyzes the hydrolysis of N-linked sulfate esters from heparan sulfate.

[0043] In some embodiments, the fusion protein herein further comprises a linker between the amino acid sequence of SGSH and the carboxy terminus of the amino acid sequence of the immunoglobulin heavy chain.

[0044] In some embodiments, the present specification provides a pharmaceutical composition comprising a therapeutically effective amount of a fusion antibody described herein, and a pharma- ceutically acceptable excipient.

[0045] In some embodiments, the present specification provides an isolated polynucleotide encoding a fusion antibody described herein. In some embodiments, the isolated polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the present specification provides a vector comprising the isolated polynucleotide provided herein. In some embodiments, the present specification provides a vector comprising the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the present specification provides a host cell comprising the vector described herein. In some embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell. In some embodiments, the present specification provides a method for treating SGSH deficiency in the central nervous system of a subject in need of treatment, the method comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody having SGSH activity, the fusion antibody comprising (a) an immunoglobulin heavy chain and an amino acid sequence of SGSH, and (b) an immunoglobulin light chain. In some embodiments, the amino acid sequence of SGSH is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin heavy chain. In some embodiments, the present disclosure provides a method for treating SGSH deficiency in the central nervous system of a subject in need of treatment, the method comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody having SGSH activity, the fusion antibody comprising (a) an immunoglobulin light chain and a fusion protein comprising an amino acid sequence of SGSH, and (b) an immunoglobulin heavy chain. In some embodiments, the amino acid sequence of SGSH is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor. In some embodiments, the endogenous BBB receptor is a human insulin receptor. In some embodiments, the fusion antibody is an antibody that binds to an endogenous BBB receptor. In some embodiments, the fusion antibody is an antibody that binds to a human insulin receptor. In some embodiments, the fusion antibody catalyzes the hydrolysis of N-linked sulfate esters from heparan sulfate.

[0046] In some embodiments, the present disclosure provides methods and compositions for treating a subject suffering from an enzyme deficiency in the CNS. In some embodiments, the methods described herein feature delivering an enzyme deficient in mucopolysaccharidosis type III (MPS-III) to the CNS by systemically administering a therapeutically effective amount of a bifunctional fusion antibody or protein. In some embodiments, the bifunctional fusion antibody comprises an antibody against an endogenous blood-brain barrier (BBB) ​​receptor and an amino acid sequence of an enzyme deficient in MPS-III. In some embodiments, the bifunctional fusion antibody is a human insulin antibody (HIR Ab) genetically fused to the enzyme. In some embodiments, the fusion antibody binds to the extracellular domain of the insulin receptor and is transported across the BBB into the CNS while retaining enzymatic activity. In some embodiments, the fusion antibody acts as a molecular Trojan horse that binds to the endogenous insulin receptor on the BBB and transports the enzyme into the brain. In some embodiments, the therapeutically effective systemic dose of the fusion antibody used for systemic administration is based, in part, on the specific CNS uptake properties of the fusion antibody from peripheral blood, as described herein.

[0047] In some embodiments, the present disclosure provides a method for treating an enzyme deficiency in the central nervous system of a subject in need of treatment, the method comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody, the fusion antibody comprising an amino acid sequence of an immunoglobulin heavy chain, an amino acid sequence of an enzyme deficient in MPS-III, and an amino acid sequence of an immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor (e.g., human insulin receptor). In some embodiments, the amino acid sequence of the enzyme is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin heavy chain.

[0048] In some embodiments, the enzyme deficient in MPS-III is a lysosomal enzyme.

[0049] In some embodiments, the enzyme deficient in MPS-III is N-sulfoglucosamine sulfohydrolase (SGSH), α-N-acetylglucosaminidase (NAGLU), heparin-α-glucosaminide N-acetyltransferase (HGSNAT), or N-acetylglucosamine-6-sulfatase (GNS).

[0050] In some embodiments, the fusion antibody is post-translationally modified by sulfatase modifier type 1 (SUMF1). In some embodiments, the post-translational modification comprises a cysteine ​​to formylglycine conversion. In some embodiments, the fusion antibody comprises formylglycine.

[0051] In some embodiments, the fusion antibody catalyzes the hydrolysis of N-linked sulfate esters from heparan sulfate, catalyzes the hydrolysis of N-acetyl-D-glucosamine residues in N-acetyl-α-D-glucosaminide, catalyzes the acetylation of glucosamine residues in heparan sulfate, or catalyzes the hydrolysis of the 6-sulfate group of heparan sulfate.

[0052] In some embodiments, the enzyme retains at least 20% of its activity compared to its activity alone. In some embodiments, the enzyme and the immunoglobulin each retain at least 20% of its activity compared to its activity alone. In some embodiments, the enzyme retains at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% of its activity compared to its activity alone. In some embodiments, the enzyme and the immunoglobulin each retain at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% of its activity compared to its activity alone.

[0053] In some embodiments, at least about 10 μg of said enzyme is delivered to the brain. In some embodiments, at least about 20 μg of enzyme is delivered to the brain. In some embodiments, at least about 30 μg of enzyme is delivered to the brain. In some embodiments, at least about 40 μg of enzyme is delivered to the brain. In some embodiments, at least about 50 μg of enzyme is delivered to the brain. In some embodiments, at least about 100 μg of enzyme is delivered to the brain. In some embodiments, at least about 200 μg of enzyme is delivered to the brain. In some embodiments, at least about 300 μg of enzyme is delivered to the brain. In some embodiments, at least about 400 μg of enzyme is delivered to the brain. In some embodiments, at least about 500 μg of enzyme is delivered to the brain. In some embodiments, at least about 1000 μg of enzyme is delivered to the brain. In some embodiments, at least about 5 μg of enzyme is delivered to the brain. In some embodiments, at least about 1 μg of enzyme is delivered to the brain. In some embodiments, at least about 0.5 μg of enzyme is delivered to the brain. In some embodiments, at least about 0.1 μg of enzyme is delivered to the brain.

[0054] In some embodiments, when standardized per 50 kg body weight, at least about 200 μg of enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 250 μg of enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 300 μg of enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 400 μg of enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 500 μg of enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 1000 μg of enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 2000 μg of enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 150 μg of enzyme is delivered to the brain. In some embodiments, when standardized per 50 kg body weight, at least about 100 μg of enzyme is delivered to the brain. In some embodiments, at least about 50 μg of enzyme is delivered to the brain when standardized per 50 kg of body weight. In some embodiments, at least about 10 μg of enzyme is delivered to the brain when standardized per 50 kg of body weight.

[0055] In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.5 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.6 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.7 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.8 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.9 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 1 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 2 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 5 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.4 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.3 mg / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 0.2 mg / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 0.1 mg / Kg body weight.

[0056] In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 1000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 1500 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 2000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 3000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 4000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 5000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 10,000 units / Kg body weight. In some embodiments, a therapeutically effective dose of a fusion antibody comprises at least about 15,000 units / Kg body weight. In some embodiments In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 20,000 units / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 25,000 units / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 900 units / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 800 units / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 700 units / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 600 units / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 500 units / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 400 units / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 300 units / Kg body weight. In some embodiments, the therapeutically effective dose of the fusion antibody comprises at least about 200 units / Kg body weight. In some embodiments, a therapeutically effective dose of the fusion antibody comprises at least about 100 units / Kg body weight.

[0057] In some embodiments, the enzymatic specific activity of the fusion antibody is at least 1000 units / mg protein. In some embodiments, the enzymatic specific activity of the fusion antibody is at least 1500 units / mg. In some embodiments, the enzymatic specific activity of the fusion antibody is at least 2000 units / mg. In some embodiments, the enzymatic specific activity of the fusion antibody is at least 3000 units / mg. In some embodiments, the enzymatic specific activity of the fusion antibody is at least 4000 units / mg. In some embodiments, the enzymatic specific activity of the fusion antibody is at least 5000 units / mg. In some embodiments, the enzymatic specific activity of the fusion antibody is at least 10,000 units / mg. In some embodiments, the enzymatic specific activity of the fusion antibody is at least 12,000 units / mg. In some embodiments, the enzymatic specific activity of the fusion antibody is at least 15,000 units / mg.

[0058] In some embodiments, systemic administration is parenteral, intravenous, subcutaneous, intramuscular, intranasal, intraarterial, transdermal, or by inhalation.

[0059] In some embodiments, the fusion antibody is a chimeric antibody. In some embodiments, the fusion antibody is a humanized antibody.

[0060] In some embodiments, the immunoglobulin heavy chain is an IgG immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is an IgG1 class immunoglobulin heavy chain.

[0061] In some embodiments, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1 containing no more than four single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2 containing no more than six single amino acid mutations, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3 containing no more than three single amino acid mutations, where the single amino acid mutation is a substitution, deletion, or insertion.

[0062] In another embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1 containing not more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2 containing not more than 6 single amino acid mutations, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3 containing not more than 3 single amino acid mutations.

[0063] In another embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1 containing not more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2 containing not more than 6 single amino acid mutations, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3 containing one single amino acid mutation.

[0064] In another embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1 containing one single amino acid mutation, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2 containing one single amino acid mutation, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3 containing one single amino acid mutation.

[0065] In another embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3.

[0066] In a further embodiment, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3.

[0067] In some embodiments, the immunoglobulin light chain is an immunoglobulin light chain of the kappa or lambda class.

[0068] In some embodiments, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4 containing no more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5 containing no more than 5 single amino acid mutations, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6 containing no more than 5 single amino acid mutations, where the single amino acid mutation is a substitution, deletion, or insertion.

[0069] In another embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4 containing not more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5 containing not more than 5 single amino acid mutations, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6 containing not more than 5 single amino acid mutations.

[0070] In another embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4 containing not more than 3 single amino acid mutations, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5 containing not more than 3 single amino acid mutations, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6 containing not more than 3 single amino acid mutations.

[0071] In another embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4 containing one single amino acid mutation, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5 containing one single amino acid mutation, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6 containing one single amino acid mutation.

[0072] In another embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6.

[0073] In a further embodiment, the immunoglobulin light chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6.

[0074] In some embodiments, the immunoglobulin heavy chain of the fusion antibody comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3, and the immunoglobulin light chain comprises a CDR1 corresponding to the amino acid sequence of SEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:5, and a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6.

[0075] In some embodiments, the immunoglobulin heavy chain of the fusion antibody is at least 90% identical to SEQ ID NO:7 and the amino acid sequence of the light chain immunoglobulin is at least 90% identical to SEQ ID NO:8.

[0076] In some embodiments, the immunoglobulin heavy chain of the fusion antibody is at least 95% identical to SEQ ID NO:7 and the amino acid sequence of the light chain immunoglobulin is at least 95% identical to SEQ ID NO:8.

[0077] In some embodiments, the immunoglobulin heavy chain of the fusion antibody comprises SEQ ID NO:7 and the amino acid sequence of the light chain immunoglobulin comprises SEQ ID NO:8.

[0078] In some embodiments, the enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21. In some embodiments, the enzyme comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21. In some embodiments, the enzyme comprises the amino acid sequence of SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21.

[0079] In some embodiments, the fusion antibody herein crosses the BBB by binding an endogenous BBB receptor-mediated transport system. In some embodiments, the fusion antibody crosses the BBB via an endogenous BBB receptor selected from the group consisting of insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and insulin-like growth factor (IGF) receptor. In some embodiments, the fusion antibody crosses the BBB by binding the insulin receptor.

[0080] In some embodiments, systemic administration is parenteral, intravenous, subcutaneous, intramuscular, intranasal, intraarterial, transdermal, or by inhalation.

[0081] In some embodiments, the enzyme deficiency in the central nervous system is Mucopolysaccharidosis type IIIA (MPS-IIIA), Mucopolysaccharidosis type IIIB (MPS-IIIB), Mucopolysaccharidosis type IIIC (MPS-IIIC), or Mucopolysaccharidosis type IIID (MPS-IIID).

[0082] In some embodiments, the present disclosure provides a method for treating an enzyme deficiency in the central nervous system of a subject in need thereof, the method comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody, the fusion antibody comprising (a) an immunoglobulin light chain and an amino acid sequence of an enzyme deficient in Mucopolysaccharidosis Type III (MPS-III), and (b) an immunoglobulin heavy chain, and crossing the blood-brain barrier (BBB). In some embodiments, the amino acid sequence of the enzyme is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin light chain.

[0083] In some embodiments, the present disclosure provides a method for treating an enzyme deficiency in the central nervous system of a subject in need of treatment, the method comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody, the fusion antibody comprising (a) a fusion protein comprising an amino acid sequence at least 90% identical to SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, and (b) an immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor. In some embodiments, the endogenous BBB receptor is the human insulin receptor. In some embodiments, the fusion antibody catalyzes the hydrolysis of N-linked sulfate esters from heparan sulfate, catalyzes the hydrolysis of N-acetyl-D-glucosamine residues in N-acetyl-α-D-glucosaminides, catalyzes the acetylation of glucosamine residues of heparan sulfate, or catalyzes the hydrolysis of the 6-sulfate group of heparan sulfate. In some embodiments, the fusion protein comprises an amino acid sequence at least 95% identical to SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22.

[0084] In some embodiments, the present disclosure provides a fusion antibody comprising (a) a fusion protein comprising an amino acid sequence at least 90% identical to SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, and (b) an immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor. In some embodiments, the endogenous BBB receptor is a human insulin receptor. In some embodiments, the fusion antibody is an antibody that binds to an endogenous BBB receptor. In some embodiments, the fusion antibody is an antibody that binds to a human insulin receptor. In some embodiments, the fusion antibody catalyzes the hydrolysis of N-linked sulfate esters from heparan sulfate, catalyzes the hydrolysis of N-acetyl-D-glucosamine residues in N-acetyl-α-D-glucosaminides, catalyzes the acetylation of glucosamine residues of heparan sulfate, or catalyzes the hydrolysis of the 6-sulfate group of heparan sulfate. In some embodiments, the fusion protein comprises an amino acid sequence at least 95% identical to SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22.

[0085] In some embodiments, the present disclosure provides (a) a fusion protein comprising an immunoglobulin heavy chain and an amino acid sequence of an enzyme deficient in mucopolysaccharidosis type III (MPS-III), and (b) a fusion antibody comprising an immunoglobulin light chain. In some embodiments, the amino acid sequence of the enzyme is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin heavy chain. In some embodiments, the present disclosure provides (a) a fusion protein comprising an immunoglobulin light chain and an amino acid sequence of an enzyme deficient in mucopolysaccharidosis type III (MPS-III), and (b) a fusion antibody comprising an immunoglobulin heavy chain. In some embodiments, the amino acid sequence of the enzyme is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor. In some embodiments, the endogenous BBB receptor is the human insulin receptor. In some embodiments, the fusion antibody is an antibody that binds to an endogenous BBB receptor. In some embodiments, the fusion antibody is an antibody that binds to the human insulin receptor. In some embodiments, the fusion antibody catalyzes the hydrolysis of N-linked sulfate esters from heparan sulfate, catalyzes the hydrolysis of N-acetyl-D-glucosamine residues in N-acetyl-α-D-glucosaminide, catalyzes the acetylation of glucosamine residues in heparan sulfate, or catalyzes the hydrolysis of the 6-sulfate group of heparan sulfate.

[0086] In some embodiments, the fusion protein herein further comprises a linker between the amino acid sequence of the enzyme and the carboxy terminus of the amino acid sequence of the immunoglobulin heavy chain.

[0087] In some embodiments, the present specification provides a pharmaceutical composition comprising a therapeutically effective amount of a fusion antibody described herein, and a pharma- ceutically acceptable excipient.

[0088] In some embodiments, the present specification provides an isolated polynucleotide encoding a fusion antibody described herein. In some embodiments, the isolated polynucleotide comprises a nucleic acid sequence of SEQ ID NO:14, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. In some embodiments, the present specification provides a vector comprising an isolated polynucleotide herein. In some embodiments, the present specification provides a vector comprising a nucleic acid sequence of SEQ ID NO:14, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. In some embodiments, the present specification provides a host cell comprising a vector described herein. In some embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell.

[0089] In some embodiments, the present disclosure provides a method for treating an enzyme deficiency in the central nervous system of a subject in need of treatment, the method comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody, the fusion antibody comprising (a) an immunoglobulin heavy chain and a fusion protein comprising an amino acid sequence for an enzyme deficient in mucopolysaccharidosis type III (MPS-III), and (b) an immunoglobulin light chain. In some embodiments, the amino acid sequence for the enzyme is covalently linked to the carboxy terminus of the amino acid sequence for the immunoglobulin heavy chain. In some embodiments, the present disclosure provides a method for treating an enzyme deficiency in the central nervous system of a subject in need of treatment, the method comprising systemically administering to the subject a therapeutically effective dose of a fusion antibody, the fusion antibody comprising (a) an immunoglobulin light chain and a fusion protein comprising an amino acid sequence for an enzyme deficient in mucopolysaccharidosis type III (MPS-III), and (b) an immunoglobulin heavy chain. In some embodiments, the amino acid sequence for the enzyme is covalently linked to the carboxy terminus of the amino acid sequence for the immunoglobulin light chain. In some embodiments, the fusion antibody binds to the extracellular domain of an endogenous BBB receptor. In some embodiments, the endogenous BBB receptor is the human insulin receptor. In some embodiments, the fusion antibody is an antibody that binds to an endogenous BBB receptor. In some embodiments, the fusion antibody is an antibody that binds to the human insulin receptor. In some embodiments, the fusion antibody catalyzes the hydrolysis of N-linked sulfate esters from heparan sulfate, catalyzes the hydrolysis of N-acetyl-D-glucosamine residues in N-acetyl-α-D-glucosaminides, catalyzes the acetylation of glucosamine residues of heparan sulfate, or catalyzes the hydrolysis of the 6-sulfate group of heparan sulfate.

