Anti-transferrin receptor fusion proteins and methods of use thereof

Abstract

Certain embodiments provide fusion proteins comprising an iduronate 2-sulfatase (IDS) amino acid sequence, an amino acid sequence of an IDS variant, or a catalytically active fragment thereof, and an anti-transferrin receptor (TfR) antibody described herein, and methods of use thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 148,543, filed February 11, 2021. The entire contents of the above-referenced applications are incorporated herein by reference. [Background technology]

[0002] Hunter syndrome or MPS II is a rare X-linked recessive disorder caused by IDS gene mutations. Insufficient iduronate 2-sulfatase (IDS) activity leads to accumulation of glycosaminoglycans (GAGs) heparan sulfate (HS) and dermatan sulfate (DS) and lysosomal dysfunction in multiple organs and tissues. Approximately two-thirds of patients exhibit a neuropathic phenotype (nMPS II). Recombinant forms of IDS have been approved to treat Hunter syndrome, but have little effect on the brain due to the difficulty of delivering the recombinant enzyme across the blood-brain barrier (BBB). One possible approach involves a technique that utilizes anti-transferrin receptor antibodies to facilitate the passage of large molecules such as IDS across the BBB. Summary of the Invention [Means for solving the problem]

[0003] Thus, a particular embodiment is a fusion protein comprising: (a) an iduronate 2-sulfatase (IDS) amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof; and (b) comprising an anti-transferrin receptor (TfR) antibody (e.g., an anti-human TfR antibody) described herein; The fusion protein is provided wherein the fusion protein comprises at least about 6 mol / mol sialic acid:fusion protein and / or comprises about 1.5 to about 2.5 mol / mol mannose 6-phosphate (M6P):fusion protein. In certain embodiments, the fusion protein comprises about 15 to 16 mol / mol sialic acid:fusion protein (i.e., about 15 to about 16 mol / mol sialic acid:fusion protein) and / or comprises about 1.5 to about 2.5 mol / mol M6P:fusion protein (i.e., about 1.5 to about 2.5 mol / mol M6P:fusion protein). In certain embodiments, the fusion protein comprises about 15 to 16 mol / mol sialic acid:fusion protein (i.e., about 15 to about 16 mol / mol sialic acid:fusion protein) and / or comprises about 1.5 to about 2.3 mol / mol M6P:fusion protein (i.e., about 1.5 to about 2.3 mol / mol M6P:fusion protein).

[0004] In certain embodiments, the fusion protein comprises: (a) an iduronate 2-sulfatase (IDS) amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof; and (b) an anti-human transferrin receptor antibody, i) heavy chain complementarity determining region 1 (CDR-H1) comprising GYSFTNY (SEQ ID NO: 13), or a sequence having one or two substitutions compared to SEQ ID NO: 13; ii) a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15), or a sequence having one or two substitutions compared to SEQ ID NO: 15; iii) a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16), or a sequence having one or two substitutions compared to SEQ ID NO: 16; iv) a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7), or a sequence having one or two substitutions compared to SEQ ID NO: 7; v) a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8), or a sequence having one or two substitutions compared to SEQ ID NO: 8; and vi) an antibody comprising a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9), or a sequence having one or two substitutions compared to SEQ ID NO: 9; The fusion protein contains at least about 6 mol / mol sialic acid:fusion protein (eg, about 15-16 mol / mol sialic acid:fusion protein (ie, about 15-about 16 mol / mol sialic acid:fusion protein)).

[0005] In certain other embodiments, the fusion protein comprises: (a) an iduronate 2-sulfatase (IDS) amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof; and (b) an anti-human transferrin receptor antibody, i) heavy chain CDR-H1 comprising GYSFTNY (SEQ ID NO: 13), or a sequence having one or two substitutions compared to SEQ ID NO: 13; ii) a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15), or a sequence having one or two substitutions compared to SEQ ID NO: 15; iii) a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16), or a sequence having one or two substitutions compared to SEQ ID NO: 16; iv) a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7), or a sequence having one or two substitutions compared to SEQ ID NO: 7; v) a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8), or a sequence having one or two substitutions compared to SEQ ID NO: 8; and vi) an antibody comprising a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9), or a sequence having one or two substitutions compared to SEQ ID NO: 9; The fusion protein comprises about 1.5 to about 2.5 mol / mol M6P:fusion protein. In a specific embodiment, the fusion protein comprises about 1.5 to about 2.3 mol / mol M6P:fusion protein (i.e., about 1.5 to about 2.3 mol / mol M6P:fusion protein).

[0006] Certain embodiments also provide a pharmaceutical composition comprising a fusion protein described herein and a pharma- ceutically acceptable excipient.

[0007] Certain embodiments provide host cells comprising the fusion proteins described herein.

[0008] Certain embodiments provide methods of producing a fusion protein described herein, comprising expressing in a host cell under conditions suitable for producing the fusion protein one or more polynucleotides operable to express 1) a light chain described herein and 2) a heavy chain linked to an IDS amino acid sequence described herein.

[0009] Certain embodiments provide fusion proteins produced by the methods described herein.

[0010] Certain embodiments provide a method of reducing the level of one or more GAG ​​species in a subject in need thereof, comprising administering to the subject a fusion protein described herein or a pharmaceutical composition described herein (e.g., administering a therapeutically effective amount as described herein).

[0011] Certain embodiments provide a method of treating Hunter Syndrome in a subject in need thereof, comprising administering to the subject a fusion protein described herein or a pharmaceutical composition described herein (e.g., administering a therapeutically effective amount described herein).

[0012] Certain embodiments also provide a method of reducing the level of one or more GAG ​​species in a subject in need thereof, comprising administering to the subject a fusion protein at a dose of about 1 mg / kg, 3 mg / kg, or 10 mg / kg, wherein the fusion protein comprises: (a) an iduronate 2-sulfatase (IDS) amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof; and (b) an anti-human transferrin receptor antibody, i) heavy chain complementarity determining region 1 (CDR-H1) comprising GYSFTNY (SEQ ID NO: 13), or a sequence having one or two substitutions compared to SEQ ID NO: 13; ii) a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15), or a sequence having one or two substitutions compared to SEQ ID NO: 15; iii) a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16), or a sequence having one or two substitutions compared to SEQ ID NO: 16; iv) a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7), or a sequence having one or two substitutions compared to SEQ ID NO: 7; v) a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8), or a sequence having one or two substitutions compared to SEQ ID NO: 8; and vi) Includes antibodies comprising a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9), or a sequence having one or two substitutions compared to SEQ ID NO: 9.

[0013] Certain embodiments provide a method of treating Hunter Syndrome in a subject in need thereof, comprising administering to the subject a fusion protein at a dose of about 1 mg / kg, 3 mg / kg, or 10 mg / kg, wherein the fusion protein comprises: (a) an iduronate 2-sulfatase (IDS) amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof; and (b) an anti-human transferrin receptor antibody, i) heavy chain complementarity determining region 1 (CDR-H1) comprising GYSFTNY (SEQ ID NO: 13), or a sequence having one or two substitutions compared to SEQ ID NO: 13; ii) a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15), or a sequence having one or two substitutions compared to SEQ ID NO: 15; iii) a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16), or a sequence having one or two substitutions compared to SEQ ID NO: 16; iv) a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7), or a sequence having one or two substitutions compared to SEQ ID NO: 7; v) a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8), or a sequence having one or two substitutions compared to SEQ ID NO: 8; and vi) Includes antibodies comprising a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9), or a sequence having one or two substitutions compared to SEQ ID NO: 9.

[0014] Certain embodiments also provide processes and intermediates disclosed herein that are useful for preparing fusion proteins as described herein. [Brief description of the drawings]

[0015] [Figure 1] Schematic diagram of an IgG:IDS fusion protein comprising an anti-transferrin receptor antibody, with each heavy chain fused to an IDS enzyme. [Diagram 2] Figure 1 shows the binding of IgG:IDS to the mannose-6-phosphate receptor (M6PR). ELISA binding assays were performed using various concentrations of IgG:IDS to generate representative binding curves. [Diagram 3] Figure 1 shows the cellular potency of IgG:IDS in MPSII fibroblasts. A representative cellular potency curve (averaged across cell lines) of IgG:IDS was generated using an S35-sulfate accumulation assay. [Figure 4] Pharmacokinetic (PK) profiles of IgG:IDS in serum from TfRmu / huKI mice (n=3 / group) receiving a single intravenous dose of 1, 3, or 10 mg / kg IgG:IDS. Terminal samples were collected 0.5, 4, 8, and 24 hours after dosing. [Diagram 5] Figure 1 shows the PK profile of IgG:IDS in the brain of TfRmu / huKI mice (n=3 / group) receiving a single intravenous dose of 1, 3, or 10 mg / kg IgG:IDS. Terminal samples were collected 0.5, 4, 8, and 24 hours after dosing. [Figure 6]Figure 1 shows the PK profile of IgG:IDS in the liver of TfRmu / huKI mice (n=3 / group) receiving a single intravenous dose of 1, 3, or 10 mg / kg IgG:IDS. Terminal samples were collected 0.5, 4, 8, and 24 hours after dosing. [Figure 7A] Figure 1 shows total GAG levels in liver from Ids KO, TfRmu / huKI mice (n=5 / group) administered a single intravenous dose of 1, 3 or 10 mg / kg IgG:IDS. GAG levels were measured 7 days after administration and compared to vehicle-treated and TfRmu / huKI mice. n=5 / group. Graphs show mean ± SEM and p-value: One-way ANOVA with Tukey's multiple comparison test. ****p<0.0001. [Figure 7B] Figure 1 shows heparan sulfate (HS) levels in liver from Ids KO, TfRmu / huKI mice (n=5 / group) administered a single intravenous dose of 1, 3 or 10 mg / kg IgG:IDS. GAG levels were measured 7 days after administration and compared to vehicle-treated and TfRmu / huKI mice. n=5 / group. Graphs show mean ± SEM and p-value: One-way ANOVA with Tukey's multiple comparison test. ****p<0.0001. [Figure 7C] Dermatan sulfate (DS) levels in liver from Ids KO, TfRmu / huKI mice (n=5 / group) administered a single intravenous dose of 1, 3 or 10 mg / kg IgG:IDS. GAG levels were measured 7 days after administration and compared to vehicle-treated and TfRmu / huKI mice. n=5 / group. Graphs show mean±SEM and p-value: One-way ANOVA with Tukey's multiple comparison test. ****p<0.0001. [Figure 8A]Figure 1 shows total GAG levels in CSF from Ids KO, TfRmu / huKI mice (n=5 / group) administered a single intravenous dose of 1, 3 or 10 mg / kg IgG:IDS. GAG levels were measured 7 days after administration and compared to vehicle-treated and TfRmu / huKI mice. n=5 / group. Graphs show mean ± SEM and p-values: one-way ANOVA with Tukey's multiple comparison test. **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 8B] Figure 1 shows heparan sulfate (HS) levels in CSF from Ids KO, TfRmu / huKI mice (n=5 / group) administered a single intravenous dose of 1, 3 or 10 mg / kg IgG:IDS. GAG levels were measured 7 days after administration and compared to vehicle-treated and TfRmu / huKI mice. n=5 / group. Graphs show mean ± SEM and p-values: One-way ANOVA with Tukey's multiple comparison test. **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 8C] Dermatan sulfate (DS) levels in CSF from Ids KO, TfRmu / huKI mice (n=5 / group) administered a single intravenous dose of 1, 3 or 10 mg / kg IgG:IDS. GAG levels were measured 7 days after administration and compared to vehicle-treated and TfRmu / huKI mice. n=5 / group. Graphs show mean ± SEM and p-values: One-way ANOVA with Tukey's multiple comparison test. **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 9A] Figure 1 shows total GAG levels in brains from Ids KO, TfRmu / huKI mice (n=5 / group) administered a single intravenous dose of 1, 3 or 10 mg / kg IgG:IDS. GAG levels were measured 7 days after administration and compared to vehicle-treated and TfRmu / huKI mice (n=5 / group). Graphs show mean ± SEM and p-values: one-way ANOVA with Tukey's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 9B]Figure 1 shows heparan sulfate (HS) levels in brains from Ids KO, TfRmu / huKI mice (n=5 / group) administered a single intravenous dose of 1, 3 or 10 mg / kg IgG:IDS. GAG levels were measured 7 days after administration and compared to vehicle-treated and TfRmu / huKI mice (n=5 / group). Graphs show mean ± SEM and p-values: one-way ANOVA with Tukey's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 9C] Dermatan sulfate (DS) levels in brains from Ids KO, TfRmu / huKI mice (n=5 / group) administered a single intravenous dose of 1, 3, or 10 mg / kg IgG:IDS. GAG levels were measured 7 days after administration and compared to vehicle-treated and TfRmu / huKI mice (n=5 / group). Graphs show mean ± SEM and p-values: one-way ANOVA with Tukey's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hunter syndrome, specifically the neurocognitive phenotype, remains a significant unmet medical need. Described herein is the evaluation of a specific enzyme replacement therapy called IgG:IDS, which is a fusion protein containing an IDS enzyme amino acid sequence that has the ability to cross the BBB and treat specific CNS symptoms of Hunter syndrome. As described in the Examples, production of IgG:IDS results in specific post-translational modifications (PTMs) of the fusion protein, including sialic acid and mannose 6-phosphate (M6P) modifications. These PTMs can affect peripheral (e.g., serum) exposure to the fusion protein and facilitate proper transport of the fusion protein to specific cells within the CNS, as well as intracellular trafficking. Additionally, therapeutic response to IgG:IDS can be assessed by measuring the levels of glycosaminoglycan (GAG) species present in a physiological sample from a subject. These levels can be used to identify and evaluate a therapeutically effective dose of IgG:IDS.