[0090] Citation by reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0091] The novel features of the present invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present invention are utilized, and the accompanying drawings, in which:

[0092] [Figure 1] Schematic diagram of the "molecular Trojan horse" strategy, in which the fusion antibody contains an antibody against the extracellular domain of an endogenous BBB receptor (R), which acts as a molecular Trojan horse (TH), and SGSH, a lysosomal enzyme (E). Once inside brain cells behind the BBB, the SGSH portion of the fusion antibody then converts heparan sulfate (S) into degradable products (P).

[0093] [Diagram 2] A typical HIR Ab-SGSH fusion antibody is formed by fusing the amino terminus of mature SGSH with the carboxyl terminus of the CH3 region of the heavy chain of an HIR Ab.

[0094] [Diagram 3] Ethidium bromide stained agarose gel of 1.5 kb human SGSH cDNA (lane 3) generated by PCR from human liver cDNA and SGSH-specific primers (Table 2). Lanes 1 and 2: Lambda HindIII-digested DNA standard and PhiX174 HaeIII-digested DNA standard, respectively.

[0095] [Figure 4]A genetically engineered tandem vector (TV) encoding four separate tandem expression cassettes encoding the heavy chain (HC) fusion gene, the light chain (LC) gene, the dihydrofolate reductase (DHFR) gene, and the neomycin resistance (Neo) gene, designated TV-HIRMAb-SGSH. Other elements include the ampicillin resistance gene (amp) and an origin of replication (ori). The heavy and light chain expression cassettes are flanked on the 5' side by a cytomegalovirus (CMV) promoter and on the 3' side by a bovine growth hormone (BGH) poly-A sequence. The DHFR expression cassette is flanked on the 5' side by a simian virus (SV) 40 promoter and on the 3' side by a hepatitis B virus (HBV) poly-A sequence.

[0096] [Diagram 5] Amino acid sequence of the immunoglobulin heavy chain variable region from a representative human insulin receptor antibody against the extracellular domain of the human insulin receptor. The underlined sequences are the signal peptide, CDR1, CDR2, and CDR3, respectively. The heavy chain constant region from human IgG1 is shown in italics.

[0097] [Figure 6] Amino acid sequence of the immunoglobulin light chain variable region from a representative human insulin receptor antibody against the extracellular domain of the human insulin receptor. The underlined sequences are the signal peptide, CDR1, CDR2, and CDR3, respectively. The constant region from the human kappa light chain is shown in italics.

[0098] [Figure 7] 1 is a table showing the CDR1, CDR2, and CDR3 amino acid sequences from the heavy and light chains of exemplary human insulin receptor antibodies against the extracellular domain of the human insulin receptor.

[0099] [Figure 8] Amino acid sequence of SGSH (NP_000190), without the first 20 amino acid signal peptide (mature SGSH).

[0100] [Figure 9] Amino acid sequence of an exemplary human insulin receptor antibody heavy chain fusion with mature human SGSH. The underlined sequences are, respectively, the IgG signal peptide, CDR1, CDR2, CDR3, and the peptide linker (Ser-Ser-Ser) connecting the carboxy terminus of the heavy chain to the amino terminus of SGSH. The italicized sequence corresponds to the heavy chain constant region from human IgG1. The bolded sequence corresponds to human SGSH.

[0101] [Figure 10] Reducing SDS-PAGE of molecular weight standards (lanes 1 and 4), purified HIRMAb (lane 2), and purified HIRMAb-SGSH fusion protein (lane 3).

[0102] [Figure 11] Western blots using either anti-human (h) IgG primary antibody (left panel) or anti-human SGSH primary antiserum (right panel). Immunoreactivity of the HIRMAb-SGSH fusion protein is compared to chimeric HIRMAb and recombinant SGSH. Both the HIRMAb-SGSH fusion protein and the HIRMAb have identical light chains in anti-hIgG Westerns. The HIRMAb-SGSH fusion heavy chain reacts with both anti-hIgG and anti-human SGSH antibodies, whereas the HIRMAb heavy chain reacts only with anti-hIgG antibodies. Recombinant SGSH reacts only with anti-human SGSH antibodies.

[0103] [Figure 12] Binding of either the chimeric HIRMAb or the HIRMAb-SGSH fusion protein to the HIR extracellular domain (ECD) was saturable: after normalizing for molecular weight differences, the ED50 of HIRMAb-SGSH binding to the HIR ECD (0.33 ± 0.05 nM) was comparable to the ED50 of chimeric HIRMAb binding (0.19 ± 0.02 nM).

[0104] [Figure 13](A) The SGSH fluorometric enzymatic assay is a two-step assay. The substrate 4-methylumbelliferyl-α-DN-sulfoglucosaminide (MU-α-GlcNS) is converted by SGSH to methylumbelliferyl-α-D-glucosaminide (MU-α-GlcNH2), which is then converted to the fluorescent product 4-methylumbelliferone (4-MU) by the second step enzyme α-glucosaminidase. (B) Linear generation of 4-MU product by addition of 30-300 ng of HIRMAb-SGSH fusion protein. Data are the mean ± standard deviation of triplicates.

[0105] [Figure 14] Plasma concentrations (ng / mL) of TCA-precipitable [125I]-HIRMAb-SGSH fusion protein are plotted versus time over 140 min in adult rhesus monkeys after a single intravenous injection of 19 μg / kg of fusion protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0106] Detailed Description of the Invention The blood-brain barrier (BBB) ​​is a significant barrier preventing the delivery of systemically administered lysosomal enzymes (e.g., recombinant SGSH) to the central nervous system. The methods and compositions described herein address key factors in delivering therapeutically relevant levels of enzymes deficient in Mucopolysaccharidosis Type III (MPS-III), e.g., SGSH, NAGLU, HGSNAT, GNS, across the BBB and into the CNS: 1) modification of the MPS-III deficient enzyme to enable it to cross the BBB via endogenous BBB on transporters, and 2) the amount and rate of uptake of the systemically administered modified enzyme into the CNS by retaining enzyme activity after the modifications necessary to effect BBB transport. Various embodiments of the methods and compositions described herein address these factors by (1) providing a fusion antibody that includes an enzyme (i.e., a protein with SGSH activity) fused with or without an intervening sequence to an immunoglobulin (heavy or light chain) against the extracellular domain of an endogenous BBB receptor, and (2) establishing a therapeutically effective systemic dose of the fusion antibody based on uptake and specific activity in the CNS. In some embodiments, the antibody against the endogenous BBB receptor is an antibody against the human insulin receptor (HIR Ab).

[0107] Accordingly, provided herein are compositions and methods for treating enzyme (e.g., SGSH) deficiencies in the central nervous system by systemically administering to a subject in need of treatment a therapeutically effective dose of a bifunctional BBB receptor Ab-enzyme fusion antibody, which has enzymatic activity and selectively binds to the extracellular domain of an endogenous BBB receptor transporter, such as the human insulin receptor.

[0108] (definition) As used herein, "treatment" or "treating" includes achieving a therapeutic effect and / or a prophylactic effect. A therapeutic effect refers to the eradication or amelioration of the underlying disorder or condition being treated. For example, in an individual affected with MPS-IIIA, a therapeutic effect can include a partial or complete halt in the progression of the disorder, or a partial or complete reversal of the disorder. A therapeutic effect can also be achieved by the eradication or amelioration of one or more physiological or psychological symptoms associated with the underlying condition, such that an improvement is observed in the patient, despite the fact that the patient may still be affected by the condition. A prophylactic effect of treatment can include preventing a condition, slowing the progression of a condition (e.g., slowing the progression of a lysosomal storage disorder), or reducing the likelihood of a condition occurring. As used herein, "treating" or "treatment" includes prevention.

[0109] As used herein, the term "effective amount" may be an amount sufficient to achieve a beneficial or desired result in the CNS when administered systemically, e.g., a beneficial or desired clinical outcome, or improved cognition, memory, mood, or other desired CNS outcome. An effective amount may also be an amount that provides a prophylactic effect, e.g., an amount that delays, reduces, or eliminates the onset of a pathological or undesirable condition, such as, but not limited to, mental retardation, hearing loss, and neurodegeneration. An effective amount may be administered in one or more administrations. In terms of treatment, an "effective amount" of a composition herein is an amount sufficient to alleviate, ameliorate, stabilize, reverse, or slow the progression of a disorder, e.g., a neurological disorder. An "effective amount" may be an amount of any composition herein used alone or in combination with one or more agents used to treat a disease or disorder. Within the meaning of this embodiment, an "effective amount" of a therapeutic agent will be determined by the patient's physician or veterinarian. Such an amount will be readily established by one of skill in the art and will provide a therapeutic effect when administered according to this embodiment. Factors that influence what a therapeutically effective amount will be include the specific enzyme activity of the fusion antibody administered, its absorption profile (e.g., its rate of uptake into the brain), the time since onset of the disorder, and the age, health, presence of other disease conditions, and nutritional status of the individual being treated. Additionally, other medications that the patient may be taking will affect the determination of the therapeutically effective amount of the therapeutic agent to be administered.

[0110] As used herein, a "subject" or "individual" is an animal, e.g., a mammal. In some embodiments, a "subject" or "individual" is a human. In some embodiments, the subject is afflicted with MPS-IIIA.

[0111] In some embodiments, a pharmaceutical composition comprising a fusion antibody is "administered peripherally" or "peripherally administered." As used herein, these terms refer to any form of administration of an agent, e.g., a therapeutic agent, to an individual that is not administered directly to the CNS, i.e., that contacts the non-brain side of the blood-brain barrier. As used herein, "peripheral administration" includes intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, transdermal, by inhalation, transbuccal, intranasal, rectal, oral, parenteral, sublingual, or nasal administration.

[0112] A "pharmaceutical acceptable carrier" or "pharmaceutical acceptable excipient" herein refers to any carrier that does not itself induce the production of antibodies harmful to an individual ingesting the composition. Such carriers are well known to those skilled in the art. A thorough discussion of pharmaceutical acceptable carriers / excipients can be found in Remington's Pharmaceutical Sciences, Gennaro, AR, ed., 20th edition, 2000: Williams and Wilkins PA, USA. Exemplary pharmaceutical acceptable carriers can include salts, such as mineral acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.), and salts of organic acids (e.g., acetate, propionate, malonate, benzoate, etc.). For example, the compositions described herein may be provided in liquid form and may be formulated in saline-based aqueous solutions of various pHs (5-8) with or without 0.01-1% surfactants, such as polysorbate-80, or carbohydrate additives, such as mannitol, sorbitol, or trehalose. Commonly used buffers include histidine buffer, acetate buffer, phosphate buffer, or citrate buffer.

[0113] "Recombinant host cell" or "host cell" refers to a cell that contains an exogenous polynucleotide, regardless of the method used for insertion, such as direct uptake, transduction, f-mating, or other methods known in the art for generating recombinant host cells. The exogenous polynucleotide may be maintained as a nonintegrated vector, such as a plasmid, or may be integrated into the host genome.

[0114] The terms "polypeptide," "peptide," and "protein" refer to a polymer of amino acid residues. The terms "polypeptide" and "protein" refer to amino acids that are linked together by covalent peptide bonds, and are used interchangeably herein; that is, a description of a polypeptide applies equally to a description of a peptide and to a description of a protein, and vice versa. These terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues are unnatural amino acids, e.g., amino acid analogs. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens) in which the amino acid residues are linked by covalent peptide bonds.

[0115] The term "amino acid" refers to natural and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the natural amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a natural amino acid, i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. The analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a natural amino acid.

[0116] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to herein by their commonly accepted one-letter symbols.

[0117] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof, in either single- or double-stranded form. Unless otherwise specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), analogs of DNA used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0118] The terms "isolated" and "purified" refer to a material that is substantially or essentially removed from or concentrated in its natural environment. For example, an isolated nucleic acid can be separated from the nucleic acids that normally flank it, or from other nucleic acids or components (proteins, lipids, etc.) in a sample. In another example, a polypeptide is purified if it is substantially removed from or concentrated in its natural environment. Methods for purifying and isolating nucleic acids and proteins are well known in the art.

[0119] Blood-brain barrier In one embodiment, the present disclosure provides compositions and methods that utilize an enzyme (e.g., SGSH) that is deficient in MPS-III fused to an immunoglobulin that can cross the blood-brain barrier (BBB) ​​via receptor-mediated transport on endogenous BBB receptors / transporters. A typical endogenous transporter for targeting is the insulin receptor on the BBB. The BBB insulin receptor mediates the transport of blood insulin and certain peptidomimetic monoclonal antibodies (MAbs), such as HIRMAbs, into the brain. Other endogenous transporters that can be targeted by either endogenous ligands or peptidomimetic MAbs include the BBB transferrin receptor, the BBB insulin-like growth factor (IGF) receptor, the BBB leptin receptor, or the BBB low-density lipoprotein (LDL) receptor. The compositions and methods are useful for transporting SGSH from peripheral blood across the blood-brain barrier into the CNS. As used herein, "blood-brain barrier" refers to the barrier between the peripheral circulation and the brain and spinal cord, which is formed by tight junctions in the brain capillary endothelial cell membranes, creating an extremely tight barrier that limits the transport of molecules into the brain: the BBB is so tight that it can even restrict molecules as small as urea, which has a molecular weight of 60 Da. The blood-brain barrier in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retina barrier in the retina are adjacent capillary barriers in the central nervous system (CNS) and are collectively referred to as the blood-brain barrier or BBB.

[0120] The BBB limits the development of new neurotherapeutic, diagnostic, and research tools for the brain and CNS. Most macromolecular therapeutics, e.g., recombinant proteins, antisense drugs, gene medicines, purified antibodies, or RNA interference (RNAi)-based drugs, do not cross the BBB in pharmacologically relevant amounts. It is generally assumed that small molecule drugs can cross the BBB, but in reality, <2% of all small molecule drugs are active in the brain due to the lack of transport across the BBB. To cross the BBB in pharmacologically relevant amounts, a molecule must be lipid soluble and have a molecular weight less than 400 Daltons (Da), and most small molecules do not possess these two molecular characteristics. Thus, most molecules with therapeutic, diagnostic, or research potential do not cross the BBB in pharmacologically active amounts. Invasive transcranial drug delivery strategies, e.g., intracerebroventricular (ICV) injection, intracerebral (IC) administration, and convection-enhanced diffusion (CED), are used to circumvent the BBB. Transcranial drug delivery to the brain is expensive, invasive, and largely ineffective. The ICV route, also called the intrathecal (IT) route, delivers SGSH only to the ependymal or meningeal surface of the brain, rather than into the brain parenchyma as is typical for drugs administered by the ICV route. IC administration of enzymes such as SGSH only results in localized delivery, because protein diffusion efficiency in the brain is very low. Similarly, drug penetration by diffusion is limited, so the CED route only results in localized delivery in the brain near the catheter tip.

[0121] The methods described herein provide an alternative to these highly invasive and generally unsatisfactory methods of bypassing the BBB, which allows functional SGSH to cross the BBB from the peripheral blood into the CNS upon systemic administration of the HIRMAb-SGSH fusion antibody compositions described herein. The methods described herein take advantage of the expression of insulin receptors (e.g., human insulin receptors) on the BBB to transport the desired bifunctional HIRMAb-SGSH fusion antibodies from the peripheral blood into the CNS.

[0122] Endogenous receptors Certain endogenous small molecules in blood, such as glucose or amino acids, are water-soluble and can still permeate the BBB by carrier-mediated transport (CMT) on certain BBB carrier systems. For example, glucose permeates the BBB via CMT on the GLUT1 glucose transporter. Amino acids, including therapeutic amino acids such as L-DOPA, permeate the BBB via CMT on the LAT1 large neutral amino acid transporter. Similarly, certain endogenous macromolecules in blood, such as insulin, transferrin, insulin-like growth factor, leptin, or low-density lipoprotein, can permeate the BBB by receptor-mediated transcytosis (RMT) on certain BBB receptor systems. For example, insulin permeates the BBB via RMT on the insulin receptor. Transferrin permeates the BBB via RMT on the transferrin receptor. Insulin-like growth factor can permeate the BBB via RMT on the insulin-like growth factor receptor. Leptin can permeate the BBB via RMT on the leptin receptor. Low density lipoproteins can penetrate the BBB via transport on the low density lipoprotein receptor.

[0123] The BBB has been shown to have specific receptors that allow the transport of several macromolecules from the blood to the brain, including the insulin receptor. In particular, the insulin receptor is suitable as a transporter for the HIR Ab-SGSH fusion antibodies described herein. The HIR-SGSH fusion antibodies described herein bind to the extracellular domain (ECD) of the human insulin receptor.

[0124] The insulin receptor and its extracellular insulin-binding domain (ECD) have been extensively characterized structurally and functionally in the art. See, e.g., Yip et al. (2003), J Biol. Chem, 278(30):27329-27332, and Whittaker et al. (2005), J Biol Chem, 280(22):20932-20936. The amino acid and nucleotide sequences of the human insulin receptor can be found in GenBank Accession No. NM_000208.