[0017] Fusion proteins Thus, a particular embodiment is a fusion protein comprising: (a) an iduronate 2-sulfatase (IDS) amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof; and (b) Provided are fusion proteins comprising an anti-transferrin receptor (TfR) antibody (e.g., an anti-human TfR antibody) as described herein. As described herein, such fusion proteins can include specific PTMs, including specific molecular weights or amount ranges of specific PTMs, such as sialic acid and / or M6P (e.g., amounts or ranges described herein).

[0018] In certain embodiments, the anti-TfR antibody specifically binds to TfR. In some embodiments, the antibody specifically binds to human TfR shows cross-reactivity with one or more proteins of another species that correspond to human TfR (i.e., TfR orthologues). For example, in some embodiments, the anti-TfR antibody specifically binds to an epitope on TfR that is conserved between species (e.g., structurally conserved between species), for example, conserved between non-human primates and human species (e.g., structurally conserved between non-human primates and human species). In some embodiments, the anti-TfR receptor antibody can exclusively bind to human TfR.

[0019] In some embodiments, the anti-TfR antibody is an anti-human TfR antibody. In some embodiments, the anti-human TfR antibody comprises one or more complementarity determining region (CDR) sequences described herein. In certain embodiments, the anti-human TfR antibody comprises (1) a heavy chain variable region sequence, a heavy chain sequence, or a fusion polypeptide comprising a heavy chain and an IDS amino acid sequence, and / or (2) a light chain variable region sequence or a light chain sequence described herein. For example, Table 6 shows the sequence numbers of each amino acid sequence contained in CDR1 to CDR3 of the light chain variable region and CDR1 to CDR3 of the heavy chain variable region of an anti-hTfR antibody contained in IgG:IDS (see Example 1). However, Table 6 shows these amino acid sequences only as examples, and does not limit the amino acid sequence of each CDR to the amino acid sequence of Table 6. Rather, they may be a region of an amino acid sequence containing any of these listed sequences, or any amino acid sequence consisting of three or more consecutive amino acids containing a part of these sequences.

[0020] Thus, in certain embodiments, the anti-human TfR antibody is: a) a heavy chain complementarity determining region 1 (CDR-H1) comprising GYSFX1NY (SEQ ID NO: 12), or an amino acid sequence having one or two substitutions compared to SEQ ID NO: 12, wherein X1 is M or T; b) a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15), or an amino acid sequence having one or two substitutions compared to SEQ ID NO: 15; c) a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16), or an amino acid sequence having one or two substitutions compared to SEQ ID NO: 16; d) a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7), or an amino acid sequence having one or two substitutions compared to SEQ ID NO: 7; e) a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8), or an amino acid sequence having one or two substitutions compared to SEQ ID NO: 8; and f) Includes antibodies comprising a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9), or an amino acid sequence having one or two substitutions compared to SEQ ID NO: 9.

[0021] In some embodiments, the CDR has one amino acid substitution relative to the reference sequence. In some embodiments, the CDR has two amino acid substitutions relative to the reference sequence. In some embodiments, the amino acid substitution(s) is / are a conservative substitution.

[0022] Thus, in some embodiments, the anti-human TfR antibody is: a) a heavy chain CDR-H1 comprising GYSFX1NY (SEQ ID NO: 12), or an amino acid sequence having one substitution relative to SEQ ID NO: 12, wherein X1 is M or T; b) a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15), or an amino acid sequence having one substitution compared to SEQ ID NO: 15; c) a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16), or an amino acid sequence having one substitution compared to SEQ ID NO: 16; d) a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7), or an amino acid sequence having one substitution compared to SEQ ID NO: 7; e) a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8), or an amino acid sequence having one substitution compared to SEQ ID NO: 8; and f) A light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9), or an amino acid sequence having one substitution compared to SEQ ID NO: 9.

[0023] In some embodiments, the anti-human TfR antibody comprises a heavy chain CDR-H1 comprising GYSFX1NY (SEQ ID NO: 12), where X1 is M or T; a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15); a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16); a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7); a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8); and a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9).

[0024] In some embodiments, the anti-human TfR antibody comprises: a heavy chain CDR-H1 consisting of GYSFX1NY (SEQ ID NO: 12), where X1 is M or T; a heavy chain CDR-H2 consisting of YPGGDY (SEQ ID NO: 15); a heavy chain CDR-H3 consisting of SGNYDEVAY (SEQ ID NO: 16); a light chain CDR-L1 consisting of RSSQSLVHSNGNTYLH (SEQ ID NO: 7); a light chain CDR-L2 consisting of KVSNRFS (SEQ ID NO: 8); and a light chain CDR-L3 consisting of SQSTHVPWT (SEQ ID NO: 9).

[0025] In some embodiments, the anti-human TfR antibody comprises: a heavy chain CDR-H1 comprising GYSFTNY (SEQ ID NO: 13), a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15), a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16), a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7), a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8), and a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9).

[0026] In some embodiments, the anti-human TfR antibody comprises: a heavy chain CDR-H1 consisting of GYSFTNY (SEQ ID NO: 13), a heavy chain CDR-H2 consisting of YPGGDY (SEQ ID NO: 15), a heavy chain CDR-H3 consisting of SGNYDEVAY (SEQ ID NO: 16), a light chain CDR-L1 consisting of RSSQSLVHSNGNTYLH (SEQ ID NO: 7), a light chain CDR-L2 consisting of KVSNRFS (SEQ ID NO: 8), and a light chain CDR-L3 consisting of SQSTHVPWT (SEQ ID NO: 9).

[0027] In some embodiments, the anti-human TfR antibody comprises: a heavy chain CDR-H1 comprising GYSFMNY (SEQ ID NO: 14), a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15), a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16), a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7), a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8), and a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9).

[0028] In some embodiments, the anti-human TfR antibody comprises: a heavy chain CDR-H1 consisting of GYSFMNY (SEQ ID NO: 14), a heavy chain CDR-H2 consisting of YPGGDY (SEQ ID NO: 15), a heavy chain CDR-H3 consisting of SGNYDEVAY (SEQ ID NO: 16), a light chain CDR-L1 consisting of RSSQSLVHSNGNTYLH (SEQ ID NO: 7), a light chain CDR-L2 consisting of KVSNRFS (SEQ ID NO: 8), and a light chain CDR-L3 consisting of SQSTHVPWT (SEQ ID NO: 9).

[0029] In some embodiments, the anti-human TfR antibody comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity) to DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGQGTKVEIK (SEQ ID NO: 6).

[0030] In some embodiments, a light chain variable region having at least 90% sequence identity to SEQ ID NO:6 contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions (e.g., conservative substitutions) or insertions but retains the ability to specifically bind to TfR. In some embodiments, the light chain variable region contains 1, 2 or 3 substitutions (e.g., conservative substitutions) in SEQ ID NO:6.

[0031] In some embodiments, the anti-human TfR antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0032] In some embodiments, the anti-human TfR antibody comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO:6.

[0033] In some embodiments, the anti-human TfR antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity) to EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWLGWVRQMPGKGLEWMGDIYPGGDYPTYSEKFKVQVTISADKSISTAYLQWSSLKASDTAMYYCARSGNYDEVAYWGQGTLVTVSS (SEQ ID NO: 11).

[0034] In some embodiments, a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:11 contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions (e.g., conservative substitutions) or insertions but retains the ability to specifically bind to TfR. In some embodiments, the heavy chain variable region contains 1, 2 or 3 substitutions (e.g., conservative substitutions) in SEQ ID NO:11.

[0035] In some embodiments, the anti-human TfR antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11.

[0036] In some embodiments, the anti-human TfR antibody comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:11.

[0037] In some embodiments, the anti-human TfR antibody comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO:6, and further comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO:11.

[0038] In some embodiments, the anti-human TfR antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:6, and further comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11.

[0039] In some embodiments, the anti-human TfR antibody comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO:6, and further comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:11.

[0040] As described herein, the anti-human TfR antibody can be a substantially full-length antibody, such as an IgG antibody, such as an IgG1 antibody, or other antibody class or isotype as defined herein (e.g., an IgG2, IgG3, or IgG4 antibody). Thus, in certain embodiments, the antibody comprises at least one heavy chain constant region and / or at least one light chain constant region (e.g., kappa or lambda).

[0041] In certain embodiments, the anti-human TfR antibody comprises a light chain constant region. In certain embodiments, the anti-human TfR antibody comprises a light chain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:5).

[0042] In some embodiments, a light chain having at least 90% sequence identity to SEQ ID NO:5 contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions) or insertions but retains the ability to specifically bind to TfR. In some embodiments, the light chain contains one, two or three substitutions (e.g., conservative substitutions) in SEQ ID NO:5.

[0043] In some embodiments, the anti-human TfR antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:5.

[0044] In some embodiments, the anti-human TfR antibody comprises a light chain consisting of the amino acid sequence of SEQ ID NO:5.

[0045] In certain embodiments, the anti-human TfR antibody is an IgG1 antibody that comprises a constant region(s) from IgG1. For example, in certain embodiments, the anti-human TfR antibody comprises an IgG1 Fc region (see, e.g., SEQ ID NO: 24). Thus, in some embodiments, the anti-human TfR antibody comprises a heavy chain that comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the following:EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWLGWVRQMPGKGLEWMGDIYPGGDYPTYSEKFKVQVTISADKSISTAYLQWSSLKASDTAMYYCARSGNYDEVAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 10).

[0046] In some embodiments, a heavy chain having at least 90% sequence identity to SEQ ID NO:10 contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions) or insertions but retains the ability to specifically bind to TfR. In some embodiments, the heavy chain contains one, two or three substitutions (e.g., conservative substitutions) in SEQ ID NO:10.

[0047] In some embodiments, the anti-human TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:10.

[0048] In some embodiments, the anti-human TfR antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO:10.

[0049] In some embodiments, the anti-human TfR antibody comprises a light chain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO:5, and further comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO:10.

[0050] In some embodiments, the anti-human TfR antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:5, and further comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:10.

[0051] In some embodiments, the anti-human TfR antibody comprises a light chain consisting of the amino acid sequence of SEQ ID NO:5 and further comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO:10.

[0052] In certain embodiments, an iduronate 2-sulfatase (IDS) amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof is linked directly or via a linker to the C-terminus of an antibody heavy chain (i.e., generating a heavy chain fusion polypeptide).

[0053] In some embodiments, the IDS amino acid sequence (i.e., the IDS amino acid sequence, the IDS variant amino acid sequence, or a catalytically active fragment thereof) is linked directly (e.g., by a peptide bond) to the C-terminus of the antibody heavy chain. In certain other embodiments, the IDS amino acid sequence is linked to the C-terminus of the antibody heavy chain by a linker (e.g., a peptide linker). The peptide linker may be configured to allow rotation of the IDS enzyme relative to the anti-hTfR antibody to which it is bound and / or is resistant to digestion by proteases. The peptide linker may contain natural amino acids, unnatural amino acids, or combinations thereof. In some embodiments, the peptide linker may be a flexible linker containing amino acids such as, for example, Gly, Asn, Ser, Thr, Ala, etc. Such linkers are designed using known parameters and may be of any length and contain any number of repeat units of any length (e.g., repeat units of Gly and Ser residues). In certain embodiments, the peptide linker is 1 to 50 amino acids in length, 1 to 40 amino acids in length, 1 to 30 amino acids in length, 1 to 20 amino acids in length, 1 to 15 amino acids in length, 1 to 10 amino acids in length, or 1 to 5 amino acids in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length). In certain embodiments, the peptide linker is composed of glycine or serine, for example, composed of a single amino acid, either glycine or serine, and is a sequence comprising the amino acid sequence Gly-Ser (SEQ ID NO: 23), the amino acid sequence Gly-Gly-Ser (SEQ ID NO: 22), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 19), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 20), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 21), or 1 to 10 or 2 to 5 (e.g., 2, 3, 4, or 5) of any of these amino acid sequences linked consecutively.In some embodiments, the peptide linker may include a protease cleavage site, cleavable by enzymes present, for example, in the central nervous system.

[0054] In certain embodiments, the IDS amino acid sequence is linked to the C-terminus of the heavy chain by a peptide linker comprising an amino acid sequence selected from the group consisting of one glycine residue, one serine residue, GGGGS (SEQ ID NO: 19), GGGGGS (SEQ ID NO: 20), SGGGG (SEQ ID NO: 21), GGS (SEQ ID NO: 22), GS (SEQ ID NO: 23), and an amino acid sequence consisting of 2 to 10 consecutively linked amino acids of any of the aforementioned sequences. In certain embodiments, the peptide linker consists of an amino acid sequence selected from the group consisting of a single glycine residue, a single serine residue, GGGGS (SEQ ID NO: 19), GGGGGS (SEQ ID NO: 20), SGGGG (SEQ ID NO: 21), GGS (SEQ ID NO: 22), GS (SEQ ID NO: 23), and an amino acid sequence consisting of 2 to 10 consecutively linked amino acids of any of the aforementioned sequences.