[0125] Antibodies that bind to the insulin receptor-mediated transport system One non-invasive approach to deliver enzymes missing in MPS-III (e.g., SGSH) to the CNS is to fuse SGSH to an antibody that selectively binds to the ECD of the insulin receptor. The insulin receptor expressed on the BBB can then act as a vector to transport SGSH across the BBB. Certain ECD-specific antibodies can mimic endogenous ligands and thus cross the cell membrane barrier via transport on specific receptor systems. Such insulin receptor antibodies act as molecular "Trojan horses" or "THs," as shown diagrammatically in Figure 1. SGSH alone does not normally cross the blood-brain barrier (BBB). However, when SGSH is fused to a TH, the enzyme can cross the BBB and brain cell membranes by transport on endogenous BBB receptors expressed on both the BBB and brain cell membranes in the brain, such as IR (Figure 1).

[0126] Thus, despite the fact that antibodies and other macromolecules are normally excluded from the brain, if they have specificity for a receptor expressed on the BBB, such as the extracellular domain of the insulin receptor, they can be useful vehicles to deliver molecules into the brain parenchyma. In some embodiments, the HIR Ab-SGSH fusion antibody binds an outer surface epitope on the human BBB HIR, and this binding allows the fusion antibody to cross the BBB via a transport reaction mediated by the human BBB insulin receptor.

[0127] The term "antibody" refers to an immunoglobulin, either natural or partially or wholly synthetically produced. The term also includes any polypeptide or protein having a binding domain that is, or is homologous to, an antigen-binding domain. CDR-grafted antibodies are also contemplated by the term.

[0128] "Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies among the heavy chains of the various immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable region ("VH") followed by multiple constant regions ("CH"). Each light chain has at one end a variable region ("VL") and at the other end a constant region ("CL"), with the light chain constant region aligned with the first heavy chain constant region and the light chain variable region aligned with the heavy chain variable region. Certain amino acid residues are believed to form an interface between the light chain variable region and the heavy chain variable region.

[0129] The term "variable region" refers to a protein domain that differs significantly in sequence between family members (i.e., between different isoforms or in different species). In the context of antibodies, the term "variable region" refers to the variable region of an antibody that is responsible for the binding and specificity of each particular antibody to a particular antigen. However, variability is not uniformly distributed throughout the variable region of an antibody. It is concentrated in three parts, called hypervariable regions, in both the light and heavy chain variable regions. The more highly conserved parts of the variable regions are called "framework regions" or "FRs." Unmodified heavy and light chain variable regions each contain four FRs (FR1, FR2, FR3, and FR4, respectively), which are primarily in a β-sheet structure and are connected by three hypervariable regions that form loops that connect and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chains, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669). The constant regions are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity.

[0130] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable region directly binds to an antigen in a complementary manner and contains amino acid residues from three "complementarity determining regions" or "CDRs", known as CDR1, CDR2, and CDR3, respectively.

[0131] In the light chain variable region, the CDRs generally correspond to approximately residues 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3); in the heavy chain variable region, the CDRs generally correspond to approximately residues 31-35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), and / or residues from the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk, J. Mol. Biol. 196:901 917(1987)).

[0132] As used herein, "variable framework region" or "VFR" refers to framework residues that form part of an antigen-binding pocket or groove and / or may contact an antigen. In some embodiments, the framework residues form a loop that is part of the antigen-binding pocket or groove. The amino acid residues in the loop may or may not contact the antigen. In some embodiments, the loop amino acids of a VFR are determined by examining the three-dimensional structure of an antibody, antibody heavy chain, or antibody light chain. The three-dimensional structure may be analyzed for solvent accessible amino acid positions, since such positions are more likely to form loops and / or provide antigen contact in the antibody variable region. Some solvent accessible positions may tolerate amino acid sequence diversity, while others (e.g., structural positions) may have less diversity. The three-dimensional structure of an antibody variable region may be derived from a crystal structure or protein modeling. In some embodiments, a VFR comprises, consists essentially of, or consists of amino acid positions corresponding to amino acid positions 71-78 of the heavy chain variable region, positions defined by Kabat et al., 1991. In some embodiments, the VFR forms part of framework region 3, which is located between CDRH2 and CDRH3. The VFR may form a loop that is well positioned to contact the target antigen or may form part of the antigen-binding pocket.

[0133] Depending on the amino acid sequence of the constant region of the immunoglobulin heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions (Fc) corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0134] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called "kappa" and "lambda," based on the amino acid sequences of their constant regions.

[0135] With respect to the antibodies or fusion antibodies described herein, the terms "selectively bind," "selectively binding," "specifically binds," or "specifically binding" refer to the binding of an antibody or fusion antibody to its target antigen with a dissociation constant (Kd) of about 10. -6 M or less, i.e., 10 -7 ,10 -8 ,10 -9 ,10 -10 ,10 -11 or 10 -12 It's M.

[0136] As used herein, the term "antibody" is understood to mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nature Biotech. 23(9) 1126-1129 (2005)). Non-limiting examples of such antibodies include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains, (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544 546), and (vi) an isolated complementarity determining region (CDR). Furthermore, the two domains VL and VH of the Fv fragment, although encoded by separate genes, can be linked using recombinant techniques by a synthetic linker that allows them to be produced as a single-chain protein in which the VL and VH regions combine to form a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423 426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879 5883, and Osbourn et al. (1998) Nat. Biotechnol. 16:778). Such single-chain antibodies are also intended to be encompassed by the term antibody. Any VH and VL sequence of a specific scFv can be combined with cDNA or genomic sequences of human immunoglobulin constant regions to generate expression vectors encoding complete IgG molecules or other isotypes. The VH and VL can also be used to generate Fab, Fv, or other fragments of immunoglobulins, using either protein chemistry or recombinant DNA techniques.Other forms of single chain antibodies, such as diabodies, are also encompassed.

[0137] The "F(ab')2" and "Fab'" portions can be generated by treating immunoglobulins (monoclonal antibodies) with proteases such as pepsin and papain, and include antibody fragments generated by digesting immunoglobulins near the disulfide bonds present between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds present between the hinge regions in each of the two H chains to generate two homologous antibody fragments, in which the L chain consisting of VL (L chain variable region) and CL (L chain constant region), and the H chain fragment consisting of VH (H chain variable region) and CHγ1 (γ1 region in the constant region of the H chain) are linked at their C-terminal regions via disulfide bonds. Each of these two homologous antibody fragments is called Fab'. Pepsin cleaves IgG downstream of the disulfide bond between the hinge regions in each of the two H chains to produce an antibody fragment slightly larger than the Fab' fragment connected at the hinge region described above. This antibody fragment is called F(ab')2.

[0138] Fab fragments also contain the constant region of the light chain and the first constant region (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. As used herein, Fab'-SH is the designation for Fab' in which the cysteine ​​residues of the constant regions bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are known.

[0139] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable region in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A total of six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable region (or even half of an Fv containing only the three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although with a lower affinity than the complete binding site.

[0140] "Single-chain Fv" or "sFv" antibody fragments comprise the VH, VL, or both the VH and VL domains of an antibody, where both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269 315 (1994).

[0141] "Chimeric" antibodies include antibodies from a combination of different mammals. The mammals may be, for example, rabbit, mouse, rat, goat, or human. Combinations of different mammals include combinations of fragments from human and mouse origin.

[0142] In some embodiments, the antibodies described herein are monoclonal antibodies (MAbs), typically chimeric human-mouse antibodies obtained by humanization of mouse monoclonal antibodies. The antibodies are obtained, for example, from transgenic mice that are "engineered" to produce specific human antibodies in response to antigen challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice are capable of synthesizing human antibodies specific to human antigens, and can be used to produce human antibody-secreting hybridomas.

[0143] For use in humans, the HIR Abs preferably contain sufficient human sequences such that they are not significantly immunogenic when administered to humans, e.g., about 80% human and about 20% mouse, about 85% human and about 15% mouse, about 90% human and about 10% mouse, about 95% human and 5% mouse, more than about 95% human and less than about 5% mouse, or 100% human sequences. More highly humanized forms of HIR MAbs can also be designed, which have activity comparable to mouse HIR Abs and can be used in the embodiments described herein. See, e.g., U.S. Patent Application Publication No. 20040101904, filed November 27, 2002, and U.S. Patent Application Publication No. 20050142141, filed February 17, 2005. A humanized antibody against the human BBB insulin receptor with sufficient human sequence for use in this embodiment is described, for example, in Boado et al. (2007), Biotechnol Bioeng, 96(2):381-391.

[0144] In typical embodiments, the HIR antibody or fusion antibody derived therefrom (e.g., HIR-SGHS, HIR-NGLU, HIR-HGSNAT, HIR-GNS) comprises an immunoglobulin heavy chain comprising a CDR corresponding to at least one sequence of the HC CDRs set forth in Figure 7 (SEQ ID NOs: 1-3), or variants thereof, such as an HC CDR1 corresponding to the amino acid sequence of SEQ ID NO: 1 containing not more than 1, 2, 3, 4, 5, or 6 single amino acid mutations, an HC CDR2 corresponding to the amino acid sequence of SEQ ID NO: 2 containing not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single amino acid mutations, or an HC CDR3 corresponding to the amino acid sequence of SEQ ID NO: 3 containing not more than 1 or 2 single amino acid mutations, wherein the single amino acid mutation is a substitution, deletion, or insertion.

[0145] In another embodiment, the HIR Ab or fusion Ab (e.g., HIR Ab-SGHS, HIR Ab-NGLU, HIR Ab HGSNAT, HIR Ab-GNS) comprises an immunoglobulin HC whose amino acid sequence is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or any other percent identical up to 100%) to SEQ ID NO:7 (shown in FIG. 5).

[0146] In some embodiments, the HIR Ab or fusion Ab (e.g., HIR Ab-SGHS, HIR Ab-NGLU, HIR Ab-HGSNAT, HIR Ab-GNS) comprises an immunoglobulin light chain that includes a CDR corresponding to the sequence of at least one of the LC CDRs set forth in Figure 7 (SEQ ID NOS:4-6), or a variant thereof, such as an LC CDR1 corresponding to the amino acid sequence of SEQ ID NO:4 containing no more than 1, 2, 3, 4 or 5 single amino acid mutations, an LC CDR2 corresponding to the amino acid sequence of SEQ ID NO:5 containing no more than 1, 2, 3 or 4 single amino acid mutations, or an LC CDR3 corresponding to the amino acid sequence of SEQ ID NO:6 containing no more than 1, 2, 3, 4 or 5 single amino acid mutations.

[0147] In another embodiment, the HIR Ab or fusion Ab (e.g., HIR Ab-SGHS, HIR Ab-NGLU, HIR Ab-HGSNAT, HIR Ab-GNS) comprises an immunoglobulin LC whose amino acid sequence is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or any other percent identical up to 100%) to SEQ ID NO:8 (shown in FIG. 6).

[0148] In yet another embodiment, the HIR Ab or fusion Ab (e.g., HIR Ab-SGHS, HIR Ab-NGLU, HIR Ab-HGSNAT, HIR Ab-GNS) comprises both a heavy and light chain corresponding to any of the HIR heavy chains and HIR light chains described above.

[0149] The HIR antibodies herein may be glycosylated or non-glycosylated. If the antibody is glycosylated, any pattern of glycosylation that does not significantly affect the function of the antibody may be used. Glycosylation may occur in a pattern that is specific to the cell in which the antibody is made and may vary between cell types. For example, the glycosylation pattern of a monoclonal antibody produced by a mouse myeloma cell may differ from the glycosylation pattern of a monoclonal antibody produced by a transfected Chinese Hamster Ovary (CHO) cell. In some embodiments, the antibody is glycosylated in the pattern produced by a transfected Chinese Hamster Ovary (CHO) cell.

[0150] Those skilled in the art will appreciate that with current technology, a vast number of sequence variants of candidate or known HIR Abs can be readily generated (e.g., in vitro) and screened for binding to a target antigen, e.g., the ECD of the human insulin receptor, or isolated epitopes thereof. For an example of ultra-high throughput screening of antibody sequence variants, see, e.g., Fukuda et al. (2006) "In vitro evolution of single-chain antibodies using mRNA display" Nuc. Acid Res., 34(19) (published online). See also Chen et al. (1999) "In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site" Prot Eng, 12(4): 349-356. The insulin receptor ECD can be purified, for example, as described in Coloma et al. (2000) Pharm Res, 17:266-274, and used to screen HIR Abs, and HIR Ab sequence variants of known HIR Abs.

[0151] Thus, in some embodiments, a genetically engineered HIR Ab with a desired level of human sequence is fused to an enzyme that is deficient in MPS-III (e.g., SGSH) to generate a recombinant fusion antibody that is a bifunctional molecule. For example, the HIR Ab-SGSH fusion antibody (i) binds to the extracellular domain of the human insulin receptor, (ii) catalyzes the hydrolysis of sulfate ester bonds in heparan sulfate, (iii) can cross the BBB via transport on the BBB HIR, and retain SGSH activity once in the brain following peripheral administration.

[0152] N-Sulfoglucosamine sulfohydrolase (SGSH) Systemic administration of recombinant SGSH (e.g., by intravenous injection) is not expected to rescue the deficiency of SGSH in the CNS of patients affected by MPS-IIIA. SGSH does not cross the BBB, and the lack of transport of the enzyme across the BBB prevents it from having a meaningful therapeutic effect in the CNS after peripheral administration. However, the inventors have found that SGSH fusion (e.g., by a covalent linker) with an antibody that crosses the BBB, such as HIR Ab, allows the enzyme to enter the CNS from the blood via a non-invasive peripheral administration route (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, or even oral administration). Administration of HIR Ab-SGSH fusion antibody allows delivery of SGSH activity from the peripheral blood into the brain. This specification describes determining a systemic dose of HIR Ab-SGSH fusion antibody that is therapeutically effective for treating SGSH deficiency in the CNS. As described herein, an appropriate systemic dose of the HIR Ab-SGSH fusion antibody is established based on quantitative determination of the CNS uptake characteristics and enzymatic activity of the HIR Ab-enzyme fusion antibody.

[0153] Heparan sulfate is a sulfated glycosaminoglycan synthesized in oligodendrocytes within the central nervous system. As used herein, SGSH (e.g., the human SGSH sequence set forth in GenBank Accession No. NP_000190) refers to any natural or artificial enzyme capable of catalyzing the hydrolysis of N-linked sulfate esters from heparan sulfate.

[0154] SGSH is a member of the sulfatase family that requires specific post-translational modifications for expression of SGSH enzyme activity. The activity of the SGSH enzyme is activated when Cys-70 (of the intact SGSH protein including the signal peptide) is converted to a formylglycine residue by sulfatase modifier type 1 (SUMF1), also called formylglycine generating enzyme (FGE). In some embodiments, the fusion antibody comprising SGSH is post-translationally modified by sulfatase modifier type 1 (SUMF1). In some embodiments, the post-translational modification comprises the conversion of a cysteine ​​to formylglycine. In some embodiments, the fusion antibody comprises SGSH comprising a formylglycine residue.

[0155] In some embodiments, SGSH has an amino acid sequence that is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95, or any other percent identical or less than 100%) to the amino acid sequence of human SGSH, which is a 502 amino acid protein set forth in GenBank NP_000190, or a 482 amino acid subsequence thereof that lacks the 20 amino acid signal peptide and corresponds to SEQ ID NO:9 (Figure 8). The structure-function relationship of human SGSH has been studied, and Cys-70 is a conserved residue in sulfatases for post-translational modification, as described in Scott et al. (1995), "Cloning of the sulphamidase gene and identification of mutations in Sanfilippo A syndrome", Nature Genetics, 11:465-467. The Asp-51 residue is involved in divalent cation binding as described by Gliddon et al. (2004), "Purification and characterization of recombinant murine sulfamidase," Molec. Genet. Metab. 83:239-245. N-linked glycosylation sites are present at Asn-41, Asn-142, Asn-151, Asn-264, and Asn-413 as described by DiNatale et al. (2001), "Heparan N-sulfatase: in vitro mutagenesis of potential N-glycosylation sites," Biochem. Biophys. Res. Comm. 280:1251-1257, which includes a 20-amino acid signal peptide according to the amino acid numbering system.

[0156] In some embodiments, SGSH has an amino acid sequence that is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95, or any other percent identical up to 100%) to SEQ ID NO:9 (shown in FIG. 8). Sequence variants of a standard SGSH sequence, such as SEQ ID NO:9, can be generated, for example, by random mutagenesis of the entire sequence or specific subsequences corresponding to specific domains. Alternatively, site-directed mutagenesis can be performed in an iterative manner, while avoiding mutations to residues known to be essential for the function of SGSH, such as those mentioned above. Furthermore, in generating multiple variants of the SGSH sequence, mutation tolerance prediction programs can be used to greatly reduce the number of non-functional sequence variants that would be generated by strict random mutagenesis. Various programs (e.g., SIFT, PolyPhen, PANTHER PSEC, PMUT, and TopoSNP) that predict the effect of amino acid substitutions in protein sequences on protein function are described, for example, in Henikoff et al. (2006), "Predicting the Effects of Amino Acid Substitutions on Protein Function," Annu. Rev. Genomics Hum. Genet., 7:61-80. SGSH sequence variants can be screened for SGSH activity / retention of SGSH activity by fluorometric enzyme assays known in the art (Karpova et al. (1996): A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type A (MPS IIIA), J. Inher. Metab. Dis. 19: 278-285). Thus, those skilled in the art will appreciate that a large number of usable SGSH sequence variants can be obtained by generating and screening a highly diverse "library" of SGSH sequence variants, as described above, by methods routine in the art.