[0055] In some embodiments, the IDS amino acid sequence is linked to the C-terminus of the heavy chain by a Gly-Ser linker (SEQ ID NO:23).

[0056] Thus, in certain embodiments, the fusion protein described herein comprises an anti-human TfR antibody and an IDS amino acid sequence linked to the C-terminus of the heavy chain by a peptide linker (i.e., a heavy chain fusion polypeptide) comprising, in N'- to C'-terminus order: a heavy chain (SEQ ID NO: 10), a Gly-Ser linker (SEQ ID NO: 23), and an IDS amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof. In certain embodiments, the fusion protein comprises an anti-human TfR antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain linked to the IDS amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof by a peptide linker comprising, in N'- to C'-terminus order: SEQ ID NO: 10, a Gly-Ser linker (SEQ ID NO: 23), and an IDS amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof.

[0057] In certain embodiments, the IDS amino acid sequence is a wild-type IDS amino acid sequence or a catalytically active variant or fragment of a wild-type IDS, e.g., wild-type human IDS. In some embodiments, a catalytically active variant or fragment of an IDS enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the activity of the wild-type IDS enzyme.

[0058] In some embodiments, the IDS enzyme, or catalytically active variant or fragment thereof, present in the fusion protein described herein retains at least 25% of its activity compared to its activity when not bound to an anti-TfR antibody. In some embodiments, the IDS enzyme, or catalytically active variant or fragment thereof, retains at least 10%, or at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of its activity compared to its activity when not bound to an anti-TfR antibody. In some embodiments, the IDS enzyme, or catalytically active variant or fragment thereof, retains at least 80%, 85%, 90%, or 95% of its activity compared to its activity when not bound to an anti-TfR antibody. In some embodiments, fusion to an anti-TfR antibody does not reduce the activity of the IDS enzyme, or catalytically active variant or fragment thereof. In some embodiments, fusion to an anti-TfR antibody does not reduce the activity of the IDS enzyme.

[0059] In certain embodiments, the IDS amino acid sequence comprises an amino acid sequence having at least about 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 1, 2, 3 or 4. In certain embodiments, the IDS amino acid sequence comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, 2, 3 or 4. In certain embodiments, the IDS amino acid sequence comprises SEQ ID NO: 1, 2, 3 or 4. In certain embodiments, the IDS amino acid sequence consists of SEQ ID NO: 1, 2, 3 or 4.

[0060] In certain embodiments, the IDS amino acid sequence comprises SEQ ID NO: 3 or 4. In certain embodiments, the IDS amino acid sequence comprises SEQ ID NO: 3. In certain embodiments, the IDS amino acid sequence consists of SEQ ID NO: 3. In certain embodiments, the IDS amino acid sequence comprises SEQ ID NO: 4. In certain embodiments, the IDS amino acid sequence consists of SEQ ID NO: 4.

[0061] Thus, in certain embodiments, the fusion protein comprises an anti-human TfR antibody comprising a heavy chain linked to an IDS amino acid sequence (i.e., a heavy chain fusion polypeptide) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17 or 18. In certain embodiments, the heavy chain linked to the IDS amino acid sequence (i.e., a heavy chain fusion polypeptide) comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 17 or 18. In certain embodiments, the heavy chain linked to the IDS amino acid sequence comprises SEQ ID NO: 17 or 18. In certain embodiments, the heavy chain linked to the IDS amino acid sequence consists of SEQ ID NO: 17 or 18.

[0062] In certain embodiments, the fusion protein comprises an anti-human TfR antibody comprising a heavy chain linked to an IDS amino acid sequence (i.e., a heavy chain fusion polypeptide) that comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the heavy chain linked to the IDS amino acid sequence comprises SEQ ID NO: 17. In certain embodiments, the heavy chain linked to the IDS amino acid sequence consists of SEQ ID NO: 17.

[0063] In certain embodiments, the anti-human TfR antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:5 and a heavy chain linked to an IDS amino acid sequence comprising the amino acid sequence of SEQ ID NO:17. In certain embodiments, the anti-human TfR antibody comprises a light chain consisting of SEQ ID NO:5 and a heavy chain linked to an IDS amino acid sequence consisting of SEQ ID NO:17.

[0064] In certain embodiments, the fusion protein comprises an anti-human TfR antibody comprising a heavy chain linked to an IDS amino acid sequence (i.e., a heavy chain fusion polypeptide) that comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In certain embodiments, the heavy chain linked to the IDS amino acid sequence comprises SEQ ID NO: 18. In certain embodiments, the heavy chain linked to the IDS amino acid sequence consists of SEQ ID NO: 18.

[0065] In certain embodiments, the anti-human TfR antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:5 and a heavy chain linked to an IDS amino acid sequence comprising the amino acid sequence of SEQ ID NO:18. In certain embodiments, the anti-human TfR antibody comprises a light chain consisting of SEQ ID NO:5 and a heavy chain linked to an IDS amino acid sequence consisting of SEQ ID NO:18.

[0066] Certain embodiments provide a fusion protein as described herein.

[0067] In a further aspect, a fusion protein according to any of the above embodiments, or an anti-human TfR antibody contained therein, may incorporate any of the features described below, either alone or in combination.

[0068] Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of a fusion protein / antibody described herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0069] In certain embodiments, antibody variants possess some, but not all, effector functions, making them desirable candidates for applications in which antibody half-life in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Primary cells for mediating ADCC include NK cells, which express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3, page 464, of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA, and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0070] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0071] In a specific embodiment, Pro329 of the wild-type human Fc region is substituted with glycine, arginine, serine, or an amino acid residue large enough to disrupt the proline sandwich within the Fc / Fcγ receptor interface formed between proline 329 of Fc and tryptophan residues Trp87 and Trp110 of FcγRIII (Sondermann et al.: Nature 406, 267-273 (20 July 2000)). In a further embodiment, the at least one additional amino acid substitution in the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S, and in yet another embodiment, the at least one additional amino acid substitution is L234A and L235A in a human IgG1 Fc region, or S228P and L235E in a human IgG4 Fc region (U.S. Pat. No. 8,969,526, incorporated by reference in its entirety).

[0072] In certain embodiments, the polypeptide comprises an Fc variant of a wild-type human IgG Fc region, the polypeptide having Pro329 of the human IgG Fc region replaced with glycine, the Fc variant comprising at least two additional amino acid substitutions at L234A and L235A of the human IgG1 Fc region, or S228P and L235E of the human IgG4 Fc region, the residues being numbered according to the EU index of Kabat (US Pat. No. 8,969,526, which is incorporated by reference in its entirety). In certain embodiments, for downregulation of ADCC to at least 20% of that induced by a polypeptide comprising a wild-type human IgG Fc region, and / or downregulation of ADCP, the polypeptide comprising the substitutions of P329G, L234A, and L235A exhibits reduced affinity for human FcγRIIIA and FcγRIIA. (US Pat. No. 8,969,526, which is incorporated by reference in its entirety).

[0073] In one particular embodiment, a polypeptide comprising an Fc variant of a wild-type human Fc polypeptide comprises a triple mutation, i.e., an amino acid substitution at position Pro329, an L234A mutation, and an L235A mutation (P329 / LALA) (U.S. Patent No. 8,969,526, which is incorporated by reference in its entirety). In some embodiments, the polypeptide comprises the following amino acid substitutions: P329G, L234A, and L235A. In some embodiments, the polypeptide comprises the following amino acid substitutions: P329S, L234A, and L235A.

[0074] Certain antibody variants with improved or diminished binding to FcRs are known in the art and described herein (see, e.g., US 6737056, WO 2004 / 056312, and Shields et al., (2001) J. Biol. Chem. 9(2):6591-6604).

[0075] In a specific embodiment, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0076] In some embodiments, changes are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 1999 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0077] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which are responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0078] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), US 5648260, US 5624821, and WO 1994 / 29351.

[0079] Post-translational modifications: sialic acid and mannose-6-phosphate Post-translational modification (PTM) is a biological process involving the modification of amino acid side chains, carboxyl groups or terminal amino acids by covalent or enzymatic modification. Such modifications may include, for example, glycosylation, sialylation, phosphorylation or acetylation of one or more amino acids in a protein. The PTMs of a particular protein may not be predictable based on sequence alone and may vary based on the protein production and purification method. PTMs have been shown to be important in determining protein function and stability. For example, glycoform heterogeneity may affect, for example, antibody stability, pharmacokinetics (PK), efficacy, immunogenicity and / or Fc receptor binding.

[0080] The IgG:IDS fusion proteins described herein contain specific PTMs, including sialic acid and mannose 6-phosphate modifications. These PTMs facilitate proper trafficking of the fusion proteins to specific cells in the CNS, as well as intracellular trafficking. Additionally, the PTMs may affect the exposure (e.g., pharmacokinetics) of the fusion proteins in vivo. For example, mannose-6-phosphate (M6P) glycans that bind to the mannose-6-phosphate receptor (M6PR) have been shown to be involved in cellular uptake and lysosomal targeting.

[0081] Methods for assessing the molecular weight of a particular modification present on a protein of interest, such as sialic acid or M6P, are known in the art. A non-limiting example of such an assay is described in Example 3, where liquid chromatography-mass spectrometry is used to determine the mol / mol M6P:fusion protein and mol / mol SA:fusion protein. Various methods for examining M6PR binding / affinity are also known in the art. For example, a fluorescent enzyme assay as described in Example 2 may be used.

[0082] In certain embodiments, the fusion proteins described herein comprise at least about 6 mol / mol sialic acid:fusion protein. In certain embodiments, the fusion proteins described herein comprise at least about 8 mol / mol sialic acid:fusion protein. In certain embodiments, the fusion proteins described herein comprise at least about 10 mol / mol sialic acid:fusion protein. In certain embodiments, the fusion proteins described herein comprise at least about 12 mol / mol sialic acid:fusion protein. In certain embodiments, the fusion proteins described herein comprise at least about 14 mol / mol sialic acid:fusion protein. In certain embodiments, the fusion proteins described herein comprise at least about 15 mol / mol sialic acid:fusion protein. In certain embodiments, the fusion proteins described herein comprise from about 6 to about 19 mol / mol sialic acid:fusion protein (i.e., from about 6 to about 19 mol / mol sialic acid:fusion protein). In certain embodiments, the fusion proteins described herein comprise about 6 to about 16 mol / mol sialic acid:fusion protein (i.e., about 6 to about 16 mol / mol sialic acid:fusion protein). In some embodiments, the fusion proteins described herein comprise about 6 mol / mol sialic acid:fusion protein. In certain embodiments, the fusion proteins described herein comprise about 12 to about 19 mol / mol sialic acid:fusion protein (i.e., about 12 to about 19 mol / mol sialic acid:fusion protein). In certain embodiments, the fusion proteins described herein comprise about 15 to 16 mol / mol sialic acid:fusion protein (i.e., about 15 to about 16 mol / mol sialic acid:fusion protein). In some embodiments, the fusion proteins described herein comprise about 15 mol / mol sialic acid:fusion protein. In some embodiments, the fusion proteins described herein comprise about 16 mol / mol sialic acid:fusion protein.

[0083] In certain embodiments, the fusion proteins described herein comprise about 1.5 to about 2.5 mol / mol M6P:fusion protein (i.e., about 1.5 to about 2.5 mol / mol M6P:fusion protein). In certain embodiments, the fusion proteins described herein comprise about 1.5 to about 2.3 mol / mol M6P:fusion protein (i.e., about 1.5 to about 2.3 mol / mol M6P:fusion protein). In some embodiments, the fusion proteins described herein comprise about 1.5 mol / mol M6P:fusion protein. In some embodiments, the fusion proteins described herein comprise about 1.7 mol / mol M6P:fusion protein. In some embodiments, the fusion proteins described herein comprise about 2.3 mol / mol M6P:fusion protein.

[0084] Affinity, binding kinetics, concentration / exposure and functional characterization The fusion proteins described herein may have a wide range of binding affinities. In some embodiments, the fusion proteins have affinities for TfR in the range of about 1 pM to 2500 pM. For example, in some embodiments, the fusion proteins have affinities for TfR in the range of about 1 pM to 500 pM. In some embodiments, the affinity for TfR is in the range of about 10 pM to 250 pM. In some embodiments, the affinity for TfR is in the range of about 50 pM to 200 pM. For example, the affinity for TfR may be about 25 pM, 50 pM, 100 pM, 150 pM, or 200 pM. In some embodiments, the TfR affinity is for human TfR (hTfR).

[0085] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are known in the art (see, e.g., the Examples). These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet™ (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity, binding kinetics, and / or cross-reactivity. Methods for performing ELISA assays are known in the art and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, binding kinetic exclusion is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, BioLayer interferometry assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity. Non-limiting examples of methods for determining binding affinity, as well as methods for determining the specific activity of IgG:IDS fusion proteins are described in Example 2.