[0157] Percent sequence identity is determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992). Briefly, two amino acid sequences are aligned and the alignment score is optimized using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (supra). The percent homology is then calculated as ([total number of perfect matches] / [length of the longer sequence + number of gaps introduced into the longer sequence to align the two sequences])(100).

[0158] Those skilled in the art will appreciate that many established algorithms are available for aligning two amino acid sequences. The Pearson and Lipman "FASTA" similarity search algorithm is a suitable protein alignment method for determining the level of homology shared by the amino acid sequences disclosed herein and those of other peptides. The FASTA algorithm is described in Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA characterizes sequence similarity by first identifying regions shared by a test sequence and a query sequence (e.g., SEQ ID NO:9 or SEQ ID NO:16) that have either the highest density of homology (when the ktup variable is 1) or homologous pairs (when ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The 10 regions with the highest density of homology are then rescored by comparing all pairwise amino acid similarities using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only the residues contributing to the highest score. If there are several regions with scores above a "cutoff" value (calculated by a predefined formula based on the length of the sequences and the ktup value), the first trimmed regions are examined to determine whether they can be joined to form an approximate alignment that includes gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which takes into account amino acid insertions and deletions.Exemplary parameters for FASTA analysis are: ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62. These parameters can be introduced into the FASTA program by modifying the scoring matrix file ("SMATRIX") as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

[0159] The present embodiment also includes proteins having conservative amino acid changes compared to the amino acid sequences disclosed herein. For example, among the common amino acids, "conservative amino acid substitutions" are described by substitutions within each of the following groups of amino acids: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartic acid and glutamic acid, (5) glutamine and asparagine, and (6) lysine, arginine, and histidine. The BLOSUM62 table is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915 (1992)). Thus, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present embodiment. Although it is possible to design amino acid substitutions based solely on chemical properties (as described above), the term "conservative amino acid substitution" preferably refers to a substitution represented by a BLOSUM62 value greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2, or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

[0160] It will also be understood that the amino acid sequence may include additional residues, such as additional N-terminal or C-terminal amino acids, and still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence retains sufficient biological protein activity to function in the compositions and methods of this embodiment.

[0161] α-N-acetylglucosaminidase (NAGLU) Systemic administration of recombinant NAGLU (e.g., by intravenous injection) is not expected to rescue the deficiency of NAGLU in the CNS of patients affected by MPS-IIIA. NAGLU does not cross the BBB, and the lack of transport of the enzyme across the BBB prevents it from having a meaningful therapeutic effect in the CNS after peripheral administration. However, the inventors have found that fusion of NAGLU (e.g., by a covalent linker) to an antibody that crosses the BBB, such as HIR Ab, allows the enzyme to enter the CNS from the blood via a non-invasive peripheral administration route (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, or even oral administration). Administration of HIR Ab-NAGLU fusion antibody allows delivery of NAGLU activity from the peripheral blood into the brain. This specification describes determining a systemic dose of HIR Ab-NAGLU fusion antibody that is therapeutically effective for treating NAGLU deficiency in the CNS. As described herein, an appropriate systemic dose of the HIR Ab-NAGLU fusion antibody is established based on quantitative determination of the CNS uptake characteristics and enzymatic activity of the HIR Ab-enzyme fusion antibody.

[0162] As used herein, NAGLU (e.g., the human NAGLU sequence set forth in GenBank Accession No. NP_000254) refers to any natural or artificial enzyme capable of catalyzing the hydrolysis of N-acetyl-D-glucosamine residues in N-acetyl-α-D-glucosaminides.

[0163] In some embodiments, NAGLU has an amino acid sequence that is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or any other percent identical or less than 100%) to the amino acid sequence of human NAGLU, a protein set forth in GenBank NP_000254. In some embodiments, NAGLU has an amino acid sequence that is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or any other percent identical or less than 100%) to SEQ ID NO: 17. Sequence variants of a standard NAGLU sequence, such as SEQ ID NO: 17, can be generated, for example, by random mutagenesis of the entire sequence or specific subsequences corresponding to specific domains. Alternatively, site-directed mutagenesis can be performed in an iterative manner, avoiding mutations to residues known to be essential for the function of NAGLU, such as those described above. Furthermore, in generating multiple variants of the NAGLU sequence, mutation tolerance prediction programs can be used to greatly reduce the number of non-functional sequence variants that would be generated by strict random mutagenesis methods. Various programs that predict the effect of amino acid substitutions in protein sequences on protein function (e.g., SIFT, PolyPhen, PANTHER PSEC, PMUT, and TopoSNP) are described, for example, in Henikoff et al. (2006), "Predicting the Effects of Amino Acid Substitutions on Protein Function," Annu. Rev. Genomics Hum. Genet., 7:61-80. NAGLU sequence variants can be screened for NAGLU activity / retention of NAGLU activity by fluorometric enzymatic assays known in the art. Thus, one skilled in the art will appreciate that a large number of usable NAGLU sequence variants can be obtained by generating and screening a highly diverse "library" of NAGLU sequence variants, as described above, by methods routine in the art.

[0164] Heparin-α-glucosaminide N-acetyltransferase (HGSNAT) Systemic administration of recombinant HGSNAT (e.g., by intravenous injection) is not expected to rescue the HGSNAT deficiency in the CNS of patients affected by MPS-IIIA. HGSNAT does not cross the BBB, and the lack of transport of the enzyme across the BBB prevents it from having a meaningful therapeutic effect in the CNS after peripheral administration. However, the inventors have found that HGSNAT can be fused (e.g., by a covalent linker) to an antibody that crosses the BBB, such as an HIR Ab, allowing the enzyme to enter the CNS from the blood via a non-invasive peripheral administration route (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, or even oral administration). Administration of an HIR Ab-HGSNAT fusion antibody allows delivery of HGSNAT activity from the peripheral blood into the brain. This specification describes determining a systemic dose of an HIR Ab-HGSNAT fusion antibody that is therapeutically effective for treating HGSNAT deficiency in the CNS. As described herein, an appropriate systemic dose of the HIR Ab-HGSNAT fusion antibody is established based on quantitative determination of the CNS uptake characteristics and enzymatic activity of the HIR Ab-enzyme fusion antibody.

[0165] As used herein, HGSNAT (eg, the human HGSNAT sequence set forth in GenBank Accession No. NP_689632) refers to any natural or artificial enzyme capable of catalyzing the acetylation of glucosamine residues from heparan sulfate.

[0166] In some embodiments, HGSNAT has an amino acid sequence that is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95, or any other percent identical or less than 100%) to the amino acid sequence of human HGSNAT, a protein set forth in GenBank NP_689632. In some embodiments, HGSNAT has an amino acid sequence that is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95, or any other percent identical or less than 100%) to SEQ ID NO: 19. Sequence variants of standard HGSNAT sequences, such as SEQ ID NO: 19, can be generated, for example, by random mutagenesis of the entire sequence or specific subsequences corresponding to specific domains. Alternatively, site-directed mutagenesis can be performed in an iterative manner, while avoiding mutations to residues known to be essential for the function of HGSNAT, such as those described above. Furthermore, in generating multiple variants of the HGSNAT sequence, mutation tolerance prediction programs can be used to greatly reduce the number of non-functional sequence variants that would be generated by strict random mutagenesis. Various programs that predict the effect of amino acid substitutions in a protein sequence on protein function (e.g., SIFT, PolyPhen, PANTHER PSEC, PMUT, and TopoSNP) are described, for example, in Henikoff et al. (2006), "Predicting the Effects of Amino Acid Substitutions on Protein Function," Annu. Rev. Genomics Hum. Genet., 7:61-80. HGSNAT sequence variants can be screened for HGSNAT activity / retention of HGSNAT activity by fluorometric enzymatic assays known in the art. Thus, one skilled in the art will appreciate that a large number of usable HGSNAT sequence variants can be obtained by generating and screening a highly diverse "library" of HGSNAT sequence variants as described above, by methods routine in the art.

[0167] Systemic administration of recombinant GNS (e.g., by intravenous injection) is not expected to rescue the defect of GNS in the CNS of patients affected by MPS-IIIA. GNS does not cross the BBB, and the lack of transport of the enzyme across the BBB prevents it from having a meaningful therapeutic effect in the CNS after peripheral administration. However, the inventors have found that when GNS is fused (e.g., by a covalent linker) to an antibody that crosses the BBB, such as HIR Ab, the enzyme can enter the CNS from the blood following a non-invasive peripheral administration route (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, or even oral administration). Administration of HIR Ab-GNS fusion antibody allows the delivery of GNS activity from the peripheral blood into the brain. This specification describes determining a systemic dose of HIR Ab-GNS fusion antibody that is therapeutically effective for treating GNS deficiency in the CNS. As described herein, an appropriate systemic dose of the HIR Ab-GNS fusion antibody is established based on quantitative determination of the CNS uptake characteristics and enzymatic activity of the HIR Ab-enzyme fusion antibody.

[0168] As used herein, GNS (eg, the human GNS sequence set forth in GenBank Accession No. NP_002067) refers to any natural or artificial enzyme capable of catalyzing the hydrolysis of the 6-sulfate group from heparan sulfate.

[0169] In some embodiments, GNS has an amino acid sequence that is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95, or any other percent identical or less than 100%) to the amino acid sequence of human GNS, a protein set forth in GenBank NP_002067. In some embodiments, GNS has an amino acid sequence that is at least 50% identical (i.e., at least 55, 60, 65, 70, 75, 80, 85, 90, 95, or any other percent identical or less than 100%) to SEQ ID NO:21. Sequence variants of a standard GNS sequence, such as SEQ ID NO:21, can be generated, for example, by random mutagenesis of the entire sequence or specific subsequences corresponding to specific domains. Alternatively, site-directed mutagenesis can be performed iteratively, while avoiding mutations to residues known to be essential for the function of GNS, such as those described above. Furthermore, in generating multiple variants of the GNS sequence, mutation tolerance prediction programs can be used to greatly reduce the number of non-functional sequence variants that would be generated by strict random mutagenesis methods. Various programs that predict the effect of amino acid substitutions in protein sequences on protein function (e.g., SIFT, PolyPhen, PANTHER PSEC, PMUT, and TopoSNP) are described, for example, in Henikoff et al. (2006), "Predicting the Effects of Amino Acid Substitutions on Protein Function," Annu. Rev. Genomics Hum. Genet., 7:61-80. GNS sequence variants can be screened for GNS activity / retention of GNS activity by fluorometric enzymatic assays known in the art. Thus, one skilled in the art will appreciate that a large number of usable GNS sequence variants can be obtained by generating and screening a highly diverse "library" of GNS sequence variants, as described above, by methods routine in the art.

[0170] composition The bifunctional fusion antibodies described herein have been found to retain a high percentage of the activities of their separate constituent proteins, such as antibodies capable of crossing the BBB (e.g., HIR Ab) and binding to the extracellular domains of endogenous receptors on the BBB (e.g., IR ECD), and the enzymatic activity of enzymes that are missing in MPS-III (e.g., SGSH). The construction of cDNAs and expression vectors encoding any of the proteins described herein, as well as their expression and purification, are well known to those of skill in the art and are described in detail herein, for example, in Examples 1-3, and in Boado et al. (2007), Biotechnol Bioeng 96:381-391, U.S. Patent Application Serial No. 11 / 061,956, and U.S. Patent Application Serial No. 11 / 245,710.

[0171] Described herein are bifunctional fusion antibodies comprising an antibody against an endogenous BBB receptor (e.g., HIR Ab), capable of crossing the BBB, and fused to an SGSH, as described herein, wherein the antibody against an endogenous BBB receptor is capable of crossing the blood-brain barrier, and each of the SGSHs retains, on average, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of their activity compared to their activity alone. In some embodiments, provided herein are HIR Ab-SGSH fusion antibodies, in which the HIR Ab and SGSH each retain, on average, at least about 50% of their activity compared to their activity alone. In some embodiments, provided herein are HIR Ab-SGSH fusion antibodies, in which the HIR Ab and SGSH each retain, on average, at least about 60% of their activity compared to their activity alone. In some embodiments, the present disclosure provides HIR Ab-SGSH fusion antibodies, in which the HIR Ab and SGSH each retain, on average, at least about 70% of their activity compared to their activity alone. In some embodiments, the present disclosure provides HIR Ab-SGSH fusion antibodies, in which the HIR Ab and SGSH each retain, on average, at least about 80% of their activity compared to their activity alone. In some embodiments, the present disclosure provides fusion HIR Ab-SGSH fusion antibodies, in which the HIR Ab and SGSH each retain, on average, at least about 90% of their activity compared to their activity alone. In some embodiments, the HIR Ab retains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of its activity compared to its activity alone, and the SGSH retains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of its activity compared to its activity alone.Thus, described herein are compositions comprising bifunctional HIR Ab-SGSH fusion antibodies capable of crossing the BBB, wherein each of the component HIR Ab and SGSH retain, on average, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of their activity, i.e., HIR binding and SGSH activity, respectively, as part of the fusion antibody, compared to their activity as separate proteins. HIR Ab-SGSH fusion antibody refers to a fusion protein comprising any of the HIR antibodies and SGSH described herein.

[0172] In any embodiment herein, the HIR Ab may be replaced with an antibody against an endogenous BBB receptor as described herein, such as an antibody against the transferrin receptor, leptin receptor, lipoprotein receptor or insulin-like growth factor (IGF) receptor, or other similar endogenous BBB receptor-mediated transport system.

[0173] In the fusion antibodies described herein, the covalent bond between the antibody and SGSH may be to the carboxy or amino terminus of the antibody heavy or light chain and to the amino or carboxy terminus of the SGSH, so long as the fusion antibody is capable of binding to the ECD of the IR and crossing the blood-brain barrier and the SGSH retains a therapeutically useful portion of its activity. In some embodiments, the covalent bond is between the HC of the antibody and the SGSH, or between the LC of the antibody and the SGSH. Any suitable bond can be used, such as the carboxy terminus of the light chain and the amino terminus of the SGSH, the carboxy terminus of the heavy chain and the amino terminus of the SGSH, the amino terminus of the light chain and the amino terminus of the SGSH, the amino terminus of the heavy chain and the amino terminus of the SGSH, the carboxy terminus of the light chain and the carboxy terminus of the SGSH, the carboxy terminus of the heavy chain and the carboxy terminus of the SGSH, or the carboxy terminus of the heavy chain and the carboxy terminus of the SGSH. In some embodiments, the bond is from the carboxy terminus of the HC to the amino terminus of the SGSH.

[0174] The SGSH may be fused or covalently attached to the targeting antibody (e.g., MAb, HIR-MAb) by a linker. The linkage between the terminal amino acids may be achieved by an intervening peptide linker sequence that forms part of the fused amino acid sequence. The peptide sequence linker may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acids in length. In some embodiments, including some preferred embodiments, the peptide linker is less than 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in length. In some embodiments, including some preferred embodiments, the peptide linker is at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids in length. In some embodiments, the SGSH is directly attached to the targeting antibody and is therefore 0 amino acids in length. In some embodiments, there is no linker connecting the SGSH to the targeting antibody.

[0175] In some embodiments, the linker comprises glycine, serine, and / or alanine residues in any combination or order. In some examples, the combined percentage of glycine, serine, and alanine residues in the linker is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the total number of residues in the linker. In some preferred embodiments, the combined percentage of glycine, serine, and alanine residues in the linker is at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the total number of residues in the linker. In some embodiments, a combination of any number of amino acids (including natural or synthetic amino acids) can be used in the linker. In some embodiments, a three amino acid linker is used. In some embodiments, the linker has the sequence Ser-Ser-Ser. In some embodiments, the two amino acid linker comprises glycine, serine, and / or alanine residues in any combination or order (e.g., Gly-Gly, Ser-Gly, Gly-Ser, Ser-Ser, Ala-Ala, Ser-Ala, or Ala-Ser linkers). In some embodiments, the two amino acid linker consists of one glycine, serine, and / or alanine residue and another amino acid (e.g., Ser-X, where X is any known amino acid). In yet other embodiments, the two amino acid linker consists of any two amino acids (e.g., XX), except gly, ser, or ala.

[0176] As described herein, in some embodiments, the linker is greater than two amino acids in length. As further described herein, the linker may also include glycine, serine, and / or alanine residues in any combination or order. In some embodiments, the linker consists of one glycine, serine, and / or alanine residue, as well as other amino acids (e.g., Ser-nX, where X is any known amino acid and n is the number of amino acids). In yet other embodiments, the linker consists of any two amino acids (e.g., XX). In some embodiments, the any two amino acids are Gly, Ser, or Ala, in any combination or order, and within a variable number of intervening amino acids. In one embodiment, the linker consists of at least one Gly. In one embodiment, the linker consists of at least one Ser. In one embodiment, the linker consists of at least one Ala. In some embodiments, the linker consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Gly, Ser, and / or Ala residues. In a preferred embodiment, the linker comprises Gly and Ser in any combination or number of repeats, such as (Gly4Ser)3 or other variations.