[0086] Concentrations of the fusion proteins described herein in peripheral organs (e.g., liver), brain and / or plasma can be measured, for example, using a human transferrin receptor (hTfR) knock-in mouse model. Such models can be used, for example, to measure maximum brain concentration (C max ) and / or to measure and / or compare brain exposure, e.g., C max This can be used to determine whether the human apical TfR (TfR ms / hu) The generation of mouse knock-in models is described in U.S. Pat. No. 10,143,187. Fusion proteins can be administered to mouse models (e.g., TfR) for assessment of brain and / or plasma concentration or exposure of the fusion protein. ms / hu ). Plasma samples can be obtained from the mice after an appropriate period of time, followed by perfusion of the vasculature with an appropriate solution. After perfusion, peripheral organs or brains (or portions thereof) can be extracted, homogenized, and lysed. The concentration of the drug in the plasma and / or tissue (e.g., brain) lysates can then be determined using standard methods known to those skilled in the art. As a non-limiting example, concentrations can be measured using an ELISA-based assay as described in Example 4. A standard curve can be generated by administering a series of doses to the knock-in mouse model.

[0087] The fusion proteins described herein (e.g., IgG:IDS) can also be evaluated in a variety of functional assays in which the ability of the fusion protein to modulate the phenotype or behavior of a target cell is examined. A non-limiting example of an in vitro functional assay is described in Example 3, in which the cellular potency of an IgG:IDS fusion protein was evaluated in primary MPS II patient fibroblasts.

[0088] IDS activity may also be assessed by assaying a sample, such as a cell sample, tissue sample, or liquid sample (e.g., CSF or urine), for the amount of one or more glycosaminoglycans (GAGs) (e.g., heparan sulfate, dermatan sulfate, or total GAGs) that accumulate as a result of IDS deficiency. The amount of heparan and dermatan sulfate is determined by digesting the GAGs present in the sample with heparinase and chondroitinase. The resulting disaccharides can then be assayed by mass spectrometry (e.g., LC-MS / MS). Samples with high levels of heparan and dermatan sulfate accumulation have increased amounts of heparan and dermatan sulfate-derived disaccharides. Thus, the level of disaccharides is inversely proportional to IDS enzyme activity. Mass spectrometry (e.g., LC-MS / MS) assays can be performed on any sample in which GAGs accumulate, including cell samples, tissue samples, and liquid samples. Such samples can be evaluated to monitor the activity of the IDS-containing proteins described herein, for example, administered to cells in vitro or, in some embodiments, administered to a subject in vivo. The subject can be an animal, such as a rodent, e.g., a mouse, or a non-human primate. In some embodiments, the subject is a human patient, such as a patient with Hunter syndrome undergoing treatment with IDS therapy, and the assay is used to monitor IDS activity in the patient. In some embodiments, the human patient is undergoing treatment with a fusion protein described herein. An exemplary protocol for such an assay is described in Example 4.

[0089] Fusion protein / antibody production Methods for preparing fusion proteins comprising an anti-TfR antibody linked to an IDS amino acid sequence are known in the art and are also described in the Examples section below.

[0090] For example, genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. Accordingly, certain embodiments provide a polynucleotide comprising a nucleotide sequence encoding an antibody or fusion protein / polypeptide described herein. In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL (e.g., light chain) and an amino acid sequence comprising an antibody heavy chain linked to an IDS amino acid sequence via a peptide linker or peptide bond, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL (e.g., light chain) and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody heavy chain linked to an IDS amino acid sequence via a peptide linker or peptide bond. In one embodiment, the host cell is eukaryotic, for example, a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (eg, a Y0, NS0, Sp20 cell).

[0091] Accordingly, certain embodiments provide methods of producing a fusion protein as described herein, comprising expressing in a host cell under conditions suitable for producing the fusion protein one or more polynucleotides operable to express 1) a light chain as described herein and 2) a heavy chain linked to an IDS amino acid sequence as described herein. Certain embodiments also provide fusion proteins produced by such methods.

[0092] For recombinant production of fusion proteins, for example, nucleic acids encoding the fusion proteins / polypeptides described herein are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody / fusion protein).

[0093] Suitable host cells for cloning or expressing vectors encoding fusion proteins include prokaryotic or eukaryotic cells as described herein. For example, fusion proteins can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the fusion protein can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0094] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed by SV40, human embryonic kidney lines (e.g., HEK293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK, buffalo rat hepatocytes (BRL 3A), human lung cells (WI38), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals Other useful mammalian host cell lines include TRI cells, MRC5 cells, and FS4 cells, described in NYAcad.Sci.383:44-68 (1982). - Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0095] Compositions and Formulations Guidance for preparing formulations for use in the present disclosure can be found in many handbooks on pharmaceutical preparations and formulations known to those skilled in the art.

[0096] In some embodiments, a pharmaceutical composition comprises a fusion protein as described herein and further comprises one or more pharma- ceutically acceptable carriers and / or excipients. For example, in certain embodiments, the fusion protein is contained within a pharmaceutical composition that further comprises a pharma- ceutically acceptable excipient. Pharmaceutically acceptable carriers include any solvent, dispersion medium, or coating that is physiologically compatible with the active agent and does not interfere with or inhibit the activity of the active agent.

[0097] In some embodiments, the carrier is suitable for intravenous, intrathecal, intracerebroventricular, intramuscular, intraperitoneal or subcutaneous administration. Pharmaceutically acceptable carriers can include one or more physiologically acceptable compounds that act, for example, to stabilize the composition or to increase or decrease the absorption of the polypeptide. Physiologically acceptable compounds can include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of active substances, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are also available in the art.

[0098] The pharmaceutical compositions described herein can be manufactured, for example, by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. The following methods and excipients are exemplary.

[0099] As disclosed above, the fusion proteins described herein can be formulated for parenteral administration, for example, by injection, by bolus injection or continuous infusion. For injection, the fusion protein can be formulated into a formulation by dissolving, suspending or emulsifying in an aqueous or non-aqueous solvent, for example, vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, and, if necessary, together with commonly used additives such as solubilizers, isotonicity agents, suspending agents, emulsifying agents, stabilizers and preservatives. In some embodiments, the fusion protein can be formulated in an aqueous solution, such as a physiologically compatible buffer, non-limiting examples of which include Hank's solution, Ringer's solution and physiological saline buffer. The formulation for injection can be provided in a unit dosage form, for example, in ampoules or multi-dose containers, with the addition of a preservative. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. In some embodiments, the composition is a lyophilized composition. For example, the lyophilized composition (e.g., lyophilized powder or cake) may further comprise one or more excipients selected from the group consisting of salts for adjusting osmolarity and / or pH (e.g., NaCl, NaH2PO4, Na2HPO4, NaOH, HCl), cryoprotectants (e.g., trehalose, sucrose), polymers (e.g., polyether polymers or block copolymers such as polyethylene glycol, polypropylene glycol, polyethylene-polypropylene glycol), and bulking agents (e.g., mannitol, glycine). The lyophilized composition may be reconstituted into an injectable liquid form (e.g., reconstituted via water, saline, or dextrose 5% water) prior to use. In certain embodiments, the lyophilized composition ... + , Cl - , PO4 3- , sucrose, and block copolymer polyethylene-polypropylene glycol.

[0100] Typically, pharmaceutical compositions for use in in vivo administration are sterile, which can be achieved by methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0101] Doses and desired drug concentrations of the pharmaceutical compositions described herein may vary depending on the particular use envisaged. Certain suitable dosage amounts are described herein.

[0102] How to use Insufficient or absent IDS enzyme activity results in accumulation of glycosaminoglycans (GAGs) heparan sulfate (HS, e.g., D0A0 and D0S0) and dermatan sulfate (DS, e.g., D0a4) in the CNS and periphery, and lysosomal dysfunction in multiple organs and tissues. Accordingly, certain embodiments provide a method of reducing the levels of total GAGs or one or more GAG ​​species in a subject in need thereof, comprising administering to the subject a fusion protein described herein (i.e., an IgG:IDS fusion protein described herein) or a pharmaceutical composition comprising such a fusion protein. In certain embodiments, a therapeutically effective amount of the fusion protein or a pharmaceutical composition comprising the fusion protein (e.g., about 1 mg / kg to about 10 mg / kg, e.g., about 1 mg / kg, 3 mg / kg, or 10 mg / kg of protein) is administered to the subject.

[0103] The concentration of total GAG or one or more GAG ​​species can also be assessed in a subject with Hunter syndrome (e.g., in a physiological sample from the subject, e.g., in a post-treatment sample from a subject administered a fusion protein (e.g., IgG:IDS) described herein). Such assessments may be used to assess disease activity or treatment response to the fusion protein (e.g., IgG:IDS) described herein, including identifying and assessing safe and therapeutically effective doses of the fusion protein (e.g., IgG:IDS). Quantification of GAG concentration levels can be performed using methods known in the art, for example, using a liquid chromatography mass spectrometry (LCMS) assay (see, e.g., Example 4).

[0104] In certain embodiments, the concentration of one or more GAG ​​species can be assessed and compared to a baseline level or control. The control or baseline level can vary, and it is within the skill of one of ordinary skill in the art to identify an appropriate control or baseline level depending on the parameter being assessed. For example, in certain embodiments, the term "control" can refer to a healthy subject or a subject without an IDS deficiency, or a sample therefrom. Alternatively, the term "control" can refer to a subject with an IDS deficiency (e.g., an IDS KO mouse or a Hunter syndrome patient) who was not administered the fusion protein. "Control" can also refer to a subject (or a sample therefrom) with an IDS deficiency prior to treatment. Similarly, "baseline level" can refer to a level or range of levels measured, for example, in a healthy subject, or a subject without an IDS deficiency. In certain other embodiments described herein, "baseline level" can also refer to a level or range of levels in a subject with an IDS deficiency who was not administered the fusion protein, or a subject prior to administration of the fusion protein. In certain embodiments, the control value or baseline level can be established using data from a population of control subjects. In some embodiments, the population of subjects is matched to the test subjects according to one or more subject characteristics, such as age or sex. In some embodiments, the control value is established using samples (e.g., blood or PBMC-containing samples) from the same type of subject population used to assess the lipid / protein levels of the test subjects. In certain embodiments, certain assessments are performed, for example, 7 days, 4 weeks, 12 weeks, 6 months, 12 months, and / or 18 months after initiation of treatment.

[0105] In certain embodiments, administration of the fusion protein described herein reduces the level of one or more GAG ​​species in a sample from a subject compared to a control.In certain embodiments, administration of the fusion protein described herein reduces the level of one or more GAG ​​species in a sample from a subject by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to a control.In certain embodiments, the control is a subject with IDS deficiency who has not been administered the pharmaceutical composition.In certain embodiments, the control is a subject before treatment.

[0106] As used herein, the phrase "sample" or "physiological sample" is meant to refer to a biological sample obtained from a subject that contains an analyte of interest, such as a GAG species. Thus, the sample may be evaluated, for example, at the molecular, lipid, or protein level. In certain embodiments, the physiological sample comprises tissue, cerebrospinal fluid (CSF), urine, blood, serum, or plasma. In certain embodiments, the sample comprises tissue, such as brain, liver, kidney, lung, or spleen. The sample may comprise a fluid. In certain embodiments, the sample comprises CSF. In certain embodiments, the sample comprises blood and / or plasma. In certain embodiments, the sample comprises serum. In certain embodiments, the sample comprises CNS tissue (e.g., brain tissue).

[0107] In certain embodiments, administration of the fusion protein described herein reduces the level of one or more GAG ​​species in the subject's organ, tissue or body fluid (e.g., blood, plasma, serum, CSF or urine) compared to a control. In certain embodiments, the level of one or more GAG ​​species in the subject's organ, tissue or body fluid is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to a control. In certain embodiments, the control is a subject with IDS deficiency (e.g., Hunter syndrome) who has not been administered the pharmaceutical composition. In certain embodiments, the control is a subject prior to treatment. For example, in certain embodiments, administration of a fusion protein described herein reduces the level of one or more GAG ​​species in the CSF of a subject by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more (e.g., at least about 30%, 40%, 50%, 60% or more), where the reduction is compared to the level of the corresponding one or more GAG ​​species in the CSF of the subject before administration. In certain embodiments, administration reduces the level of one or more GAG ​​species (e.g., HS, DS or total GAG) in the CSF of the subject by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 70%, where the reduction is compared to the level of the corresponding one or more GAG ​​species (e.g., HS, DS or total GAG) in the CSF of the subject before administration.

[0108] In certain embodiments, administration of the fusion protein described herein reduces the level of one or more GAG ​​species in the CNS of a subject compared to a control.In certain embodiments, the level of one or more GAG ​​species in the CNS of a subject is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more (e.g., at least about 20%, 30%, 40% or more) compared to a control.

[0109] In certain embodiments, administration of the fusion protein described herein reduces the level of one or more GAG ​​species in the CSF of a subject compared to a control.In certain embodiments, the level of one or more GAG ​​species in the CSF of a subject is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more (e.g., at least about 30%, 40%, 50%, 60% or more) compared to a control.In certain embodiments, the reduction of one or more GAG ​​species in the CSF of a subject is compared to the CSF level of one or more GAG ​​species of the subject before administration.