[0177] Linkers for use in this embodiment may be designed by using any method known in the art. For example, there are several publicly available programs for determining optimal amino acid linkers in fusion protein design. Publicly available computer programs (e.g., the LINKER program) that automatically generate optimal linker amino acid sequences based on the protein sequence and user input of a linker of desired length may be used in the present methods and compositions. Often, the programs can use observed tendencies of natural linkers connecting protein subdomains to predict optimal protein linkers for use in protein design. In some cases, the programs use other methods to predict optimal linkers. Examples of several programs suitable for predicting linkers for this embodiment have been described in the art, see, for example, Xue et al. (2004) Nucleic Acids Res. 32, W562-W565 (web server version providing an internet link to the LINKER program to assist in the design of linker sequences for constructing functional fusion proteins), George and Heringa, (2003), Protein Engineering, 15(11):871-879 (providing an internet link to the linker program and describing the rational design of protein linkers), Argos, (1990), J. Mol. Biol. 211:943-958, Arai et al. (2001) Protein Engineering, 14(8):529-532, Crasto and Feng, (2000) Protein Engineering 13(5):309-312.

[0178] The peptide linker sequence may contain a protease cleavage site, but this is not necessary for activity of SGSH, and indeed an advantage of these embodiments is that once across the BBB, the bifunctional HIR Ab-SGSH fusion antibody is partially or fully active for both transport and activity without cleavage. Figure 9 shows an exemplary embodiment of the amino acid sequence of a HIR Ab-SGSH fusion antibody (SEQ ID NO: 10) in which the HC is fused at its carboxy terminus to the amino terminus of SGSH via a three amino acid "Ser-Ser-ser" linker. In some embodiments, the fused SGSH sequence lacks its 20 amino acid signal peptide, as shown in Figure 8.

[0179] In some embodiments, the HIR Ab-SGSH fusion antibodies herein comprise both a HC and a LC. In some embodiments, the HIR Ab-SGSH fusion antibodies are monovalent antibodies. In other embodiments, as described in the Examples section herein, the HIR Ab-SGSH fusion antibodies are bivalent antibodies.

[0180] In some embodiments, the HIR Ab used as part of the HIR Ab-SGSH fusion antibody may or may not be glycosylated, and in some embodiments the antibody is glycosylated, e.g., with a glycosylation pattern resulting from synthesis in a CHO cell.

[0181] As used herein, "activity" includes physiological activity (eg, ability to cross the BBB and / or therapeutic activity), binding affinity of the HIR Ab to the IR ECD, or enzymatic activity of the SGSH.

[0182] Transport of the HIR Ab-SGSH fusion antibody across the BBB can be compared to transport of the HIR Ab alone across the BBB by standard methods. For example, pharmacokinetics and brain uptake of the HIR Ab-SGSH fusion antibody in a model animal, e.g., a mammal such as a primate, can be used. Similarly, standard models for determining SGSH activity can be used to compare the function of SGSH alone and as part of a HIR Ab-SGSH fusion antibody. See, e.g., Example 4, which demonstrates the enzymatic activity of SGSH versus the HIR Ab-SGSH fusion antibody. Binding affinity to the IR ECD can be compared for the HIR Ab-SGSH fusion antibody versus the HIR Ab alone. See, e.g., Example 4 herein.

[0183] The present specification also includes pharmaceutical compositions comprising one or more HIR Ab-SGSH fusion antibodies described herein and a pharma- ceutical acceptable excipient. A thorough discussion of pharma- ceutical acceptable carriers / excipients can be found in Remington's Pharmaceutical Sciences, Gennaro, AR, ed., 20th edition, 2000: Williams and Wilkins PA, USA. The pharmaceutical compositions of this embodiment include compositions suitable for administration via any peripheral route, including intravenous, subcutaneous, intramuscular, intraperitoneal injection, oral, rectal, buccal, pulmonary, transdermal, intranasal, or any other suitable route of peripheral administration.

[0184] The compositions herein are particularly suitable for injection, e.g., as pharmaceutical compositions for intravenous, subcutaneous, intramuscular, or intraperitoneal administration. The aqueous compositions herein include an effective amount of the compositions of this embodiment, which may be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, e.g., a human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, as well as isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as it is incompatible with the active ingredient, the use of any conventional media or agent in the therapeutic compositions is contemplated. Supplementary active ingredients can also be included in the composition.

[0185] Typical pharma- ceutically acceptable carriers for injectable compositions can include salts, such as mineral acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.), and salts of organic acids (e.g., acetate, propionate, malonate, benzoate, etc.). For example, the compositions herein may be provided in liquid form and formulated in saline-based aqueous solutions of various pHs (5-8) with or without surfactants such as 0.01-1% polysorbate-80, or carbohydrate additives such as mannitol, sorbitol, or trehalose. Commonly used buffers include histidine, acetate, phosphate, or citrate buffers. In some embodiments, the pharmaceutical compositions herein include a monosaccharide, such as glucose or dextrose. For example, the fusion antibody can be administered in a solution of about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20% or more of dextrose and / or glucose. In some embodiments, the composition can contain glucose and / or dextrose at a concentration of about 5% (w / v or v / v). For example, under normal conditions of storage and use, where plasma and CSF glucose levels are significantly altered to indicate hypoglycemia, these preparations can contain preservatives to prevent the growth of microorganisms. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, such as sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0186] Preparations for human administration will meet sterility, pyrogenicity, general safety, and purity standards required by the FDA and other regulatory agency standards. The active compound will generally be formulated for parenteral administration, e.g., formulated for injection via intravenous, intramuscular, subcutaneous, intralesional, or intraperitoneal routes. The preparation of aqueous compositions containing the active element or active ingredient will be known to those of skill in the art in light of this disclosure. Typically, such compositions can be prepared as injectables, either liquid solutions or liquid suspensions, or solid forms suitable for use in preparing solutions or suspensions by adding liquid prior to injection, or the preparations can be emulsified.

[0187] Sterile injectable solutions are prepared by combining the active compound in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze-drying techniques which yield a powder of the active ingredient and any additional desired ingredient from its previously sterile-filtered solution.

[0188] Once formulated, the solutions will be administered systemically in a therapeutically active amount based on the criteria set forth herein, as appropriate for the dosage formulation. The formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above, although drug release capsules and the like can also be used.

[0189] The appropriate quantity, number of treatments, and unit dose of the pharmaceutical composition to be administered will vary depending on the CNS uptake characteristics of the HIR Ab-SGSH fusion antibody, as described herein, as well as the subject to be treated, the subject's condition, and the desired effect. In any event, the person administering will determine the appropriate dose for the individual subject.

[0190] In addition to compounds formulated for parenteral administration, e.g., intravenous or intramuscular injection, other alternative methods of administration of the present embodiment may be used, including, but not limited to, intradermal administration (see U.S. Patent Nos. 5,997,501, 5,848,991, and 5,527,288), pulmonary administration (see U.S. Patent Nos. 6,361,760, 6,060,069, and 6,041,775), buccal administration (see U.S. Patent Nos. 6,375,975, and 6,284,262), transdermal administration (see U.S. Patent Nos. 6,348,210, and 6,322,808), and transmucosal administration (see U.S. Patent No. 5,656,284). Such methods of administration are well known in the art. Intranasal administration of the present embodiment, such as nasal solutions or sprays, aerosols, or inhalants, may also be used. Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are manufactured to be similar in many respects to nasal mucus. Thus, aqueous nasal solutions are usually isotonic and slightly buffered to maintain a pH of 5.5 to 6.5. In addition, the formulation may contain, if necessary, an antimicrobial preservative similar to that used in eye drops and appropriate drug stabilizers. A variety of commercially available nasal solutions are known, which contain, for example, antibiotics and antihistamines and are used to prevent asthma.

[0191] Further formulations suitable for other modes of administration include suppositories and pessaries. Rectal pessaries or suppositories can also be used. Suppositories are solid dosage forms of various weights and shapes, usually containing a drug, for insertion into the rectum or urethra. After insertion, the suppository softens, melts or dissolves in the intracavity fluid. Conventional binders and carriers for suppositories generally include, for example, polyalkylene glycols or triglycerides, and such suppositories can be formed from mixtures containing the active ingredient in any suitable range, for example, 0.5% to 10%, preferably 1% to 2%.

[0192] Oral formulations contain such commonly employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders. In certain embodiments, oral pharmaceutical compositions will contain an inert diluent or an assimilable edible carrier, be enclosed in hard or soft shell gelatin capsules, be compressed into tablets, or can be incorporated directly into the food of the diet. For oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations may contain at least 0.1% of the active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75%, or between about 25 to 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

[0193] Tablets, troches, pills, capsules, etc. may contain the following: a binder, such as gum tragacanth, acacia, corn starch, or gelatin; an excipient, such as dibasic calcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid, etc.; a lubricant, such as magnesium stearate; and a sweetener, such as sucrose, lactose, or saccharin, or a flavoring, such as peppermint, oil of wintergreen, or cherry flavoring. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to materials of the above type. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. A syrup of elixir may contain the active compound sucrose as a sweetening agent, methylene and propylparabens as preservatives, a dye, and a flavoring, such as cherry or orange flavoring. In certain embodiments, oral pharmaceutical compositions may be enteric coated to protect the active ingredients from the stomach environment; enteric coating methods and formulations are well known in the art.

[0194] method The present specification describes a method for delivering an effective dose of an enzyme (e.g., SGSH) deficient in MPS-III across the BBB to the CNS by systemically administering a therapeutically effective amount of a fusion antibody described herein. In some embodiments, the fusion antibody herein is HIR Ab-SGSH. As described herein, the appropriate systemic dose for delivery of the HIR Ab-SGSH fusion antibody is based on its CNS uptake properties and SGSH specific activity. Systemic administration of the HIR Ab-SGSH fusion antibody to a subject suffering from SGSH deficiency is a useful approach to non-invasively deliver SGSH to the CNS. Additionally, the present specification describes a method for treating (e.g., therapeutically treating) MPS-III in a subject by systemically administering a therapeutically effective amount of a fusion antibody described herein. In some embodiments, the treatment is a therapeutic treatment. In some embodiments, the treatment alleviates one or more symptoms of MPS-III. In some embodiments, the treatment reduces one or more symptoms of MPS-III by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100%.

[0195] The amount of fusion antibody that is a therapeutically effective systemic dose of the fusion antibody, as described herein, depends in part on the CNS uptake characteristics of the administered fusion antibody, e.g., the proportion of a systemically administered dose that is taken up by the CNS.

[0196] In some embodiments, 1% (i.e., about 0.3%, 0.4%, 0.48%, 0.6%, 0.74%, 0.8%, 0.9%, 1.05, 1.1, 1.2, 1.3%, 1.5%, 2%, 2.5%, 3%, or any percentage between about 0.3% and about 3%) of the systemically administered HIR Ab-SGSH fusion antibody is delivered to the brain as a result of its uptake across the BBB from the peripheral blood. In some embodiments, at least 0.5% (i.e., about 0.3%, 0.4%, 0.48%, 0.6%, 0.74%, 0.8%, 0.9%, 1.05, 1.1, 1.2, 1.3%, 1.5%, 2%, 2.5%, 3%, or any of about 0.3% to about 3%) of a systemically administered dose of the HIR Ab-SGSH fusion antibody is delivered to the brain within 2 hours after systemic administration, i.e., 1.8, 1.7, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.6, 0.5, or any of about 0.5 to about 2 hours.

[0197] Thus, in some embodiments, the present specification provides a method of systemically administering a therapeutically effective amount of a fusion antibody described herein to a 5-50 kg human such that the amount of fusion antibody that crosses the BBB results in at least 0.5 ng of SGSH protein / mg protein in the subject's brain (e.g., 0.5, 1, 3, 10, 30, 50, or any other value between 0.5 and 50 ng of SGSH protein / mg protein in the subject's brain).

[0198] In some embodiments, the total number of units of enzyme (e.g., SGSH) activity delivered to the subject's brain is at least 50 milliunits per gram of brain (e.g., SGSH activity delivered of at least 100, 300, 1000, 3000, 10000, 30000, 50000, or any other total number of SGSH units between about 50 and 50,000 milliunits per gram of brain).

[0199] In some embodiments, a therapeutically effective systemic dose comprises at least 5,000, 10,000, 30,000, 100,000, 300,000, 1,000,000, 5,000,000, or any other systemic dose of about 5,000 to 5,000,000 units of enzyme (e.g., SGSH) activity.

[0200] In another embodiment, a therapeutically effective systemic dose is at least about 1000 units of enzyme (e.g., SGSH) activity per kg body weight, at least about 1000, 3000, 10000, 30000, 100000, or any other number of units of enzyme activity between about 1,000 and 100,000 units per kg body weight.

[0201] One of skill in the art will appreciate that the mass of a therapeutically effective systemic dose of a fusion antibody herein will depend in part on its enzymatic (e.g., SGSH) specific activity. In some embodiments, the specific activity of the fusion antibody is at least 1,000 U / mg protein, at least about 1500, 2500, 3500, 6000, 7500, 9000, or any other specific activity value between about 1,000 units / mg and about 10,000 units / mg.

[0202] Thus, taking into full consideration the specific activity of the fusion antibodies herein and the body weight of the subject to be treated, the systemic dose of the fusion antibody can be at least 5 mg, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 300, or any other value between about 5 mg and about 500 mg of fusion antibody (e.g., HIR Ab-SGSH).

[0203] As used herein, the term "systemic administration" or "peripheral administration" includes any method of administration that is not direct administration to the CNS, i.e., any method of administration that does not involve physical penetration or disruption of the BBB. "Systemic administration" includes, but is not limited to, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, intranasal, buccal, transdermal, rectal, pulmonary (inhalation), or oral administration. Any suitable fusion antibody may be used as described herein.

[0204] As used herein, SGSH deficiency includes one or more of the conditions known as Sanfilippo syndrome type A or MPS-IIIA. SGSH deficiency is characterized by the accumulation of heparan sulfate in the brain and other organs.

[0205] The compositions herein, e.g., HIR Ab-SGSH fusion antibodies, may be administered as part of a combination therapy. The combination therapy includes administration of the compositions of this embodiment in combination with other therapies for the treatment or alleviation of symptoms typically seen in patients suffering from SGSH deficiency. When the compositions of this embodiment are used in combination with other CNS disorder methods or compositions, any combination of the compositions of this embodiment and the additional methods or compositions may be used. Thus, for example, when the compositions of this embodiment are used in combination with other CNS disorder therapeutics, the two may be administered simultaneously, sequentially, for overlapping durations, at similar, identical, or different frequencies, etc. In some cases, compositions containing the compositions of this embodiment in combination with one or more other CNS disorder therapeutics will be used.

[0206] In some embodiments, the compositions, such as HIR Ab-SGSH fusion antibodies, are co-administered to a patient with other agents, either in the same formulation or as separate compositions. For example, the fusion antibodies herein may be formulated with other fusion proteins similarly designed to deliver recombinant proteins other than SGSH across the human blood-brain barrier. Additionally, the fusion antibodies may be formulated in combination with other macromolecules or small molecules.

[0207] The following specific examples are to be construed as merely being provided for illustrative purposes and are not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one of ordinary skill in the art can, based on the description herein, utilize the present embodiments to their fullest extent. All publications cited herein are incorporated by reference in their entirety. When reference is made to an identifier or address, such as a URL, it should be understood that the identifier may change and certain information on the Internet may come and go, but equivalent information may be found by searching the Internet. Reference thereto evidences that the information is available and publicly disseminated. EXAMPLES

[0208] Example 1. Expression and functional analysis of HIR Ab-GUSB fusion protein The lysosomal enzyme mutated in MPS-VII, also called Sly syndrome, is β-glucuronidase (GUSB). MPS-VII leads to accumulation of glycosaminoglycans in the brain. Enzyme replacement therapy (ERT) for MPS-VII does not appear to be effective in treating the brain because the GUSB enzyme does not cross the BBB. In an attempt to redesign human GUSB to cross the BBB, we initiated the HIR Ab-GUSB fusion protein project.

[0209] Amino acids Met1-Thr of the human GUSB protein (NP_000172), including a 22-amino acid signal peptide and an 18-amino acid carboxyl-terminal propeptide 651The human GUSB cDNA corresponding to was cloned by reverse transcription (RT) polymerase chain reaction (PCR) and custom oligodeoxynucleotides (ODN). The PCR product was separated by 1% agarose gel electrophoresis, and the expected major single band of ~2.0 kb corresponding to the human GUSB cDNA was isolated. The cloned human GUSB was inserted into a eukaryotic expression plasmid, and this GUSB expression plasmid was named pCD-GUSB. The complete expression cassette of the plasmid was confirmed by bi-directional DNA sequencing. Transfection of COS cells with pCD-GSUB in a 6-well format resulted in high GUSB enzyme activity in conditioned medium at 7 days (Table 1, experiment A), demonstrating that a functional human GUSB cDNA was successfully engineered. GUSB enzyme activity was determined by a fluorometric assay using commercially available 4-methylumbelliferyl-β-L-glucuronide (MUGlcU). The substrate is hydrolyzed to 4-methylumbelliferone (4-MU) by GUSB, and 4-MU is fluorometrically detected in a fluorometer using an emission wavelength of 450 nm and an excitation wavelength of 365 nm. A calibration curve was prepared with known amounts of 4-MU. The assay was performed at 37°C for 60 min, pH=4.8, and stopped by the addition of glycine-carbonate buffer (pH=10.5).