[0110] In certain embodiments, administration of the fusion protein described herein reduces the level of one or more GAG ​​species in the serum of the subject compared to a control. In certain embodiments, the level of one or more GAG ​​species in the serum of the subject is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to a control. In certain embodiments, the reduction of one or more GAG ​​species in the serum of the subject is compared to the serum level of one or more GAG ​​species of the subject before administration. In certain embodiments, administration of the fusion protein described herein reduces the level of one or more GAG ​​species in the serum of the subject to a baseline level (e.g., a level measured in a healthy subject or a subject without IDS deficiency (e.g., Hunter syndrome)).

[0111] In certain embodiments, total GAG levels are decreased. In certain embodiments, heparan sulfate levels are decreased (e.g., D0A0 and / or D0S0 levels are decreased). In certain embodiments, dermatan sulfate levels are decreased (e.g., D0a4 levels are decreased).

[0112] Certain embodiments provide a fusion protein as described herein, or a pharmaceutical composition comprising such a fusion protein, for use in a method for reducing one or more GAG ​​species, the method comprising administering the fusion protein or pharmaceutical composition to a subject in need thereof. In certain embodiments, the method comprises administering a therapeutically effective amount of the fusion protein or a pharmaceutical composition comprising the fusion protein to the subject (e.g., at a dose as described herein, such as about 1 mg / kg, 3 mg / kg, or 10 mg / kg protein dose).

[0113] Certain embodiments provide for the use of a fusion protein as described herein in the preparation of a medicament for reducing one or more GAG ​​species by administering the medicament to a subject in need thereof. In certain embodiments, the use is by administering a therapeutically effective amount of the medicament to a subject in need thereof (e.g., at a dose as described herein, such as a protein dose of about 1 mg / kg, 3 mg / kg, or 10 mg / kg).

[0114] Certain embodiments also provide a method of treating Hunter Syndrome in a subject in need thereof, comprising administering to the subject a fusion protein described herein or a pharmaceutical composition comprising such a fusion protein.

[0115] Certain embodiments provide a fusion protein as described herein, or a pharmaceutical composition comprising such a fusion protein for use in medical therapy.

[0116] Certain embodiments provide a fusion protein as described herein, or a pharmaceutical composition comprising such a fusion protein, for use in a method of treating Hunter Syndrome, the method comprising administering the fusion protein or pharmaceutical composition to a subject in need thereof.

[0117] In certain embodiments, the method includes administering to the subject a therapeutically effective amount of the fusion protein or a pharmaceutical composition comprising the fusion protein. For example, in certain embodiments, a dose as described herein, for example, about 1 mg / kg, 3 mg / kg, or 10 mg / kg of protein is administered to the subject.

[0118] Certain embodiments also provide for the use of a fusion protein as described herein in the preparation of a medicament for treatment of Hunter syndrome by administering the medicament to a subject in need of such treatment. In certain embodiments, the use is by administering a therapeutically effective amount of the medicament to the subject (e.g., at a dose as described herein, such as a protein dose of about 1 mg / kg, 3 mg / kg, or 10 mg / kg).

[0119] Administration and Effective Dosage A fusion protein described herein or a pharmaceutical composition comprising a fusion protein described herein may be administered to a subject in an effective amount or effective dose, e.g., a therapeutically effective dose.

[0120] In some embodiments, the dose (e.g., an effective dose or a therapeutically effective dose) is about 1 mg / kg to about 10 mg / kg of protein (e.g., about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or about 10 mg / kg). In some embodiments, the dose (e.g., an effective dose or a therapeutically effective dose) is about 1 mg / kg to about 3 mg / kg of protein. In some embodiments, the dose (e.g., an effective dose or a therapeutically effective dose) is about 3 mg / kg to about 10 mg / kg of protein. In certain embodiments, the dose (e.g., an effective dose or a therapeutically effective dose) is about 1 mg / kg of protein. In certain embodiments, the dose (e.g., an effective dose or a therapeutically effective dose) is about 3 mg / kg of protein. In certain embodiments, the dose (e.g., an effective dose or a therapeutically effective dose) is about 10 mg / kg of protein.

[0121] In certain embodiments, such doses described herein are therapeutically effective doses, eg, safe therapeutically effective doses (eg, doses that result in stabilization or a decrease in the subject's urinary total GAG concentration).

[0122] In certain embodiments, the pharmaceutical composition is administered weekly.

[0123] In some embodiments, the protein molecules described herein have an enzymatic activity of at least about 500 units (U) / mg, about 1,000 U / mg, or at least about 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 U / mg. In some embodiments, the enzyme activity is at least about 11,000 U / mg, or at least about 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45000 or 50,000 U / mg, or any within the range of about 500 U / mg to about 50,000 U / mg.

[0124] Dosage may vary according to several factors, including the selected route of administration, the formulation of the composition, the patient's response, the severity of the condition, the subject's weight, the subject's age, the subject's head size and / or head size to height ratio, and the prescribing physician's judgment.Dosage may be increased or decreased over time as required by individual patients.In some embodiments, patients are initially administered a low dose, and then increased to an effective dosage that the patient can tolerate.Determining an effective amount is well within the capabilities of those skilled in the art.

[0125] In various embodiments, the pharmaceutical compositions described herein are administered parenterally. In some embodiments, the pharmaceutical compositions are administered intravenously. Intravenous administration can be by infusion, for example, over a period of about 10 to about 30 minutes, or over a period of at least 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, or 10 hours. In some embodiments, the pharmaceutical composition is administered intravenously over a period of about 20 minutes to 6 hours, or about 30 minutes to 4 hours. In some embodiments, the pharmaceutical composition is administered as an intravenous bolus. A combination of infusion and bolus administration may also be used.

[0126] In certain embodiments, the pharmacokinetics (PK) of the fusion protein (e.g., IgG:IDS) is evaluated. For example, PK parameters include, but are not limited to: Cmax, trough concentration (Cmin), Tmax, area under the concentration-time curve from time 0 to the time of the last quantifiable concentration ( AUC0-最後 ), the area under the concentration-time curve over the dosing interval (AUC 0-T ), apparent end removal rate constant (λz), apparent end removal t1 / 2, and cumulative ratio.

[0127] product In another aspect, an article of manufacture is provided that includes a fusion protein as described herein. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container can hold the composition alone or in combination with another composition and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a fusion protein as described herein. The label or package insert indicates that the composition is used to treat Hunter syndrome. Additionally, the article of manufacture can include (a) a first container containing a composition that includes a fusion protein as described herein, and (b) a second container containing a composition that includes a therapeutic agent. The article of manufacture in this embodiment can further include a package insert indicating that the composition can be used to treat Hunter syndrome. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0128] Specific Definitions Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and are consistent with Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York, NY, and Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York.

[0129] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate reasonable deviations from that numerical value and values ​​known to one of ordinary skill in the art, for example, ±20%, ±10%, or ±5%, within the intended meaning of the stated value.

[0130] The term "administering" refers to a method of delivering an agent (such as, for example, an IgG:IDS fusion protein described herein), compound, or composition (e.g., a pharmaceutical composition) to a desired site of biological action. These methods include, but are not limited to, oral, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal delivery. In one embodiment, the polypeptides described herein are administered intravenously.

[0131] "Affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally measured using the dissociation constant ("Kd", "KD", or "K D "). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein.

[0132] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0133] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

[0134] Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0135] 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.

[0136] As used herein, the term "antibody" refers to a protein that has an immunoglobulin fold and specifically binds to an antigen through a variable region. As used herein, "antibody" is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has multiple binding sites, also called epitopes, that are recognized by CDRs (complementarity determining regions) on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have two or more corresponding antibodies. An antibody comprises a full-length immunoglobulin molecule or a portion of a full-length immunoglobulin molecule that comprises an antigen-binding site that immunospecifically binds to an antigen or a portion thereof of a target of interest (e.g., TfR). The immunoglobulins disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulins may be derived from any species. However, in one embodiment, the immunoglobulins are of human, murine, or rabbit origin.

[0137] The terms "antibody that binds to TfR" and "anti-TfR antibody" refer to an antibody that can bind to TfR via its variable region with sufficient affinity such that the antibody can bind to TfR for transport across the blood-brain barrier. In one embodiment, the extent to which an anti-TfR antibody binds to unrelated non-TfR proteins is less than about 10% of the binding of the antibody to TfR, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to TfR has a binding affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 M) In certain embodiments, the anti-TfR antibody binds to an epitope of TfR that is conserved among TfRs from different species.

[0138] The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TfR) via its variable region. Examples of antigen-binding fragments include, but are not limited to, Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains), F(ab')2 fragments (bivalent fragments consisting of two Fab fragments linked by a disulfide bridge at the hinge region), single chain Fvs (scFvs), disulfide-linked Fvs (dsFvs), complementarity determining regions (CDRs), VL (light chain variable region), and VH (heavy chain variable region).

[0139] As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. For the purposes of this application, a CH3 domain polypeptide refers to the segment of amino acids from about position 341 to about position 447 as numbered according to EU, and a CH2 domain polypeptide refers to the segment of amino acids from about position 231 to about position 340 as numbered according to the EU numbering scheme, not including the sequence of the hinge region. Polypeptides of CH2 and CH3 domains may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where the numbering of the CH2 domain is 1-110 and the numbering of the CH3 domain is 1-107 according to the numbering of the IMGT Scientific chart (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. Fc region, as used herein, refers to the segment of amino acids from about 231 to about 447, numbered according to the EU numbering scheme, but can include at least a portion of the hinge region of an antibody. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 25).

[0140] The term "control" or "control sample" refers to any sample appropriate for the detection technique used. A control sample may contain the product of the detection technique used or the material being tested. Furthermore, a control may be a positive control or a negative control.

[0141] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portions of the heavy and / or light chain are derived from a different source or species. In some embodiments, a chimeric antibody is a monoclonal antibody that contains variable regions derived from one source or species (e.g., mouse) and constant regions derived from another source or species (e.g., human). Methods for making chimeric antibodies have been described in the art.

[0142] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major antibody classes, IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0143] The term "complementarity determining region" or "CDR" refers to the three hypervariable regions in each chain that separate the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for antibody binding to an epitope of an antigen. The CDRs of each chain are usually referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus, and are also usually identified by the chain in which a particular CDR is located. Thus, the VH CDR3 or CDR-H3 is located in the variable region of the antibody heavy chain in which it is found, while the VL CDR1 or CDR-L1 is the CDR1 from the variable region of the antibody light chain in which it is found.

[0144] The term "cross-react," as used herein, refers to the ability of an antibody to bind to an antigen other than the antigen against which the antibody was raised. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen from a species different from the species of the antigen against which the antibody was raised. As a non-limiting example, an anti-TfR antibody described herein raised against a human TfR peptide may exhibit cross-reactivity with TfR orthologs (e.g., monkeys).

[0145] The phrase "effective amount" refers to an amount of a compound that (i) treats or prevents a particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition or disorder described herein.

[0146] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0147] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds. The epitope to which an antibody binds can be determined by epitope binning methods known in the art, such as pairwise combinatorial methods. In some embodiments, the specific site on an antigen molecule to which an antibody binds is determined by hydroxyl radical footprinting.

[0148] "Expression" refers to the transcription and / or translation in a cell of an endogenous gene, a transgene, and the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of an antisense construct, expression may refer to the transcription of antisense DNA only. Expression may also refer to the production of a protein.

[0149] The "Fab" fragment (also called F(ab)) also contains the light chain constant region and the heavy chain constant region (CH1). For example, papain digestion of an antibody produces two types of fragments: an antigen-binding fragment called a Fab fragment, which contains the variable regions of the heavy and light chains that act as a single antigen-binding domain; and a remaining portion called "Fc" because it is easier to crystallize. The Fab' fragment differs from the Fab fragment in that it also contains some residues from the carboxyl terminus of the heavy chain CH1 region, including one or more cysteine ​​residues from the hinge region of the antibody. However, the Fab' fragment is structurally equivalent to the Fab in that it is an antigen-binding fragment containing the variable regions of the heavy and light chains that together act as a single antigen-binding domain. Antigen-binding fragments that contain the variable regions of the heavy and light chains that act as a single antigen-binding domain and are equivalent to those obtained by papain digestion are referred to herein as "Fab-like antibodies", even if they are not identical to the antibody fragments produced by protease digestion. Fab'-SH is a Fab' with one or more cysteine ​​residues with free thiol groups in its constant region. F(ab') fragments are generated by cleaving the disulfide bond between the cysteine ​​residues in the hinge region of F(ab')2. Other chemically cross-linked antibody fragments are also known to those skilled in the art. Pepsin digestion of antibodies produces two fragments: one is the F(ab')2 fragment that contains two antigen-binding domains and can cross-react with the antigen, and the other is the remaining fragment (called pFc'). In this specification, antibody fragments equivalent to those obtained by pepsin digestion are referred to as "F(ab')2-like antibodies" when they contain two antigen-binding domains and can cross-react with the antigen. Such antibody fragments can also be produced, for example, by genetic engineering.

[0150] The term "Fc region" or "Fc polypeptide" herein is used to define a C-terminal region of an immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. The Fc region includes a portion of the constant region, including at least the CH2 domain and / or the CH3 domain, and may include at least a portion of the hinge region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within an Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In general, an Fc region does not contain a variable region.