[0210] A new pCD-HC-GUSB expression plasmid was designed to express a fusion protein in which the carboxyl terminus of the heavy chain (HC) of the HIR Ab is fused to the amino terminus of human GUSB without the 22 amino acid GUSB signal peptide and without the 18 amino acid carboxyl terminal GUSB propeptide. The GUSB cDNA was cloned by PCR using pCD-GUSB as a template. The forward PCR primer introduced a "CA" nucleotide to maintain the open reading frame and introduce a Ser-Ser linker between the carboxyl terminus of the CH3 region of the HIR Ab HC and the amino terminus of GUSB without the 22 amino acid signal peptide of the enzyme. The GUSB reverse PCR primer introduced a stop codon "TGA" immediately after the terminal Thr of the mature human GUSB protein. DNA sequencing of the expression cassette of pCD-HC-GUSB encompassed 4,321 nucleotides (nt), including a 714-nt cytomegalovirus (CMV) promoter, a 9-nt Kozak site (GCCGCCACC), a 3,228-nt HC-GUSB fusion protein open reading frame, and a 370-nt bovine growth hormone (BGH) transcription termination sequence. The plasmid encoded a 1,075-amino acid protein consisting of a 19-amino acid IgG signal peptide, a 443-amino acid HIRMAb HC, a 2-amino acid linker (Ser-Ser), and a 611-amino acid human GUSB without the enzyme signal peptide and carboxyl-terminal propeptide. The GUSB sequence was derived from the Leu1 region of human GUSB (NP_000172). 23 -Thr 633 The predicted molecular weight of the non-glycosylated heavy chain fusion protein is 119,306 Da, with a predicted isoelectric point (pI) of 7.83.

[0211] COS cells were plated in 6-well cluster dishes and double-transfected with pCD-LC and pCD-HC-GUSB, where pCD-LC is an expression plasmid encoding the light chain (LC) of chimeric HIR Ab. Transfection was performed using Lipofectamine 2000 at a ratio of 1:2.5 μg DNA:μL Lipofectamine 2000, and conditioned serum-free medium was harvested on days 3 and 7. However, there was no specific increase in GUSB enzyme activity after double transfection of COS cells with pCD-HC-GUSB and pCD-LC expression plasmids (Table 1, experiment B). However, the IgG in the medium was only 23±2ng / mL as measured by human IgG-specific ELISA, so the low GUSB activity in the medium could be attributed to low secretion of the HIRMAb-GUSB fusion protein. Therefore, COS cell transfection was scaled up to 10xT500 plates, and the HIRMAb-GUSB fusion protein was purified by protein A affinity chromatography. IgG Western blotting showed the expected increase in the size of the fusion protein heavy chain. However, the GUSB enzyme activity of the HIRMAb-GUSB fusion protein was low, 6.1±0.1 nmol / hr / μg protein. In contrast, the specific activity of human recombinant GUSB is 2,000 nmol / hr / μg protein [Sands et al (1994) Enzyme replacement therapy for murine mucopolysaccharidosis type VII. J Clin Invest 93, 2324-2331]. These results demonstrated that after fusing GUSB to the carboxyl terminus of the HC of the HIR Ab, the GUSB enzyme activity of the HIR Ab-GUSB fusion protein was lost by >95%. The binding affinity of the HIR Ab-GUSB fusion protein to the extracellular domain (ECD) of HIR was tested by ELISA. CHO cells permanently transfected with HIR ECD were cultured in serum-free medium (SFM), and the HIR ECD was purified by wheat germ agglutinin affinity column.HIR ECD was plated in 96-well dishes, and binding of HIR Ab and HIR Ab-GUSB fusion protein to HIR ECD was detected with biotinylated anti-human IgG (H+L) goat secondary antibody, followed by avidin and biotinylated peroxidase. The ED50, the concentration of protein giving 50% of maximum binding, was determined by nonlinear regression analysis. HIR receptor assay showed that there was no decrease in affinity for HIR after fusing the 611 amino acid GUSB to the carboxyl terminus of the HIRMAb heavy chain. The ED50 for binding of HIR Ab to HIR ECD was 0.77±0.10 nM, and the ED50 for binding of HIR Ab-GUSB fusion protein was 0.81±0.04 nM.

[0212] In summary, fusion of GUSB to the carboxyl terminus of HIR Ab HC did not decrease the binding affinity of the fusion protein to HIR, but the GUSB enzymatic activity of the fusion protein was reduced by >95%.

[0213] In an attempt to successfully produce a fusion protein of HIR Ab and GUSB, a new approach was taken in which the carboxyl terminus of mature human GUSB containing the GUSB signal peptide was fused to the amino terminus of the HC of HIR Ab. This fusion protein was named GUSB-HIR Ab. The first step was to design a new expression plasmid encoding this new fusion protein, which was named pCD-GUSB-HC. The pCD-GUSB-HC plasmid expresses a fusion protein in which the amino terminus of the heavy chain (HC) of the HIRMAb, without its 19 amino acid signal peptide, is fused to the carboxyl terminus of human GUSB with the 22 amino acid GUSB signal peptide but without the 18 amino acid carboxyl terminal GUSB propeptide. The pCD-GUSB vector was used as a template for PCR amplification of GUSB cDNA, which expresses a GUSB protein with the 22 amino acid GUSB signal peptide but lacking the 18 amino acid propeptide at the GUSB carboxyl terminus. The 18 amino acid carboxyl-terminal propeptide of GUSB in pCD-GUSB was removed by site-directed mutagenesis (SDM). 633An AfeI site was generated on the 3'-flanking region of the residues and named pCD-GUSB-AfeI. The carboxyl-terminal propeptide was then removed with AfeI (located on the 3'-noncoding region of GUSB) and HindIII. The HIRMAb HC open reading frame, including the HIRMAb HC stop codon but not the 19 amino acid IgG signal peptide, was generated by PCR using the HIRMAb HC cDNA as a template. The PCR-generated HIRMAb HC cDNA was inserted into the AfeI-HindIII sites of pCD-GUSB-AfeI to form pCD-GUSB-HC. A Ser-Ser linker between the carboxyl terminus of GUSB and the amino terminus of HIRMAb HC was introduced into the AfeI site by the PCR primers used for cloning the HIRMAb HC cDNA. DNA sequencing of the pCD-GUSB-HC expression cassette showed that the plasmid expressed a 1,078 amino acid protein consisting of a 22 amino acid GUSB signal peptide, a 611 amino acid GUSB, a 2 amino acid linker (Ser-Ser), and a 443 amino acid HIRMAb HC. The GUSB sequence was derived from the Met1 site of human GUSB (NP_000172). 1 -Thr 633 It was 100% identical.

[0214] Dual transfection of COS cells with pCD-LC and pCD-GUSB-HC expression plasmids in a 6-well format resulted in higher GUSB enzyme activity in the conditioned medium at 7 days compared to dual transfection with pCD-LC and pCD-HC-GUSB plasmids (Table 1, experiment C). However, the GUSB-HIRMAb fusion protein was also poorly secreted by the COS cells, as the concentration of human IgG in the conditioned medium at 7 days was only 13 ± 2 ng / mL as measured by ELISA. The COS cell transfection was scaled up to 10xT500 plates, and the GUSB-HIRMAb fusion protein was purified by protein A affinity chromatography. SDS-PAGE showed the expected increase in the size of the fused protein heavy chain. The GUSB enzyme activity of the purified GUSB-HIRMAb fusion protein was high, 226 ± 8 nmol / hr / μg protein, which is 37-fold higher than the specific GUSB enzyme activity of the HIRMAb-GUSB fusion protein. However, the HIR receptor assay showed that after GUSB was fused to the amino terminus of the HIRMAb heavy chain, the affinity for HIR was significantly reduced, resulting in a 95% decrease in receptor binding affinity. The ED50 for the binding of the HIR Ab to the HIR ECD was 0.25±0.03 nM, and the ED50 for the binding of the HIR Ab-GUSB fusion protein was 4.8±0.4 nM.

[0215] In summary, fusion of GUSB to the amino terminus of the HIR Ab HC resulted in retention of GUSB enzymatic activity of the fusion protein but caused a 95% reduction in binding of the GUSB-HIR Ab fusion protein to the HIR. In contrast, fusion of GUSB to the carboxyl terminus of the HIR Ab HC did not result in a reduction in the binding affinity of the HIR Ab-GUSB fusion protein to the HIR. However, the GUSB enzymatic activity of this fusion protein was reduced by >95%. These findings demonstrate the bifunctionality of IgG-enzyme fusion proteins, i.e., the unpredictable nature of a technology that fuses lysosomal enzymes to IgG molecules in such a way that the IgG moiety retains high affinity binding to its cognate antigen and high enzymatic activity. Table 1. GUSB enzyme activity in transfected COS cells [mean ± standard deviation (n = 3 dishes / time point)] [Table 1]

[0216] Example 2. Expression and functional analysis of HIR Ab-GCR fusion protein The lysosomal enzyme mutated in Gaucher disease (GD) is β-glucocerebrosidase (GCR). The neuronopathic form of GD affects the CNS, where lack of GCR enzyme activity in the brain leads to accumulation of intralysosomal inclusions in brain cells. Enzyme replacement therapy (ERT) for GD is ineffective in treating the brain because the GCR enzyme does not cross the BBB. As part of re-engineering human GCR to cross the BBB, a HIR Ab-GCR fusion protein project was designed, expressed, and tested for enzymatic activity. The amino acid Ala of the human GCR protein (NP_000148), without the 39 amino acid signal peptide, is expressed at 100 ng / mL. 40 -Gln 536 Human GCR cDNA corresponding to was custom synthesized by a commercial DNA manufacturer. GCB cDNA consisted of 1522 nucleotides (nt) containing the open reading frame of GCB, not including the signal peptide through the TGA stop codon. At the 5' end, a StuI restriction endonuclease (RE) sequence was added, and at the 3' end, a 14 nt fragment from the 3'-untranslated region of GCR mRNA followed by a HindIII RE site. Internal HindIII and StuI sites within the GCR gene were mutated without changing the amino acid sequence. The GCR gene was excised from the pUC plasmid provided by the supplier with StuI and HindIII and transformed into a eukaryotic expression plasmid encoding the HIR Ab heavy chain. The plasmid was inserted into the HpaI and HindIII sites of pCD-HC-GCR and the expression plasmid was named pCD-HC-GCR. This expression plasmid expresses a fusion protein in which the carboxyl terminus of the heavy chain (HC) of the HIR Ab is fused to the amino terminus of human GCR without the 39 amino acid GCR signal peptide, with a 3 amino acid linker (Ser-Ser-Ser) between the HC and GCR of the HIR Ab. The pCD-HC-GCR expression cassette was identified by DNA sequencing. The expression cassette was composed of 5,390 nt, including a 2134 nt CMV promoter sequence, a 2,889 nt expression cassette, and a 367BGH polyA sequence. The plasmid encoded a 963 amino acid protein consisting of a 19 amino acid IgG signal peptide, a 443 amino acid HIRMAb HC, a 3 amino acid linker (Ser-Ser-Ser), and a 497 amino acid human GCR without the enzyme signal peptide. The GCR sequence is the Als of human GCR (NP_000148). 40 -Gln 536 The predicted molecular weight of the non-glycosylated heavy chain fusion protein is 104,440 Da, with a predicted isoelectric point (pI) of 8.42.

[0217] The HIR Ab-GCR fusion protein was expressed in transiently transfected COS cells. COS cells were plated in 6-well cluster dishes and double transfected with pCD-LC, an expression plasmid encoding the light chain (LC) of the chimeric HIR Ab, and pCD-HC-GCR. Transfections were performed with Lipofectamine 2000 at a ratio of 1:2.5, ugDNA:uL Lipofectamine 2000, and conditioned serum-free medium was harvested on days 3 and 7. Secretion of the fusion protein into serum-free medium (SFM) was monitored by human IgG ELISA. Conditioned medium was clarified by depth filtration, and the HIR Ab-GCR fusion protein was purified by protein A affinity chromatography. The purity of the fusion protein was confirmed by reducing SDS-PAGE, and the fusion protein was identified by Western blotting using primary antibodies against either human IgG or human GCR. Both IgG and GCR antibodies reacted with the 130 kDa heavy chain of the HIR Ab-GCR fusion protein.

[0218] The GCR enzyme activity of the fusion protein was measured by fluorometric enzyme assay using 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) as the enzyme substrate as previously described for the enzyme assay of recombinant GCR (JB Novo, et al, Generation of a Chinese hamster ovary cell line producing recombinant human glucocerebrosidase, J. Biomed. Biotechnol., Article ID 875383, 1-10, 2012). The GCR enzyme assay was performed in citrate / phosphate buffer / pH=5.5 containing 0.25% Triton X-100 and 0.25% sodium taurocholate with a final concentration of 5 mM 4-MUG and incubated at 37°C for 60 min. The enzyme activity was stopped by adding 0.1 M glycine / 0.1 M NaOH. The GCR enzyme converts the 4-MUG substrate to the product 4-methylumbelliferone (4-MU). An assay calibration curve was constructed using 4-MU (0.03-3 nmol / tube). Enzyme activity was reported as units / mg protein, where 1 unit = 1 umol / min. The enzyme activity of recombinant human GCR is 40 units / mg (Novo et al, 2012). However, the GCR enzyme activity of the HIR Ab-GCR fusion protein was only 0.07 units / mg, a 99% decrease compared to the specific activity of recombinant GCR.

[0219] Examples 1 and 2 demonstrate the unpredictability of designing biologically active IgG-lysosomal enzyme fusion proteins. In both cases, fusion of either GUSB or GCR to the carboxyl terminus of the heavy chain of a HIR Ab results in loss of >95% of the enzyme activity. In the following examples, we describe the surprising finding that SGSH enzyme activity is preserved after fusion to the carboxyl terminus of the heavy chain of a HIR Ab.

[0220] Example 3. Construction of human HIR Ab heavy chain-SGSH fusion protein expression vector The lysosomal enzyme mutated in MPS-IIIA is SGSH. MPS-IIIA leads to the accumulation of heparan sulfate in the brain. As described by Hemsley et al. (2009) (Examination of intravenous and intra-CSF protein delivery for treatment of neurological disease), Eur. J. Neurosci., 29:1197-1214, MPS-IIIA enzyme replacement therapy is not effective for treating the brain because the SGSH enzyme does not cross the BBB. To develop a bifunctional molecule that can cross the BBB and exhibit enzymatic activity, SGSH was fused to the HIR Ab. In one embodiment, the amino terminus of mature SGSH was fused to the carboxyl terminus of each heavy chain of the HIR Ab (Figure 2).

[0221] It was unclear whether SGSH's enzymatic activity would be preserved when fused to the HIR Ab. The experience with the IgG-GUSB fusion protein described in Example 1 illustrates the unpredictable nature of the technology and the possibility that constructing an IgG-enzyme fusion protein may compromise biological activity of either the IgG or lysosomal enzyme portion. The situation with SGSH is further complicated because it is not until the protein is post-translationally modified in the endoplasmic reticulum at a Cys residue near the amino terminus (Cys-514 in SEQ ID NO: 10), that the SGSH enzyme becomes catalytically active, and it was not known whether fusing SGSH to the HIR Ab would impair this process. SGSH is a member of the sulfatase family, and its enzymatic activity is activated when a specific Cys residue is converted to a formylglycine residue by sulfatase modifier type 1 (SUMF1), also called formylglycine generating enzyme (FGE), in the endoplasmic reticulum (Fraldi et al. (2007), "SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies", Biochem. J., 403: 305-312.). Without this conversion of endogenous cysteine ​​to a formylglycine residue, the enzyme has little activity. For example, if SGSH is fused to the carboxyl terminus of the HC of an HIR Ab as part of maintaining high affinity binding of the fusion protein to the HIR, the IgG heavy chain will fold into a three-dimensional structure after translation in the host cell, and then the SGSH portion of the fusion protein will fold. It was unclear whether the SGSH portion of the HIR Ab HC-SGSH fusion protein would fold into a three-dimensional structure that would be recognized and activated by SGSH-modifying factors in the endoplasmic reticulum, resulting in the expression of full SGSH enzymatic activity in the HIR Ab-SGSH fusion protein.