[0151] The term "FcRn" refers to the fetal Fc receptor. Binding of an Fc polypeptide to FcRn reduces the clearance and extends the serum half-life of the Fc polypeptide. The human FcRn protein is a heterodimer consisting of a protein with a size of about 50 kDa similar to major histocompatibility (MHC) class I proteins and β2-microglobulin with a size of about 15 kDa.

[0152] As used herein, "FcRn binding site" refers to a region of an Fc polypeptide that binds to FcRn. In human IgG, the FcRn binding site, numbered using the EU index, includes T250, L251, M252, I253, S254, R255, T256, T307, E380, M428, H433, N434, H435, and Y436. These positions correspond to positions 20-26, 77, 150, 198, and 203-206 of SEQ ID NO:1.

[0153] As used herein, a "native FcRn-binding site" refers to a region of an Fc polypeptide that binds to FcRn and has the same amino acid sequence as a region of a native Fc polypeptide that binds FcRn.

[0154] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues that are relatively conserved within a species. The FR of a variable domain generally consists of four FR domains, FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order in a VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0155] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein.

[0156] As used herein, a "fusion protein" or "antibody fusion protein" refers to an anti-TfR antibody (e.g., an anti-human TfR antibody) linked or fused to an IDS enzyme, an IDS enzyme variant, or a catalytically active fragment thereof. As described herein, in certain embodiments, the IDS enzyme, an IDS enzyme variant, or a catalytically active fragment thereof (collectively referred to as an "IDS amino acid sequence") may be linked to the C-terminus of a heavy chain (i.e., a heavy chain fusion polypeptide). One or both heavy chains in an antibody may be linked to an IDS amino acid sequence. In certain embodiments, the fusion protein comprises an anti-TfR antibody comprising one heavy chain linked to an IDS amino acid sequence. In certain other embodiments, the fusion protein may comprise an anti-TfR antibody in which both heavy chains are each independently linked to an IDS amino acid sequence. The IDS enzyme, an IDS enzyme variant, or a catalytically active fragment thereof may be linked to the anti-TfR antibody by a peptide bond or a polypeptide linker.

[0157] As used herein, a "fusion polypeptide" or "heavy chain fusion polypeptide" refers to a heavy chain amino acid sequence linked (e.g., fused) to an IDS enzyme, an IDS enzyme variant, or a catalytically active fragment thereof. The heavy chain fusion polypeptide may be linked to the IDS enzyme, an IDS enzyme variant, or a catalytically active fragment thereof by a peptide bond or a polypeptide linker.

[0158] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0159] A "human antibody" or "fully human antibody" is one that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0160] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0161] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, with all or substantially all of its HVRs (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of its FRs corresponding to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0162] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, naturally occurring four-chain antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally contain amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs), the latter of which are the most highly sequence variable and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35B (H1), 50-65 (H2), and 95-102 (H3). (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) With the exception of CDR1 in VH, CDRs generally include amino acid residues that form hypervariable loops. CDRs also include "specificity determining residues" or "SDRs," which are residues that contact the antigen. The SDRs are contained within regions of the CDRs referred to as abbreviated CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)).Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered according to Kabat et al., supra, herein.

[0163] The term "identical" or percent "identity" in the context of two or more polypeptide sequences refers to two or more polypeptide sequences or subsequences that have amino acid residues that are identical or a certain percentage, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more identical over a particular region when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0164] For sequence comparison of polypeptides, one amino acid sequence usually serves as a reference sequence to be compared with the candidate sequence. Alignment can be performed by various methods available to those skilled in the art, such as visual alignment, or by using publicly available software with known algorithms to achieve maximum alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art. For the purpose of this application, for sequence comparison of polypeptide sequences, the BLASTP algorithm standard protein BLAST is used to align two protein sequences with default parameters.

[0165] The phrases "corresponding to," "determined with respect to," or "numbered with respect to," when used in the context of identifying a given amino acid residue in a polypeptide sequence, refer to the position of the residue in the reference sequence when the given amino acid sequence is maximally aligned and compared to a particular reference sequence. A polypeptide that is aligned to a reference sequence need not be the same length as the reference sequence.

[0166] As used herein, "iduronate sulfatase", "iduronate-2-sulfatase" or "IDS" refers to iduronate 2-sulfatase (EC 3.1.6.13), an enzyme involved in the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. Deficiency in IDS is associated with mucopolysaccharidosis II, also known as Hunter syndrome. As used herein, the term "IDS" or "IDS enzyme" is catalytically active and encompasses wild-type IDS and functional variants, including alleles and splice variants, as well as catalytically active fragments thereof, optionally as a component of a fusion protein with an anti-TfR antibody. The sequence of human IDS isoform I, the human sequence referred to as the canonical sequence, is available in UniProt entry P22304 and is encoded by the human IDS gene at Xq28. The full-length sequence is shown as SEQ ID NO:1. As used herein, a "mature" IDS sequence refers to a form of the polypeptide chain that lacks the signal and propeptide sequences of the naturally occurring full-length polypeptide chain. The amino acid sequence of a mature human IDS polypeptide is set forth as SEQ ID NO:2, which corresponds to amino acids 34-550 of the full-length human sequence. As used herein, a "truncated" IDS sequence refers to a catalytically active fragment of a naturally occurring full-length polypeptide chain. The amino acid sequence of an exemplary truncated human IDS polypeptide is set forth as SEQ ID NO:3, which corresponds to amino acids 26-550 of the full-length human sequence. The structure of human IDS has been well characterized. An exemplary structure is available under PDB accession code 5FQL. The structure is also described in Nat.Comm. 8:15786 doi:10.1038 / ncomms15786,2017. Non-human primate IDS sequences have also been described, including chimpanzee (UniProt entry K7BKV4) and rhesus macaque (UniProt entry H9FTX2). The mouse IDS sequence is available in Uniprot entry Q08890.An IDS variant has, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type IDS or fragment thereof when assayed under identical conditions. A catalytically active IDS fragment has, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length IDS or variant thereof when assayed under identical conditions.

[0167] "IDS enzyme variant" refers to functional variants, e.g., allelic variants and splice variants, of a wild-type IDS enzyme or a fragment thereof, where an IDS enzyme variant has, e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of a corresponding wild-type IDS enzyme or a fragment thereof when assayed under the same conditions.

[0168] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s).

[0169] The terms "individual," "subject," and "patient," as used interchangeably herein, refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In certain embodiments described herein, the subject is a human subject. In certain embodiments, the subject is a male subject.

[0170] Depending on the amino acid sequence of the constant domain of the heavy chain, intact antibodies can be assigned to different "classes". There are five major classes of intact immunoglobulin antibodies: 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 domains that correspond to the different classes of antibodies 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. Ig forms include hinge-modified or hingeless forms (Roux et al. (1998) J. Immunol. 161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US 2005 / 0048572; US 2004 / 0229310).

[0171] An "isolated" antibody is an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 90%, 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0172] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0173] "Isolated nucleic acid encoding an anti-TfR antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) are present in one or more locations within a host cell.

[0174] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogenous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies described herein may be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be produced by recombinant DNA methods (see, e.g., US 4816567, US 5807715). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597, for example.

[0175] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4816567, and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0176] As used herein, the term "mutant" in reference to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant." Mutations can include substitutions, insertions, and deletions.

[0177] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0178] The terms "obtaining a sample from a patient," "obtained from a patient," and similar expressions are used to refer to obtaining a sample directly from a patient, as well as obtaining a sample indirectly from a patient via an intermediate entity (e.g., obtaining a sample from a courier who obtains the sample from a nurse who obtains the sample from the patient).

[0179] The term "package insert" is used to refer to instructions typically included in the packaging of a research tool or therapeutic product that contain information about the conditions of use, indications, uses, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding such product.

[0180] The term "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained in the preparation is effective and does not contain any additional components that are unacceptably toxic to a subject to which the formulation will be administered.

[0181] The term "pharmaceutically acceptable excipient" refers to non-active pharmaceutical ingredients that are biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, buffers, carriers, or preservatives.

[0182] The terms "polypeptide" and "peptide" are used interchangeably herein and refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can contain entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids.

[0183] As used herein, the term "protein" refers to either one polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of a single-chain polypeptide. The single-chain polypeptides of a protein can be linked by covalent bonds, e.g., disulfide bonds, or by non-covalent interactions.

[0184] The terms "polynucleotide" and "nucleic acid" refer interchangeably to a chain of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules having a mixture of single- and double-stranded DNA and RNA.

[0185] As used herein, the term "binding affinity" refers to the strength of a non-covalent interaction between two molecules, e.g., between a single binding site on a polypeptide and the target to which it binds, e.g., the transferrin receptor. Thus, for example, unless otherwise indicated or clear from the context, the term may refer to a 1:1 interaction between a polypeptide and its target. Binding affinity is measured by the equilibrium dissociation constant (K D ), which can be quantified by measuring the binding rate constant (k a ,time -1 M -1 ) divided by the dissociation rate constant (k d ,time -1 ) refers to K D can be determined by measuring the kinetics of complex formation and dissociation, for example, using surface plasmon resonance (SPR) methods, such as the Biacore™ system, equilibrium exclusion assays such as KinExA®, and BioLayer interferometry (e.g., using the ForteBio® Octet® platform). As used herein, "binding affinity" refers not only to formal binding affinities, such as those that reflect a 1:1 interaction between a polypeptide and its target, but also to K values ​​that may reflect strong binding. D The apparent affinity is also included, from which the

[0186] As used herein, when referring to a TfR-binding antibody or fusion protein comprising such an antibody described herein, the term "specifically binds" or "selectively binds" to a target, e.g., TfR, refers to a binding reaction in which the TfR-binding antibody or fusion protein binds to a target with higher affinity, higher avidity, and / or higher duration than it binds to a structurally different target. In typical embodiments, the TfR-binding antibody or fusion protein has at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more affinity for a particular target, e.g., TfR, compared to an unrelated target when assayed under the same affinity assay conditions. As used herein, the terms "specifically bind," "specifically binds," or "is specific for" a particular target (e.g., TfR) refer to, for example, a binding reaction in which the TfR-binding antibody or fusion protein binds to a target with higher affinity, higher avidity, and / or higher duration than it binds to a structurally different target. -4 M or less, e.g. 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M is the equilibrium dissociation constant K of the target it binds D In some embodiments, the TfR-binding antibody or fusion protein specifically binds to an epitope on the TfR that is conserved between species (e.g., structurally conserved between species), for example, between non-human primates and human species (e.g., structurally conserved between non-human primates and human species). In some embodiments, the TfR-binding antibody or fusion protein may bind exclusively to the human TfR.

[0187] As used herein, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 26. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cattle, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term "transferrin receptor" also encompasses allelic variants of exemplary reference sequences, e.g., human sequences encoded by genes at the transferrin receptor protein 1 chromosomal locus. Full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is set forth in SEQ ID NO:27.

[0188] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention to alter the natural history of the individual being treated, and may be performed either for prophylaxis or during the clinical pathology process. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, amelioration or remission of the disease, and remission or improvement of prognosis.

[0189] The "therapeutically effective amount" of a substance / molecule disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule to induce a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or adverse effects of the substance / molecule are outweighed by therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount is less than a therapeutically effective amount, since a prophylactic dose is used in subjects before or at an early stage of disease.

[0190] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms containing transgenic cells are referred to as "transgenic organisms."

[0191] "Transformed", "transgenic" and "recombinant" refer to a host cell or organism into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome using methods commonly known in the art. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene specific primers, vector specific primers, partial mismatch primers, and the like. For example, "transformed", "transformant" and "transgenic" cells have undergone a transformation process and may contain a foreign gene integrated into their chromosomes. The term "non-transformed" refers to a normal cell that has not undergone a transformation process.

[0192] A "transgenic" organism is one having one or more cells that contain an expression vector.

[0193] The terms "variable region", "variable domain", "V region" or "V domain" refer to the domain of an antibody heavy or light chain involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, a VH or VL domain from an antibody that binds to an antigen may be used to screen a library of complementary VL or VH domains, respectively, to isolate antibodies that bind to a particular antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0194] By "variant" polypeptide is intended a polypeptide derived from a native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminus and / or C-terminus of the native protein, deletion or addition of one or more amino acids at one or more sites in the native protein, or substitution of one or more amino acids at one or more sites in the native protein. Such variants can result, for example, from genetic polymorphism or human manipulation. Methods for such manipulation are generally known in the art.

[0195] Thus, the polypeptides described herein can be modified in various ways, including amino acid substitution, deletion, truncation and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of polypeptides can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are well known in the art.

[0196] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to alterations that result in the replacement of an amino acid with another amino acid that can be classified as having similar characteristics. Examples of classifications of conservative amino acid groups defined in this way include the "charged / polar group" including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H), the "aromatic group" including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H), and the "aliphatic group" including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids can be divided into subgroups including the "positively charged subgroup" containing Lys, Arg, and His, the "negatively charged subgroup" containing Glu and Asp, and the "polar subgroup" containing Asn and Gln. In another example, the aromatic or cyclic group can be divided into subgroups including the "nitrogen ring subgroup" containing Pro, His, and Trp, and the "phenyl subgroup" containing Phe and Tyr. In another further example, the aliphatic group can be divided into subgroups, such as the "aliphatic non-polar subgroup" containing Val, Leu, Gly, and Ala, and the "aliphatic slightly polar subgroup" containing Met, Ser, Thr, and Cys. Examples of conservative mutation classifications include amino acids within the above subgroups, such as, but not limited to, amino acid substitutions of Lys for Arg, or vice versa, so that a positive charge may be maintained, Asp for Glu, or vice versa, so that a negative charge may be maintained, Thr for Ser, or vice versa, so that a free -OH may be maintained, and Asn for Gln, or vice versa, so that a free -NH2 may be maintained.In some embodiments, the hydrophobic amino acid is a naturally occurring hydrophobic amino acid, e.g., replacing the active site and preserving hydrophobicity.

[0197] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0198] "Wild-type" refers to a normal gene, or an organism found in nature without any known mutations.

[0199] The following examples are intended to be non-limiting. EXAMPLES

[0200] Example 1: Construction of IgG:IDS Cloning and Architecture The IgG:IDS molecule contains a TfR antibody linked to two IDS enzymes. One IDS enzyme is linked to the C-terminus of each Fc portion of the TfR antibody heavy chain by a peptide linker (Figure 1). The antibody constant region in the IgG:IDS was human IgG1. The heavy chain IDS sequence is represented by SEQ ID NO:17 or SEQ ID NO:18, and the light chain sequence is represented by SEQ ID NO:5.

[0201] Protein expression The IgG:IDS construct was expressed by transient transfection of ExpiCHO cells (Thermo Fisher Scientific) according to the manufacturer's instructions. Cultures were co-transfected with plasmids encoding IgG:IDS (heavy and light chains) and SUMF1 cDNA in a 2:2:1 ratio.

[0202] Protein purification IgG:IDS was purified to homogeneity from serum-free CHO cultures by a series of chromatographic steps. IgG:IDS was affinity purified using Protein A followed by anion exchange chromatography. Final fractions that were both highly pure and relatively enriched in negatively charged post-translational modifications (PTMs) (e.g., approximately 12-19 mol / mol sialic acid:fusion protein) as assessed by analytical size-exclusion chromatography (SEC) and / or microcapillary electrophoresis and LCMS were pooled, concentrated, and dialyzed into 20 mM sodium phosphate, pH 6.5 and 130 mM NaCl. Preparations were stored at 4°C or -80°C prior to use and routinely analyzed by SEC, specific activity, and endotoxin content.

[0203] Example 2: Characterization of IgG:IDS Surface Plasmon Resonance (SPR) D decision The affinity of IgG:IDS for the human transferrin receptor (hTfR) was determined by surface plasmon resonance on a Biacore™ T200 instrument using two methods similar to those previously described (Kariolis et al. 2020. Sci Transl Med 12(545):eaay1359). In method 1, to assess monovalent TfR binding affinity, IgG:IDS was captured onto a Protein A-coated Biacore™ Series S CM5 sensor chip and incubated with serial dilutions of the apical domain of hTfR (hTfR アピカル ) was injected over the captured sample. In method 2, to assess the apparent hTfR binding affinity of IgG:IDS, biotinylated full-length hTfR was immobilized on a streptavidin-coated CM5 sensor chip, followed by injection of serial dilutions of IgG:IDS. For both methods, single cycle kinetics mode was used for sample injection (association time, 90 s, dissociation time, 600 s) and binding affinities were calculated using Biacore T200 evaluation software v3.0.

[0204] Using SPR, we observed that IgG:IDS can bind to the TfR in a bivalent manner, which is strongly influenced by receptor density. アピカル It was measured to have a monovalent affinity of 2.6 nM for the receptor and an apparent affinity of approximately 100 pM for the full-length receptor.

[0205] In vitro IDS activity Specific activity of IgG:IDS was measured in a two-step fluorogenic enzymatic assay using an artificial substrate as previously described (Ullman et al. 2020. Sci Transl Med 12(545):eaay 1163). Briefly, the amount of product was calculated by fitting a 4-methylumbelliferone (leaving group) standard curve by linear regression and was confirmed to be less than 10% of total substrate cleavage. Specific activity was calculated in pmol product / min / mg protein.

[0206] Based on in vitro activity assays, the specific activity of IgG:IDS was highly active, measured to be approximately 4400 pmol of product / min / mg of protein (average of n=5 determinations).

[0207] Mannose-6-phosphate bond ELISA plates were coated overnight at 4° C. with Fc fusion of the ninth domain of human cation-independent mannose 6-phosphate receptor (ciM6PR, Uniprot P11717) at 1 μg / mL in PBS. The next day, plates were washed three times with washing buffer (PBS with 0.02% Tween®-20) and blocking buffer (PBS with 0.02% Tween®-20 and 5% BSA) was added to each well. Blocking was performed at room temperature for 1 hour, after which the plates were washed three times and IgG:IDS was added to the first column of the plate at a concentration of 25 nM. Serial three-fold dilutions were performed across the plate. The primary incubation of the binding reaction was performed at room temperature for 1 hour. After binding, plates were washed three times and binding was detected using biotinylated anti-IDS antibody diluted to 0.0625 μg / mL in sample buffer. Plates were incubated with detection antibody for 1 hour at room temperature and then washed three times. Streptavidin-HRP diluted 1:50,000 in sample buffer was then added to each well. Plates were incubated at room temperature for 30 minutes and then washed three times. ELISA was developed using TMB reagent. ELISA plates were read on a HighRes BioTek Synergy plate reader and absorbance at 450 nm was recorded.

[0208] Based on ELISA binding assays, the binding of IgG:IDS to the mannose-6-phosphate receptor was measured with an EC50 of approximately 55-76 pM. A representative binding curve of IgG:IDS is shown in Figure 2.

[0209] Example 3: Pharmacokinetic characterization of IgG:IDS S for assessing cell potency 35 -Sulfate accumulation assay The cellular activity of IgG:IDS was examined in fibroblasts derived from MPS II patients. 35 S is incorporated into newly synthesized GAGs 35S pulse-chase assay was used to evaluate the fibroblast activity of MPS II patients (GM01928, GM12366, GM01583). Primary fibroblasts from MPS II patients (GM01928, GM12366, GM01583) were obtained from Coriell. Cell S 35 Accumulation assays were performed using a method modified from Lu and co-authors (Lu et al. 2010. Bioconjug Chem 21:151-156). Briefly, fibroblasts were seeded at 25,000 cells / well in 96-well plates and grown in DMEM high glucose (Gibco) with 10% FBS (Sigma). After 3 days of culture, the medium was replaced with low sulfate F12 medium (Gibco) supplemented with 10% dialyzed fetal bovine serum and 40 mCi / mL [S35] sodium sulfate (PerkinElmer) for 96 hours. After incubation with [S35] sodium sulfate, cells were treated with IgG:IDS. After 24 hours of incubation, the medium was aspirated and cells were washed with cold PBS and lysed with 0.01 N NaOH. Incorporated S35 was measured by scintillation counting (Microbeta Trilux). EC50 curves were generated using Prism software using a log (agonist) vs response, variable slope (four parameter) fit.

[0210] IgG:IDS was found to be highly active in this assay, 35 It reduced accumulation of S-labeled substrates with cellular EC50 values ​​ranging from approximately 16 pM to 23 pM (see Table 1). The data demonstrate that IgG:IDS was effectively internalized in MPS II patient-derived cells in the low pM range, resulting in reduced substrate accumulation. A representative cellular potency curve (averaged across cell lines) for IgG:IDS is shown in Figure 3.

[0211] [Table 1]

[0212] Liquid chromatography-mass spectrometry of mannose-6-phosphate (M6P) and sialic acid (SA) Recombinant purified IgG:IDS protein (5 μg) was buffer exchanged into 50 mM ammonium acetate, pH 7.2. Normalized amounts of protein were analyzed using stable isotope labeled (SIL) as an internal standard. 13 C6 mannose-6-phosphate (SIL-M6P, Omicronbio Inc, Catalog No. MAN-05, 62.5 ng per sample) and 13 The samples were spiked with C3-sialic acid (SIL-SA, Omicronbio Inc, Cat. No. NEU-004, 200 ng per sample) and then hydrolyzed or digested for analysis. M6P was hydrolyzed from the protein using 150 μL of 6.6 M trifluoroacetic acid solution for 90 min in a 90° C. heater block with shaking. The fast vac samples were then washed once with acetonitrile (ACN) and dried. The final pellet resuspended in 50 μL ACN:water (20:80, v:v) was analyzed by LC-MS / MS. SA was digested from the protein by SialEXO Enzyme (Genovis). Briefly, 5 μg of protein was mixed with 10 μL of 20 ng / μL SIL-SA, 3 μL of 1 M Tris buffer (pH 7.5) was added, and made up to 60 μL with MiliQ water, followed by addition of 1 μL of SialEXO (10 units / μL) and incubation for 2 hours at 37° C. The SA sample was injected without further processing.

[0213] M6P and SA analysis was performed by liquid chromatography on a UHPLC Vanquish (Thermo Scientific, CA, USA) coupled to a UV / Vis and Q Exactive Orbitrap electrospray ionization mass spectrometer (Thermo Scientific, CA, USA). Samples were injected onto an ACQUITY UPLC BEH Amide 1.7 mm, 2.1×150 mm column (Waters Corporation) and analyzed by negative ionization mode mass spectrometry with a mobile phase of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid using a 4.5 min gradient starting at 60% B, then 42% B at 1.8 min, decreasing to 10% B at 2.2 min, holding until 3 min, then increasing to 60% B and holding until 4.5 min. The flow rate was 0.45 mL / min and the column temperature was 60° C.

[0214] Data were collected using parallel reaction monitoring (PRM) acquisition under negative mode including M6P and SIL-M6P internal standards. The inclusion list was 259.0224 and 265.0426 (anion) at 1.6-2.2 min for M6P and SIL-M6P, respectively, and 308.0992 and 311.175 (anion) at 1.0-1.6 min for SA and SIL-SA, respectively. The AUC ratios of M6P / SIL-M6P and SA / SIL-SA were used to calculate the molecular weights of M6P and SA, M6P:mol protein / mol and SA:mol protein / mol.

[0215] The measured M6P content of IgG:IDS ranged from approximately 1.5 mol / mol to 2.3 mol / mol M6P:protein. The measured sialic acid content of IgG:IDS was approximately 15-16 mol / mol SA:protein.

[0216] Example 4: Administration of IgG:IDS reduces GAG in the brain and CSF in IDS KO and TfR mu / hu Decreased in mice. Materials and Methods Animal Care Mice were housed under a 12-h light / dark cycle and had free access to water and standard rodent chow (LabDiet® No. 25502, irradiated).

[0217] Mouse strains Previously described Ids KO mice on a B6N background were obtained from The Jackson Laboratories (JAX line 024744). TfR harboring the human TfR apical domain knocked into the mouse receptor. mu / hu The development and characterization of the KI mouse line was previously described (U.S. Patent No. 10,143,187). mu / hu Male mice were crossed with female Ids heterozygous mice to generate Ids KO, TfR mu / hu KI mice were generated. All mice used in this study were male.

[0218] Pharmacokinetics (PK) of IgG:IDS TfR at 2-3 months of age mu / hu KI mice (n=3 / group) were injected with 1 mg / kg, 3 mg / kg or 10 mg / kg body weight IgG:IDS via the tail vein and sacrificed 0.5, 4, 8 and 24 hours after dosing. For final sample collection, animals were deeply anesthetized via intraperitoneal (ip) injection of 2.5% Avertin. Blood was collected via cardiac puncture for serum collection and allowed to clot for at least 30 minutes at room temperature. The tubes were then centrifuged at 12,700 rpm for 7 minutes at 4°C. Serum was transferred to fresh tubes and snap frozen on dry ice. Animals were perfused transcardially with ice-cold PBS using a peristaltic pump (Gilson Inc. Minipuls Evolution) and brains and livers were dissected. Liver and brain tissue (50 mg) were snap frozen on dry ice and prepared for IDS / IDS ELISA as described below.

[0219] Tissue preparation for PK analysis Tissue (50 mg) was homogenized in 10x tissue weight volume of cold 1% NP40 lysis buffer (1 mL 10% NP-40 Surfact-Amps detergent solution, 9 mL 1x PBS, 1 cOmplete protease inhibitor tablet, 1 PhosSTOP protease inhibitor tablet) using a Qiagen TissueLyzer II for 2x 3 min at 27 Hz with 3 mm stainless steel beads. The homogenate was then incubated on ice for 20 min and spun at 14,000 rpm for 20 min at 4°C. The resulting lysate was transferred to single-use aliquots and stored at -80°C.