[0222] Human SGSH cDNA was generated by polymerase chain reaction (PCR) using oligodeoxynucleotides (ODN) derived from the nucleotide sequence of human SGSH mRNA (GenBank accession #NM_000199). Using the ODNs listed in Table 2 and commercially available human liver polyA+RNA, a cDNA encoding human SGSH without its signal peptide Arg21-Leu-502 was generated by reverse transcription (RT) PCR. In the tandem vector (TV) expression plasmid, the forward (FOR) ODN primer had "CC" on the 5'-flanking region to maintain an open reading frame (orf) containing the CH3 region of human IgG1 and to introduce a Ser-Ser linker between human IgG1-CH3 and SGSH cDNA. The reverse (REV) ODN was complementary to the end of the SGSH orf and included its stop codon TGA. RT-PCR was completed and the predicted single band of ~1.5 kb corresponding to the SGSH orf cDNA was detected by agarose gel electrophoresis (Figure 3, lane 3) and gel purified. Both forward and reverse ODNs were phosphorylated for direct insertion into the expression vector. SGSH cDNA was inserted into the HpaI site of the precursor TV, pUTV-1, using T4 DNA ligase to form TV-HIRMAb-SGSH as outlined in Figure 4. pUTV-1 was linearized with HpaI and digested with alkaline phosphatase to prevent self-ligation. TV-HIRMAb-SGSH is a tandem vector that encompasses the mouse dihydrofolate reductase (DHFR) gene after both the light chain (LC) and heavy chain (HC) genes, respectively, of the HIRMAb-SGSH fusion protein. The light and heavy chain genes of the HIRMAb-SGSH fusion protein are driven by the CMV promoter and the orf is followed by the bovine growth hormone (BGH) polyadenylation sequence. The DHFR gene is under the influence of the SV40 promoter and contains the Hepatitis B virus (HBV) polyadenylation termination sequence.The DNA sequence of the TV-HIRMAb-SGSH plasmid was confirmed by bidirectional DNA sequencing performed at Sequetech Corp (Mountaign View, CA) using custom ODN synthesized by Midland (Midland, TX). The complete expression cassette of the plasmid was confirmed by sequencing both strands. The fusion of the SGSH monomer to the carboxyl terminus of each HC is shown in Figure 2. Table 2. Oligodeoxynucleotide primers used for designing RT-PCR cloning of human SGSH without the signal peptide, derived from the human SGSH mRNA sequence (GenBank NM_000199), and for designing the HIRMAb-SGSH expression vector. [Table 2]

[0223] DNA sequencing of the TV-HIRMAb-SGSH plasmid encompassed 10,000 nucleotides (nt), covering the expression cassette of the LC gene, the HC-SGSH gene, and the DHFR gene (Figure 4). Starting from the 5' end, the plasmid was constructed from a cytomegalovirus (CMV) promoter, a 9 nt complete Kozak site, GCCGCCACC (nt 1-9 of SEQ ID NO:13), a 705 nt open reading frame (orf) of LC (nt 10-714 of SEQ ID NO:13), then a bovine growth hormone (BGH) polyA sequence, then a linker sequence, then a tandem CMV promoter, then a complete Kozak site (nt 1-9 of SEQ ID NO:14), then a 2,841 nt HIRMAb HC-SGSH fusion protein orf (nt 10-2850 of SEQ ID NO:14), then a tandem BGH polyA sequence, then an SV40 promoter, then a complete Kozak site (nt 1-9 of SEQ ID NO:15), then a 564 nt DHFR orf (nt 10-573 of SEQ ID NO:15), then a Hepatitis B virus polyA sequence (Figure 4). TV encoded the 234 amino acid HIRMAb LC (SEQ ID NO: 8) containing a 20 amino acid signal peptide, and a 946 amino acid protein fusion protein of HIRMAb HC and SGSH (SEQ ID NO: 10). The fusion protein HC was constructed from a 19 amino acid IgG signal peptide, a 442 amino acid HIRMAb HC, a 3 amino acid linker (Ser-Ser-Ser), and a 482 amino acid human SGSH without the enzyme's signal peptide. The predicted molecular weight of the non-glycosylated heavy chain fusion protein is 103,412 Da and the predicted isoelectric point (pI) is 7.44. Except for the R456H polymorphism in the SGSH domain of the fusion protein, the amino acid sequence of the SGSH domain of the HC fusion protein is 100% identical to the sequence of amino acids 21-502 of human SGSH (NP_000190), where the numbering system includes the 20 amino acid signal peptide of SGSH. The residue is frequently an arginine (R or Arg) residue, although histidine (H or His) residues are also known.The R456H polymorphism does not significantly affect the enzymatic activity of SGSH (Montfort et al. (2004), Expression and functional characterization of human mutant sulfamidase in insect cells, Mol. Genet. Metab. 83: 246-251). TV also encodes the product of the selected gene DHFR, a 187 amino acid protein (SEQ ID NO: 16).

[0224] Example 4. Stable transfection of Chinese hamster ovary cells with TV-HIRMAb-SGSH Chinese hamster ovary (CHO) cells were cultured in serum-free HyQ SFM4CHO Utility medium (HyClone) containing 1xHT supplement (hypoxanthine and thymidine). CHO cells (5x10 6 10,000 live cells) were electroporated with 5 μg of PvuI-linearized TV-HIRMAb-SGSH plasmid DNA. The cell-DNA suspension was then incubated on ice for 10 min. Cells were electroporated with BioRad's preset protocol for CHO cells, i.e., square wave with a pulse of 15 msec and 160 volts. After electroporation, cells were incubated on ice for 10 min. Cell suspension was transferred to 50 mL medium and plated at 125 μl per well into 4x96-well plates (10,000 cells per well). A total of 10 electroporations and 4,000 wells were performed per study.

[0225] After electroporation (EP), the CHO cells were placed in an incubator at 37°C and 8% CO2. Due to the presence of the neo gene in TV, the transfected cell lines were initially selected with G418. Because TV-HIRMAb-SGSH also contains the DHFR gene (Figure 4), the transfected cells could also be selected by 20 nM methotrexate (MTX) and HT-deficient medium. When visible colonies were detected, approximately 21 days after EP, conditioned medium was sampled for human IgG by ELISA. Wells showing high human IgG signals by ELISA were transferred from the 96-well plate to a 24-well plate containing 1 mL of HyQ SFM4CHO-Utility. The 24-well plate was returned to the incubator at 37°C and 8% CO2. The following week, IgG ELISA was performed on the clones in the 24-well plate. This was repeated from 6-well plates to T75 flasks and finally to 60 mL and 125 mL square plastic bottles on an orbital shaker. At this stage, the final MTX concentration was 80 nM, and the IgG concentration in the medium, a measure of the HIRMAb-SGSH fusion protein in the medium, was 10 6 / mL cell density is >10 mg / L.

[0226] Clones selected for dilution cloning (DC) were removed from the orbital shaker in the incubator and transferred to a sterile hood. Cells were diluted to 500 mL in F-12K medium with 5% dialyzed fetal bovine serum (d-FBS) and penicillin / streptomycin so that 4,000 wells in a 40x96-well plate could be plated at a cell density of 1 cell per well (CPW), for a final dilution of 8 cells per mL. Once the cell suspension was prepared in the sterile hood, 125 μL aliquots were dispensed into each well of the 96-well plate using an 8-channel pipettor or precision pipettor system. Plates were returned to the incubator at 37°C and 8% CO2. Cells diluted to 1 cell / well cannot survive without serum. On day 6 or 7, the DC plates were removed from the incubator and transferred to a sterile hood where 125 μl of F-12K medium containing 5% dialyzed fetal bovine serum (d-FBS) was added to each well. At this point, the selection medium contained 5% d-FBS, 30 nM MTX, and 0.25 mg / mL geneticin. On day 21 from the first 1 CPW plating, an aliquot was removed from each of the 4,000 wells using a robotic device for human IgG ELISA. The DC plates were removed from the incubator and transferred to a sterile hood where 100 μl of medium per well of the 96-well plate was removed and transferred to a new sterile sample 96-well plate using an 8-channel pipettor or precision pipettor system.

[0227] On the 20th day after the initial 1CPW plating, 40x96-well immunoassay plates were plated with 100μL of 1μg / mL primary antibody solution, mouse anti-human IgG in 0.1M NaHCO3. The plates were incubated overnight in a 4℃ refrigerator. The next day, the ELISA plates were washed 5 times with 1xTBST and 100μL of 1μg / mL secondary antibody solution and blocking buffer were added. The plates were washed 5 times with 1xTBST. 100μL of 1mg / mL solution of 4-nitrophenyl phosphate di(2-amino-2-ethyl-1,3-propanediol) salt in 0.1M glycine buffer was added to the 96-well immunoassay plate. The plates were read on a microplate reader. The assay provided IgG output data for 4,000 wells / experiment. 24-48 of the most productive wells were selected for further expansion.

[0228] The most productive 24-well plate from 1 CPW DC was transferred to a sterile hood and gradually subcloned through 6-well dishes, T75 flasks, and 125 mL square plastic bottles on an orbital shaker. During this process, the serum was reduced to zero at the final stage of centrifugation of the cells and resuspended in SFM.

[0229] The above procedure was repeated at 0.5-1 cell / well (CPW) for the second dilution cloning. At this stage, approximately 40% of the wells showed some cell proliferation, and all wells showing proliferation also secreted human IgG. These results confirmed that only an average of 1 cell was plated per well in these procedures, and that the CHO cell lines arose from a single cell.

[0230] At a medium concentration of 10 mg / L and a cell density of 1-2 million cells / mL, the HIR Ab-SGSH fusion protein was secreted in large amounts into the medium by stably transfected CHO cells. We observed that the HIR Ab-SGSH fusion protein was produced in large amounts by stably transfected CHO cells, even though the host cells were not double-transfected with the fusion protein gene and the gene encoding SUMF1. To detect secretion of SGSH into the conditioned medium by the transfected host cells, it was necessary to cotransfect the cells transfected with the SGSH gene with the SUMF1 accessory factor (Fraldi et al. (2007), "SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies", Biochem. J., 403: 305-312). An unexpected advantage of designing SGSH and IgG-SGSH fusion proteins is that the host cells secrete the fusion proteins without the need for cotransfection with SUMF1.

[0231] CHO-derived HIRMAb-SGSH fusion protein was purified by protein A affinity chromatography. The purity of the HIRMAb-SGSH fusion protein was confirmed by reducing SDS-PAGE as shown in Figure 10. Only the HC and LC proteins are detected in both HIRMAb alone and HIRMAb-SGSH fusion protein. The fusion protein was identified by Western blotting using primary antibodies against human IgG (Figure 11, left panel) or human SGSH (Figure 11, right panel). Based on the mobility of MW standards, the molecular weight (MW) of the HIRMAb-SGSH heavy and light chains, as well as the MW of the HIRMAb heavy and light chains, were estimated by linear regression. Because SGSH is fused to the HIRMAb heavy chain, the size of the HIRMAb-SGSH fusion heavy chain (136 kDa) is larger than that of the HIRMAb heavy chain (62 kDa). Because the HIRMAb-SGSH fusion protein and the HIRMAb antibody use the same light chain, the size of the light chain (27 kDa) is identical for both proteins. Based on the mobility in SDS-PAGE of Western blots, the predicted MW of the heterotetrameric HIRMAb-SGSH fusion protein shown in Figure 2 is 325 kDa.

[0232] Example 5. Analysis of HIR-binding and SGSH activity of bifunctional IgG-SGSH fusion proteins The affinity of the fusion proteins for the HIR extracellular domain (ECD) was measured by ELISA. CHO cells permanently transfected with the HIR ECD were cultured in serum-free medium (SFM) and the HIR ECD was purified using a wheat germ agglutinin affinity column as previously described by Coloma et al. (2000) Pharm Res, 17:266-274. The HIR ECD was plated on Nunc-Maxisorb 96-well dishes and binding of the HIR Ab or HIR Ab-SGSH fusion protein to the HIR ECD was detected using a biotinylated anti-human IgG (H+L) goat secondary antibody, followed by avidin and biotinylated peroxidase (Vector Labs, Burlingame, CA). The ED50, the concentration giving 50% of the maximal binding of the HIR Ab or HIR Ab-SGSH fusion protein, was determined by nonlinear regression analysis. The ED50 for binding to HIR was 29±3 ng / mL, and the ED50 for binding of the HIR Ab-SGSH fusion protein to HIR was 107±17 ng / mL (Figure 12). The MW of the HIR Ab is 150 kDa, and the MW of the HIR Ab-SGSH fusion protein is 325 kDa. Thus, after normalizing for the difference in molecular weight, the binding of the chimeric HIR Ab or the HIR Ab-SGSH fusion protein to the HIR ECD was equivalent, with ED50s of 0.19±0.02 nM and 0.33±0.05 nM, respectively (Figure 12). These findings indicate that the affinity of the HIR Ab-SGSH fusion protein to bind to HIR is maintained, even though the SGSH molecules are fused to the carboxyl termini of both heavy chains of IgG.

[0233] SGSH enzyme activity was measured using a two-step fluorometric enzyme assay developed by Karpova et al. (1996) (A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type A (MPS IIIA), J. Inher. Metab. Dis. 19: 278-285) that uses 4-methylumbelliferyl-α-N-sulfo-D-glucosaminide (MU-αGlcNS) as the assay substrate. The substrate was custom synthesized by Sigma-Aldrich (St Louis, MO) and the structure of the substrate is outlined in FIG. 13A. The substrate is hydrolyzed by SGSH to 4-methylumbelliferyl-α-D-glucosaminide (MU-αGlcNH2), which is then hydrolyzed to the fluorescent product 4-methylumbelliferone (4-MU) by the α-glucosaminidase side-activity in commercially available yeast α-glucosidase (FIG. 13A). The assay was performed by incubating the HIRMAb-SGSH fusion protein (30, 100, or 300 ng) and MU-αGlcNS substrate in 0.03 M sodium barbital / 0.03 M sodium acetate / 0.13 M NaCl / pH=5.5 / 0.02% sodium azide for 17 h at 37°C. McIlvaine's buffer and 0.1 units of yeast α-glucosidase were added and the incubation continued for 24 h at 37°C. The reaction was stopped by adding 0.5 M sodium carbonate / pH=10.7. Fluorescence was measured using a Farrand fluorometer equipped with a 365 nm excitation filter and a 450 nm emission filter. A calibration curve was generated using 0.03 to 3.0 nmol / tube of 4-MU product to allow conversion of fluorescence units to nmol / tube. Enzyme activity was measured as units of HIRMAb-SGSH fusion protein / mg protein, with 1 unit = nmol of 4-MU product produced during the first 17 h of incubation (Karpova et al., 1996).The assay was linear with respect to the mass of the fusion protein (Figure 13B), with an average enzyme activity of 4,712 ± 388 units / mg protein. The SGSH enzyme specific activity of 60 kDa recombinant human SGSH by the same assay is 15,000 units / mg protein (Urayama et al. (2008): Mannose 6-phosphate receptor-mediated transport of sulfamidase across the blood-brain barrier in the newborn mouse. Mol Ther 16: 1261-1266.). However, when SGSH is redesigned as a 325 kDa heterotetrameric IgG-SGSH fusion protein (Figure 2), the effective MW of SGSH is 163 kDa, whereas the MW of SGSH is 60 kDa. After normalization for the molecular weight difference, the effective SGSH specific activity is 12,800 units / mg protein, which is 85% of the enzyme activity of recombinant SGSH. Thus, in contrast to the results observed with the IgG-GUSB fusion protein (Table 1), fusion of SGSH to the carboxyl terminus of the HC of the HIR Ab had only a minor effect on the enzymatic activity of the SGSH enzyme. The high SGSH enzymatic activity of the CHO-derived HIR Ab-SGSH fusion protein is surprising, since SGSH is a member of the sulfatase family that requires specific post-translational modifications for the expression of SGSH enzymatic activity. The activity of the SGSH enzyme is activated when Cys-50 is converted to a formylglycine residue by sulfatase modifier type 1 (SUMF1), also called formylglycine generating enzyme (FGE). The retention of the enzymatic activity of SGSH in the HIRMAb-SGSH fusion protein produced by stably transfected CHO cells indicates that the SGSH enzyme is activated in host cells despite being fused to the HIRMAb heavy chain.

[0234] Example 6. Amino acid linker linking SGSH and targeting antibody Mature human SGSH is fused to the carboxyl terminus of the HC of the HIR Ab with a three amino acid linker, Ser-Ser-Ser (underlined in FIG. 9). Any number of linker variations may be used in place of the Ser-Ser-Ser linker. The three amino acid linker may be retained, but the amino acid sequence may be changed to alternative amino acids, e.g., Gly-Gly-Gly, Ser-Gly-Ser, Ala-Ser-Gly, or any number of combinations of the 20 natural amino acids. Alternatively, the linker is reduced to 2, 1, or 0 amino acids. In the case of the 0 amino acid linker, the amino terminus of SGSH is fused directly to the carboxyl terminus of the HC of the HIR Ab. Alternatively, the length of the linker is extended to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids. Such linkers are well known in the art, as there are several publicly available programs for determining optimal amino acid linkers in the design of fusion proteins. Frequently used linkers include various combinations of Gly and Ser in the repeat sequence, such as (Gly4Ser)3, or other variations.

[0235] Example 7. Biological activity and lysosomal distribution of HIR Ab-SGSH fusion protein in Sanfilippo type A fibroblasts SGSH enzyme hydrolyzes sulfate groups from heparan sulfate GAGs, allowing their subsequent degradation by other enzymes within the cell. The effect of treatment with HIRMAb-SGSH fusion protein on the release of sulfate esters from GAGs was tested in Sanfilippo type A fibroblasts (MPS-IIIA fibroblasts). Cells were obtained from the Coriell Institute for Medical Research (Camden, NJ) and grown to confluence in 6-well cluster dishes, and then pulse-chase experiments with 35S were performed. For the pulse step, confluent cells were washed with phosphate-buffered saline (PBS) and incubated with 1 mL of low sulfate Ham's F12 medium containing 10% dialyzed fetal bovine serum (FBS) and 47 μCi / mL of [35S] sodium sulfate for 48 h at 37°C in a humidified incubator. The medium was aspirated, the wells were washed with PBS, and the cells were incubated with normal radioactive-free DMEM / F12 medium (1 mL / well) containing 10% FBS and various concentrations of HIRMAb-SGSH fusion protein for 48 h at 37°C in a humidified incubator. The medium was aspirated and the cells were washed with PBS. The cells were removed from the wells with 0.4 mL / well of 0.05% trypsin / EDTA for 4 min at 37°C, a step that removes any radioactivity attached to the cell surface so that it is not incorporated into lysosomal GAGs. The trypsin reaction was stopped by suspending the cells in serum-free medium and centrifuging at 1000 g. The supernatant was removed and discarded, and the cell pellet was solubilized in 0.4 mL of 1 N NaOH at 60°C for 1 h. Bicinchoninic acid (BCA) protein Protein content was measured using an assay. 35S radioactivity was measured in Ultima-gold counting solution using a Perkin Elmer liquid scintillation counter. CPM radioactivity was divided by mg protein well and data reported as CPM / mg protein (Table 3). This study shows that subtherapeutic concentrations (0.25-0.5 μg / mL) of HIRMAb-SGSH fusion protein reduced sulfate-labeled GAGs by 72%-83% in MPS-IIIA fibroblasts.