[0220] IDS: IDS ELISA and PK analysis IgG:IDS was measured in serum, liver lysates and brain lysates using an iduronate-2-sulfatase sandwich ELISA. 384-well Maxisorp plates (Thermo, Cat. No. 464718) were coated overnight with anti-IDS antibody (R&D Systems, Cat. No. AF2449) and blocked the next day with casein-PBS buffer (Thermo, Cat. No. 37528). Samples containing IgG:IDS were added to the plate and incubated for 1 h at room temperature. After subsequent washing, biotinylated anti-IDS antibody (R&D Systems, Cat. No. BAF2449) was added to capture immobilized IgG:IDS and IgG:IDS. The IDS sandwich was then detected using a streptavidin-horseradish peroxidase conjugate (Jackson Immuno Research, Cat. No. 016-030-084) followed by incubation with 3,3',5,5' tetra-methyl-benzidine (TMB) substrate (Thermo, Cat. No. 34028). The reaction was quenched with 4N hydrochloric acid (Life Technologies, Cat. No. SS04) and the plate was read on a plate spectrophotometer at an absorbance wavelength of 450 nm. TMB color development is directly proportional to the concentration of IgG:IDS in the sample. A calibration curve was generated using a 5-parameter logistic fit with an assay range of 0.00137 nM to 1 nM. Serum, liver and brain exposure of IgG:IDS was calculated using NCA from the Vortex module of Dotmatics version 4.8 (Bishop's Stortford, UK). Semi-log-linear graphs with standard deviations and tabular results were curated in Prism 8 (GraphPad, San Diego, CA).

[0221] Pharmacodynamics of IgG:IDS TfR at 2-3 months of age mu / hu KI (n=5 / group) and Ids KO, TfR mu / huMice (n=5 / group) were administered a single dose of 0, 1 mg / kg, 3 mg / kg, or 10 mg / kg body weight IgG:IDS via the tail vein and sacrificed 7 days after administration. For final sample collection, animals were deeply anesthetized via intraperitoneal 2.5% Avertin injection. For CSF collection, a sagittal incision was made at the rear of the animal's skull, the subcutaneous tissue and muscle were separated to expose the cisterna magna, and a pre-tensioned glass capillary tube was used to puncture the cisterna magna to collect CSF. CSF was transferred to a Low Protein LoBind Eppendorf tube and centrifuged at 4°C for 10 minutes. CSF was transferred to a fresh tube and flash frozen on dry ice. The absence of blood contamination in mouse CSF was confirmed by measuring the absorbance of the samples at 420 nm. Blood, serum, and tissues were obtained as described above and flash frozen on dry ice.

[0222] Tissue and liquid preparations for pharmacodynamic GAG analysis 50 mg of tissue was homogenized in 750 μL of water using a Qiagen TissueLyzer II at 30 Hz for 3 min. The homogenates were transferred to a 96-well deep plate and sonicated with 10×1 sec pulses using a 96-tip sonicator (Q Sonica). The sonicated homogenates were spun at 2,500×g for 30 min at 4° C. The resulting lysates were transferred to a clean 96-well deep plate and BCA was performed to quantify total protein. 10 μg of total protein lysate or 3 μL of CSF was used for the subsequent HS / DS digestion. Digestion was performed in a PCR plate as follows: HS and DS internal standard mixture (20 ng total) was added to each sample and mixed with heparinase I, II, III and chondriotinase B in digestion buffer for 3 h at 30° C. with shaking. After digestion, EDTA was added to each sample and the mixture was boiled at 95° C. for 10 min. The digested samples were spun for 5 min and transferred to a cellulose acetate filter plate (Millipore, MSUN03010) and spun for 5 min. The resulting flow-through was mixed with an equal volume of acetonitrile in a glass vial and analyzed by mass spectrometry as described below.

[0223] Mass spectrometry of GAGsQuantification of GAGs was performed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled to electrospray mass spectrometry (Sciex QTRAP 6500+, Sciex, Framingham, MA, USA). For each analysis, samples were injected onto an ACQUITY UPLC BEH Amide 1.7 mm, 2.1 × 150 mm column (Waters Corporation) at a flow rate of 0.55 mL / min and a column temperature of 55 °C. Mobile phases A and B consisted of water with 10 mM ammonium formate and 0.1% formic acid, and acetonitrile with 0.1% formic acid, respectively. The gradient was programmed as follows: 80% B from 0.0 to 0.5 min, 80% B to 50% B from 0.5 to 3.5 min, 50% B to 80% B from 3.5 to 4.0 min, hold at 80% B from 4.0 to 4.5 min. Electrospray ionization was performed in negative ion mode applying the following settings: curtain gas 25, collision gas set to medium, ion spray voltage -4500, temperature 600 °C, ion source gas 1 50, ion source gas 2 60. Data acquisition was performed in multiple reaction monitoring mode (MRM) using Analyst 1.6.3 (Sciex) with various dwell times of 50 (msec). Collision energy (CE) was set to -30, declustering potential (DP) to -80, entrance potential (EP) to -10, collision cell exit potential (CXP) to -10. GAGs were detected as [MH]- using the following MRM transitions: D0A0 m / z 378.1>87.0, D0S0 m / z 416.1>138.0, D0a4 m / z 458.1>300.0, D4UA-2S-GlcNCOEt-6S (Iduron Ltd, Manchester, UK) m / z 472.0 (in source fragment ion)>97.0 was used as an internal standard. Individual disaccharide species were identified based on their retention time and MRM transitions using commercially available reference standards (Iduron Ltd). GAGs were quantified by peak area ratios of D0A0 (HS), D0S0 (HS) and D0a4 (DS / CS) relative to the internal standards using MultiQuant 3.0.2 (Sciex).Reported GAG amounts were normalized to total protein levels as measured by BCA assay (Pierce). TfR. mu / hu The fold increase in KI value was calculated as follows:

number

number

[0224] Heparan sulfate (HS) and dermatan sulfate (DS) calibration curves Pure standards of D0a4 (DS / CS), D0A0 (HS), and D0S0 (HS) were dissolved in acetonitrile:water 50 / 50 (v / v) to generate 1 mg / mL stocks. An 8-point dilution curve in PBS was generated ranging from 0.12 ng to 1000 ng. The internal standard D4UA-2S-GlcNCOEt-6S (20 ng) was then added to each serial dilution. Samples were then boiled at 95°C for 10 min and then spun to pellet particulate matter. The supernatant was filtered using a 30 kD MWCO cellulose acetate filter plate (Millipore, MSUN03010) by spinning at 3364xg for 5 min at room temperature. The resulting flow-through was mixed with an equal volume of acetonitrile in a glass vial and analyzed by mass spectrometry as described above.

[0225] result The pharmacokinetic profile of IgG:IDS is shown in Figure 4-Figure 6. Serum exposure of IgG:IDS is shown in Figure 4, demonstrating a dose-dependent effect. The IgG:IDS brain PK profile is shown in Figure 5, with maximum concentrations observed 4-8 hours after dosing. IgG:IDS liver PK levels are shown in Figure 6, indicating that IgG:IDS demonstrated good liver uptake.

[0226] To investigate the pharmacodynamic effects, mu / hu KI mice were administered a single intravenous dose of 1, 3, or 10 mg / kg IgG:IDS, and 7 days later, GAG levels (total GAG levels, HS levels, and DS levels) in the liver, CSF, and brain were evaluated (see Figures 7 to 9). mu / hu The fold increase in KI total GAG levels is shown in Table 2. Ids KO, TfR in liver, CSF and brain mu / hu Percentage reduction from KI+vehicle total GAG levels is shown in Table 3. Ids KO, TfR in liver, CSF and brain mu / hu The percent reduction from KI+vehicle HS and DS levels are shown in Tables 4 and 5, respectively.

[0227] [Table 2]

[0228] [Table 3]

[0229] [Table 4]

[0230] [Table 5]

[0231] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

[0232] All publications, patents, and patent documents are incorporated by reference herein as if individually incorporated by reference. The disclosure has been described with reference to various specific preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention.

Claims

1. 1. A fusion protein comprising: (a) an iduronate 2-sulfatase (IDS) amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof; and (b) an anti-human transferrin receptor antibody, i) a heavy chain complementarity determining region 1 (CDR-H1) comprising GYSFTNY (SEQ ID NO: 13), or a sequence having one or two substitutions compared to SEQ ID NO: 13; ii) a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15), or a sequence having one or two substitutions compared to SEQ ID NO: 15; iii) a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16), or a sequence having one or two substitutions compared to SEQ ID NO: 16; iv) a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7), or a sequence having one or two substitutions compared to SEQ ID NO: 7; v) a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO: 8), or a sequence having one or two substitutions compared to SEQ ID NO: 8; and vi) a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9), or a sequence having one or two substitutions compared to SEQ ID NO: 9; The fusion protein comprises at least about 6 mol / mol sialic acid:fusion protein and / or comprises about 1.5 to about 2.5 mol / mol mannose-6-phosphate (M6P):fusion protein.

2. The fusion protein of claim 1, wherein the fusion protein comprises at least about 6 mol / mol sialic acid:fusion protein, at least about 10 mol / mol sialic acid:fusion protein, or at least about 12 mol / mol sialic acid:fusion protein.

3. 2. The fusion protein of claim 1, wherein the fusion protein comprises about 6 to about 19 mol / mol sialic acid:fusion protein; about 12 to about 19 mol / mol sialic acid:fusion protein; or about 15 to about 16 mol / mol sialic acid:fusion protein.

4. The fusion protein of claim 1, wherein the fusion protein contains about 15 mol / mol sialic acid:fusion protein, or about 16 mol / mol sialic acid:fusion protein.

5. The fusion protein of claim 1, wherein the fusion protein comprises about 1.5 to about 2.5 mol / mol of mannose-6-phosphate (M6P):fusion protein.

6. The fusion protein of claim 5, wherein the fusion protein comprises about 1.5 mol / mol M6P:fusion protein; about 1.7 mol / mol M6P:fusion protein; or about 2.3 mol / mol M6P:fusion protein.

7. The antibody comprising: a) a heavy chain CDR-H1 comprising GYSFTNY (SEQ ID NO: 13); b) a heavy chain CDR-H2 comprising YPGGDY (SEQ ID NO: 15); c) a heavy chain CDR-H3 comprising SGNYDEVAY (SEQ ID NO: 16); d) a light chain CDR-L1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 7); e) a light chain CDR-L2 comprising KVSNRFS (SEQ ID NO:8); and The fusion protein of any one of claims 1 to 6, comprising: f) a light chain CDR-L3 comprising SQSTHVPWT (SEQ ID NO: 9).

8. The antibody, a) a light chain variable region comprising an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:6; b) a light chain comprising an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5; c) a heavy chain variable region comprising an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:11; and / or d) a heavy chain comprising an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:

10. The fusion protein according to any one of claims 1 to 7, comprising:

9. The fusion protein of any one of claims 1 to 7, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:5 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

10.

10. The fusion protein of any one of claims 1 to 9, wherein the IDS amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof is linked directly or via a linker to the C-terminus of the antibody heavy chain.

11. The fusion protein of claim 10, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:5, a heavy chain linked to an IDS amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof by a peptide linker comprising, in the N'-terminal to C'-terminal direction: SEQ ID NO:10, SEQ ID NO:23, and an IDS amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof.

12. The fusion protein of any one of claims 1 to 11, wherein the IDS amino acid sequence comprises an amino acid sequence having at least about 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 1, 2, 3 or 4.

13. The fusion protein of claim 12 , wherein the heavy chain linked to the IDS amino acid sequence comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 17 or 18.

14. The fusion protein of any one of claims 1 to 6, wherein the antibody comprises a light chain comprising SEQ ID NO:5 and a heavy chain linked to an IDS amino acid sequence comprising SEQ ID NO:

18.

15. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 14 and a pharma- ceutically acceptable excipient.

16. A host cell comprising a fusion protein according to any one of claims 1 to 14.

17. 15. A method of producing the fusion protein of any one of claims 1 to 14, comprising expressing in a host cell under conditions suitable for producing the fusion protein one or more polynucleotides operable to express 1) the antibody light chain, and 2) the antibody heavy chain linked to an IDS amino acid sequence, wherein the antibody light chain and the antibody heavy chain linked to an IDS amino acid sequence are as described in any one of claims 1 to 14.

18. A fusion protein produced by the method of claim 17.

19. A composition or pharmaceutical composition for reducing the levels of one or more GAG ​​species in a subject in need thereof, comprising a fusion protein according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15. (a) administration of the composition reduces the level of one or more GAG ​​species in the CSF of the subject by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 70%, wherein the reduction is compared to the level of the corresponding one or more GAG ​​species in the CSF of the subject prior to administration of the composition; and / or (b) the composition of claim 19, wherein the GAG ​​species is heparan sulfate or dermatan sulfate.

21. The composition of claim 19 or 20(a), wherein administration of the composition reduces the level of total GAGs in the CSF of the subject by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 70%, wherein the reduction is compared to the level of total GAGs in the CSF of the subject before administration of the composition.

22. Use of a fusion protein according to any one of claims 1 to 14 in the preparation of a medicament for reducing one or more GAG ​​species by administering said medicament to a subject in need thereof.

23. A composition or pharmaceutical composition for treating Hunter syndrome in a subject in need thereof, comprising a fusion protein according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15.

24. Use of a fusion protein according to any one of claims 1 to 14 in the preparation of a medicament for treating Hunter syndrome by administering said medicament to a subject in need thereof.

25. A composition described in any one of claims 19 to 21 and 23, or a use described in claim 22 or 24, characterized in that it is administered at a dose of about 1 mg / kg, 3 mg / kg or 10 mg / kg.

26. A composition described in any one of claims 19 to 21 and 23 to 24, or a use described in claim 22 or 24, characterized in that it is administered weekly.

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