[0236] The reduction of sulfated GAGs in MPS-IIIA fibroblasts suggests that the HIRMAb-SGSH fusion protein is not only internalized by target cells but also triaged to the lysosomal compartment of target cells, which was confirmed by confocal microscopy. MPS-IIIA fibroblasts were incubated with the HIRMAb-SGSH fusion protein for 6 to 24 hours, then washed, fixed in 4% paraformaldehyde, permeabilized with Triton X-100, and incubated with rabbit primary antibody against human SGSH and mouse primary antibody against human lysosomal membrane protein type 1 (LAMP1), followed by incubation with anti-mouse IgG, donkey, Alexa Fluor-488 conjugate (green channel), and anti-rabbit IgG, donkey, Alexa Fluor-594 conjugate (red channel). The washed slides were stained with 4',6-diamidino-2-phenylindole (DAPI) (Life Technologies), washed, air-dried, and mounted in Prolong Gold Antifade (Life Technologies). Confocal microscopy was performed using a Leica TCS SP2 AOBS inverted fluorescence microscope equipped with a Leitz P1 Apo 100X oil immersion objective and a Leica confocal laser scanning adapter utilizing argon (476 and 488 nm), new diode (561 nm), and helium-neon (633 nm) lasers, respectively. SGSH immunoreactivity was detected in the red channel, and intracellular LAMP1 immunoreactivity was detected in the green channel. The overlap of SGSH and LAMP1 immunoreactivity indicates that the HIRMAb-SGSH fusion protein is triaged to the lysosomal compartment after internalization into MPSIIIA fibroblasts. There was no immunoreactivity in cells labeled with an isotype control antibody. Table 3. Reduction of GAGs in MPS-IIIA fibroblasts treated with HIRMAb-SGSH fusion protein [Table 3]

[0237] Example 8. Receptor-Mediated Transport of SGSH into the Primate Brain The BBB penetration of the HIRMAb-SGSH fusion protein in vivo was evaluated in rhesus monkeys. The HIRMAb domain of the fusion protein cross-reacts with insulin receptors of Old World primates such as rhesus monkeys, but not with insulin receptors of New World monkeys such as squirrel monkeys, or with insulin receptors of mice. Therefore, in vivo delivery to the brain was evaluated in rhesus monkeys. 125 I]-Monoiodide-Bolton Hunter The HIRMAb-SGSH fusion protein was radioactively labeled with the reagent to a specific activity of 3.7 μCi / μg. 125 The precipitateability of the [I]-HIRMAb-SGSH fusion protein was >97% for at least 8 days after labeling while stored at -70°C. Prior to labeling, the fusion protein was buffer exchanged using 0.01M sodium acetate / 140mM NaCl / pH=5.5 / 0.001% Tween-80 and Amicon Ultracel-30K centrifugal filtration units. The labeled HIRMAb-SGSH fusion protein was purified using gel filtration on a 1x28cm column of Sephadex G-25 and an elution buffer of 0.01M sodium acetate / 140mM NaCl / pH=5.5 / 0.001% Tween-80. Adult female rhesus macaques weighing 17.3 kg were studied at MPI Research (Mattawan, MI). The animals received 1200μCi of [I] over 30 seconds by intravenous bolus into the left femoral vein. 125 I]-HIRMAb-SGSH fusion protein was injected intravenously (IV). The injection dose (ID) of HIRMAb-SGSH fusion protein was 19 μg / kg. The animals were anesthetized with intramuscular ketamine. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals approved and published by the National Institutes of Health. Total plasma [ 125Femoral vein plasma was collected at 2, 5, 15, 30, 60, 90, and 140 min after intravenous drug administration for the measurement of [I] radioactivity (dpm / mL) and plasma radioactivity precipitable with 10% cold TCA. The animals were euthanized and samples of major tissues (heart, liver, spleen, lung, skeletal muscle, and omental fat) were removed, weighed, and processed for radioactivity measurements. The skull was opened and the brain removed. Samples of frontal cortical gray matter, cerebellar gray matter, and choroid plexus were removed for radioactivity measurements. A Wizard model 1470 gamma counter was used. 125 Plasma and tissue samples were analyzed for I radioactivity. Based on the specific activity of the injected fusion protein, TCA-precipitable [ 125 The radioactivity (DPM / mL) was converted to ng / mL and the plasma concentration of the fusion protein was calculated using the binomial exponential equation %ID / mL = A1e -k1t +A2e -k2t The intercepts (A1, A2) and slopes (k1, k2) were used to calculate median residence time (MRT), median volume of distribution (Vc), steady-state volume of distribution (Vss), area under the plasma concentration curve (AUC), and total body clearance (CL). For nonlinear regression analysis, the AR subroutine of BMDP Statistical Software (Statistical Solutions Ltd, Cork, Ireland) was used. Data were scaled as 1 / (%ID / mL) 2 Weighted at .

[0238] Frontal cortex samples (~2 grams) were removed for capillary depletion assay to confirm transport of HIRMAb-SGSH fusion protein across the BBB. The capillary depletion assay separates intracerebral vascular tissue from the post-vascular compartment. The post-vascular supernatant is depleted of >95% of the brain vasculature based on measurement of specific activity of enzymes specific for brain capillaries, such as gamma-glutamyltransferase or alkaline phosphatase. To separate the vascular and post-vascular compartments, brains were homogenized in 8 mL of cold PBS in a tissue grinder. Eight mL of cold 40% dextran (70 kDa, Sigma Chemical Co.) was added to the homogenate, and an aliquot of the homogenate was taken for radioactivity measurement. The homogenate was centrifuged at 3200 g for 10 min at 4°C in a fixed-angle rotor. Since the brain microvasculature is quantitatively precipitated as a pellet, the postvascular supernatant is a measure of the brain parenchyma depleted of capillaries. 125 The total [I radioactivity in the brain fraction was counted for each of the three fractions. 125 I]radioactivity (DPM / gram brain) in peripheral plasma at 140 min was 125 The volume of distribution (VD) was determined from the ratio of [I] radioactivity (DPM / μL plasma) divided by the percentage of radioactivity in the postvascular supernatant that was precipitable with 10% cold TCA.

[0239] Male rhesus monkeys, 125 I]-HIRMAb-SGSH fusion protein (1200 μCi, 324 μg) was intravenously injected, and TCA-precipitable [ 125The time course of plasma concentrations of [I]-HIRMAb-SGSH fusion protein is shown in Figure 14. At 2, 5, 15, 30, 60, 90, and 140 min after intravenous injection, the percentage of total plasma radioactivity precipitable with TCA was 96 ± 1%, 95 ± 1%, 94 ± 1%, 89 ± 1%, 84 ± 2%, 79 ± 1%, and 72 ± 2%, respectively. A binomial exponential equation was fitted to the plasma profile of the TCA-precipitable fusion protein to yield the pharmacokinetic (PK) parameters shown in Table 4. With a mean residence time of 62 ± 4 min, a total body volume of distribution (Vss) 2.5 times greater than the median volume of distribution (Vc), and a high systemic clearance rate (1.11 ± 0.03 mL / min / kg), [ 125 I]-HIRMAb-SGSH fusion protein is rapidly cleared from plasma (Table 4). Table 4. Pharmacokinetic parameters of HIRMAb-SHSH fusion protein [Table 4] Parameters calculated from plasma profiles in Figure 14 The volume of distribution (VD) of the HIRMAb-SGSH fusion protein in total brain homogenates 140 min after injection (782 ± 36 μL / g) was greater than the brain VD of a nonspecific human IgG1 isotype control antibody (20 ± 6 μL / g) (Table 5). The brain VD of the IgG1 isotype control antibody represents the brain uptake of molecules that are sequestered within the cerebral blood volume and do not cross the BBB. The VD of the HIRMAb-SGSH fusion protein in the postinjection vascular supernatant (666 ± 71 μL / g) was greater than the VD of the HIRMAb-SGSH fusion protein in the brain vascular pellet (24 ± 17 μL / g) (Table 5), indicating that the majority of the HIRMAb-SGSH fusion protein crossed the BBB and penetrated the brain parenchyma. TCA precipitation of postvascular supernatant radioactivity was 95.9±0.7% (Table 5), indicating that the radioactivity in the postvascular supernatant represents intact HIRMAb-SGSH fusion protein and not labeled metabolites. Table 5. Capillary depletion analysis of brain uptake of HIRMAb-SGSH fusion protein [Table 5] Mean ± standard deviation Fusion proteins were administered by intravenous injection, and brain measurements were performed 140 min after injection. Radioactivity in the posterior vascular supernatant was 95.9 ± 0.7% precipitable with cold 10% trichloroacetic acid. Homogenate VD against a human IgG1 isotype control antibody has been reported previously (Boado, RJ; Pardridge, WM Comparison of blood-brain barrier transport of GDNF and an IgG-GDNF fusion protein in the Rhesus monkey. Drug Metab. Disp. 2009, 37, 2299-2304).

[0240] Since the adult rhesus monkey brain weighs 100 grams, tissue uptake of the HIRMAb-SGSH fusion protein is expressed as % of injected dose (ID) per 100 grams wet tissue weight (Table 6). The primary tissues responsible for the clearance of HIRMAb-SGSH fusion protein from plasma are the liver and spleen (Table 6). Cerebral cortex uptake of the HIRMAb-SGSH fusion protein was 0.81 ± 0.07% ID / 100 grams brain (Table 6). Table 6. Tissue uptake of HIRMAb-SGSH fusion protein in rhesus monkeys [Table 6] Data are the mean ± standard deviation of triplicate samples.

[0241] In summary, this study in primates shows that the HIRMAb-SGSH fusion protein is rapidly cleared from plasma after intravenous administration due to its uptake by peripheral tissues. However, unlike SGSH alone, the HIRMAb-SGSH fusion protein rapidly penetrates the BBB (Tables 5 and 6). Conversely, SGSH alone does not cross the BBB (Urayama, A.; Grubb, JH; Sly, WS; Banks, WA Mannose 6-phosphate receptor-mediated transport of sulfamidase across the blood-brain barrier in the newborn mouse. Mol Ther. 2008, 16, 1261-1266).

[0242] Example 9. Receptor-Mediated Transport of SGSH into the Human Brain Sanfilippo type A or MPS-IIIA is a lysosomal storage disorder caused by a deficiency in the gene encoding the lysosomal enzyme sulfamidase (SGSH). The lack of SGSH leads to the accumulation of certain GAGs, such as heparan sulfate, in cells. Accumulation of heparan sulfate in the brain leads to the clinical symptoms of MPS-IIIA, including severe behavioral disorders, speech impairment typically by age 7 years, gait problems typically leading to wheelchair confinement by age 12 years, and death at a mean age of 18 years (Valstar et al. (2010): Mucopolysaccharidosis Type IIIA: clinical spectrum and genotype-phenotype correlations. Ann. Neurol. 68:876-887).

[0243] The nucleotide sequence of SGSH mRNA and the amino acid sequence of human SGSH protein are known (Scott et al. (1995), Cloning of the sulphamidase gene and identification of mutations in Sanfilippo A syndrome, Nature Genetics, 11:465-467). The sequences allow the production of recombinant SGSH for enzyme replacement therapy (ERT) of MPS-IIIA. SGSH produced by Chinese hamster ovary (CHO) cells has a specific activity of 15 units / μg enzyme (Urayama et al. (2008), Mannose 6-phosphate receptor-mediated transport of sulfamidase across the blood-brain barrier in the newborn mouse, Mol. Ther. 16: 1261-1266). SGSH specific activity was measured by the same two-step fluorometric enzyme assay described above (Karpova et al. (1996) A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type A (MPS IIIA), J. Inher. Metab. Dis. 19: 278-285). The problem with ERT of MPS-IIIA using recombinant SGSH is that, like other macromolecular drugs, SGSH does not cross the BBB and is therefore not an effective treatment for MPS-IIIA when administered intravenously (Hemsley et al. (2009) Examination of intravenous and intra-CSF protein delivery for treatment of neurological disease, Eur. J. Neurosci. 29: 1197-1214). The lack of transport of SGSH across the BBB makes it difficult to increase SGSH in the brain by systemic injection of large amounts of the enzyme. Therefore, intravenous ERT with recombinant SGSH is not expected to have beneficial effects on the brain in patients with MPS-IIIA.Direct intracerebroventricular (ICV) infusion of SGSH into the brain of dogs with MPS-IIIA resulted in the injection of the enzyme into the ventricles with the aim of bypassing the BBB (Jolly et al. (2011), Intracisternal enzyme replacement therapy in lysosomal storage diseases: routes of absorption into brain, Neuropathol Appl. Neurobiol. 37: 414-422). Drug delivery to the brain via the ICV route is well known to distribute the drug exclusively to the ependymal or meningeal surface of the brain, and this is exactly what was observed in dogs with MPS-IIIA after ICV administration of SGSH.

[0244] ICV enzyme administration is an invasive method that requires chronic implantation of catheters in the brain. The preferred approach to deliver SGSH to the brain of MPS-IIIA patients is to use intravenous infusion of a form of SGSH redesigned to cross the BBB via receptor-mediated transport (RMT). The HIRMAb-SGSH fusion protein retains high affinity binding to the human insulin receptor, which allows SGSH to penetrate the BBB and enter the brain from the blood via RMT on the endogenous BBB insulin receptor. In rhesus monkeys, the brain uptake of the HIRMAb-SGSH fusion protein is 1% of the injected dose (ID) per 100 grams of brain (Table 6). Given this level of brain uptake of the fusion protein, the SGSH enzyme activity in the brain after intravenous administration of the HIRMAb-SGSH fusion protein can be calculated and compared to normal endogenous SGSH enzyme activity in the brain. The SGSH specific activity of the SGSH fusion protein is 5000 units / mg fusion protein (Example 4), and assuming a 50 kg human receiving 5 mg / kg of IgG-SGSH fusion protein intravenously, the injected dose is equivalent to 250 mg, or 1,250,000 units of fusion protein. In rhesus monkeys, the brain uptake of the HIRMAb-SGSH fusion protein is ~1% ID / 100 grams brain (Table 6), with a rhesus monkey brain weighing 100 grams. Thus, the brain uptake of the HIRMAb-SGSH fusion protein is approximately 1% of the intravenously injected dose, which is equivalent to 12,500 units / brain in a 50 kg human receiving 3 mg / kg of fusion protein. This level of brain SGSH enzyme activity is equivalent to 12.5 units / gram brain, since a human brain weighs approximately 1,000 grams, and is equivalent to a brain SGSH enzyme activity of 0.125 units / mg brain protein, since 1 gram of brain is equivalent to approximately 100 mg of protein.SGSH enzyme activity in normal brain was 0.12 units / mg protein (Tomatsu, S.; Vogler, C.; Montano, A. M.; Gutierrez, M.; Oikawa, H.; Dung, V. C.; Orii, T.; Noguchi, A.; Sly, W. S. Murine model (Galns. tm(C76S)slu ) of MPS IVA with missense mutation at the active site cysteine ​​conserved among sulfatase proteins. Mol. Genet. Metab. 2007, 91, 251-258) to 1.4 units / mg protein (Fraldi et al. (2007) Functional correction of CNS lesions in an MPS-IIIA mouse model by intracerebral AAV-mediated delivery of sulfamidase and SUMF1 genes, Human Mol. Genet 16: 2693-2702). Thus, administration of a dose of 3 mg / kg of HIRMAb-SGSH fusion protein results in levels of SGSH enzyme activity in the brain that are 10-100% of the normal endogenous SGSH enzyme activity. In patients with lysosomal storage disorders, enzyme replacement therapy results in cellular enzyme activity at only 1-2% of that of a normal individual, and they do not develop signs and symptoms of disease (J. Muenzer and A. Fisher, Advances in the treatment of mucopolysaccharidosis type I, N. Engl J Med, 350: 1932-1934, 2004). These considerations indicate that intravenous injection of HIRMAb-SGSH fusion protein at systemic doses of approximately 3 mg / kg, or in the range of 1-10 mg / kg of HIRMAb-SGSH fusion protein, can replenish clinically significant SGSH enzyme in the human brain.

Claims

1. (a) a fusion protein comprising an immunoglobulin heavy chain and the amino acid sequence of an enzyme deficient in mucopolysaccharidosis type III (MPS-III), and (b) a fusion antibody comprising an immunoglobulin light chain and capable of crossing the blood-brain barrier (BBB).

2. The fusion antibody of claim 1, wherein the enzyme deficient in MPS-III is N-sulfoglucosamine sulfohydrolase (SGSH), α-N-acetylglucosaminidase (NAGLU), heparin-α-glucosaminide N-acetyltransferase (HGSNAT), or N-acetylglucosamine-6-sulfatase (GNS).

3. The fusion antibody of claim 1, wherein the amino acid sequence of the enzyme is covalently linked to the carboxy terminus of the amino acid sequence of the immunoglobulin heavy chain.

4. The fusion antibody of claim 1, wherein the enzyme and the immunoglobulin each retain at least 20% of their activity compared to their activity alone.

5. The fusion antibody of claim 1, which crosses the BBB via an endogenous BBB receptor selected from the group consisting of insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and IGF receptor.

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