Fusion proteins containing alpha-L-iduronidase enzyme and methods thereof

Abstract

Provided herein are proteins that can be transported across the blood-brain barrier (BBB) ​​and that include an alpha-L-iduronidase (IDUA) enzyme-Fc fusion polypeptide. Certain embodiments also provide methods of using such proteins to treat MPS I.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 291,283, filed December 17, 2021. The entire contents of the above-referenced applications are incorporated herein by reference. [Background technology]

[0002] Mucopolysaccharidosis type I (MPS I) (or Hurler syndrome) is a lysosomal storage disorder caused by genetic mutations in the IDUA gene. These mutations result in reduced or absent function of the alpha-L-iduronidase (IDUA) protein, leading to the accumulation of the glycosaminoglycans dermatan sulfate and heparan sulfate, and alterations in multiple organs and tissues, including the skeleton, heart, respiratory system, and brain. Treatment of MPS I remains largely conservative; the missing enzyme may be administered intravenously, but is largely ineffective in the brain due to the difficulty of delivering recombinant enzyme across the blood-brain barrier (BBB). Thus, more effective therapies are needed to treat MPS I symptoms and IDUA deficiency in both the CNS (central nervous system) and peripheral nerves. Summary of the Invention [Means for solving the problem]

[0003] Thus, provided herein are certain enzyme replacement therapies that have the ability to cross the BBB and treat both peripheral and CNS symptoms of MPS I. In particular, certain embodiments provide proteins comprising: (a) a first Fc polypeptide linked to an alpha-L-iduronidase (IDUA) amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; and (b) a second Fc polypeptide; the first and / or second Fc polypeptide is a modified Fc that can bind (e.g., can specifically bind) to a blood-brain barrier (BBB) ​​receptor, such as the transferrin receptor (TfR). In certain embodiments, the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide.

[0004] In certain embodiments, the first Fc polypeptide is a modified Fc that can bind (e.g., specifically binds) to the TfR. In certain embodiments, the first Fc polypeptide is a modified Fc that comprises a sequence having at least 90% identity to SEQ ID NO:28 or 98 (e.g., SEQ ID NO:28) and can specifically bind to the TfR.

[0005] In certain embodiments, the first Fc polypeptide 1) comprises a sequence having at least 90% identity to SEQ ID NO:28 or 98 (e.g., SEQ ID NO:28); 2) is capable of specifically binding to TfR; and 3) has an Ala at position 389 according to EU numbering. In certain embodiments, the first Fc polypeptide further comprises a Glu at position 380 and an Asn at position 390 according to EU numbering. In some embodiments, the first Fc polypeptide further comprises a Tyr at position 384; a Thr at position 386; a Glu at position 387; a Trp at position 388; a Thr at position 413; a Glu at position 415; a Glu at position 416; and a Phe at position 421 according to EU numbering.

[0006] In certain embodiments, the second Fc polypeptide is a modified Fc that can bind (e.g., specifically bind) to the TfR. In certain embodiments, the second Fc polypeptide is a modified Fc that comprises a sequence having at least 90% identity to SEQ ID NO: 28 or 98 (e.g., SEQ ID NO: 28) and can specifically bind to the TfR.

[0007] In certain embodiments, the second Fc polypeptide 1) comprises a sequence having at least 90% identity to SEQ ID NO:28 or 98 (e.g., SEQ ID NO:28); 2) is capable of specifically binding to TfR; and 3) has an Ala at position 389 according to EU numbering. In certain embodiments, the second Fc polypeptide further comprises a Glu at position 380 and an Asn at position 390 according to EU numbering. In certain embodiments, the second Fc polypeptide further comprises a Tyr at position 384; a Thr at position 386; a Glu at position 387; a Trp at position 388; a Thr at position 413; a Glu at position 415; a Glu at position 416; and a Phe at position 421 according to EU numbering.

[0008] Certain embodiments provide a protein comprising: a. a first Fc polypeptide linked to an alpha-L-iduronidase (IDUA) amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; and b. A second Fc polypeptide comprising a sequence having at least 90% identity to SEQ ID NO:28 and capable of specifically binding to the transferrin receptor (TfR).

[0009] In a specific embodiment, the second Fc polypeptide has an Ala at position 389 according to EU numbering.

[0010] In a specific embodiment, said second Fc polypeptide further comprises a Glu at position 380; and an Asn at position 390 according to EU numbering.

[0011] In certain embodiments, the second Fc polypeptide further comprises the following positions according to EU numbering: Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.

[0012] In certain embodiments, the protein can be transported across the blood-brain barrier of a subject.

[0013] In certain embodiments, the protein binds to TfR with an affinity of about 100 nM to about 500 nM.

[0014] In certain embodiments, the protein binds to TfR with an affinity of about 200 to about 400 nM.

[0015] In certain embodiments, the second Fc polypeptide binds to the apical domain of the TfR.

[0016] In certain embodiments, binding of the protein to the TfR does not substantially inhibit binding of transferrin to the TfR.

[0017] In certain embodiments, the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to any one of SEQ ID NOs: 39, 40, 45, 78, and 99.

[0018] In certain embodiments, the IDUA amino acid sequence comprises the amino acid sequence of any one of SEQ ID NOs: 39, 40, 45, 78, and 99.

[0019] In certain embodiments, the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to any one of SEQ ID NOs:41-44.

[0020] In certain embodiments, the IDUA amino acid sequence comprises the amino acid sequence of any one of SEQ ID NOs:41-44.

[0021] In certain embodiments, the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to any one of SEQ ID NOs:46-49.

[0022] In certain embodiments, the IDUA amino acid sequence comprises the amino acid sequence of any one of SEQ ID NOs:46-49.

[0023] In certain embodiments, the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to any one of SEQ ID NOs:79-82.

[0024] In certain embodiments, the IDUA amino acid sequence comprises the amino acid sequence of any one of SEQ ID NOs:79-82.

[0025] In certain embodiments, the first Fc polypeptide is linked to the IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof by a peptide bond or by a polypeptide linker. In certain embodiments, the first Fc polypeptide is linked to the IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof by a peptide bond. In certain embodiments, the first Fc polypeptide is linked to the IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof by a peptide linker.

[0026] In certain embodiments, the polypeptide linker is a flexible polypeptide linker.

[0027] In certain embodiments, the flexible polypeptide linker is a glycine-rich linker.

[0028] In certain embodiments, the polypeptide linker is GS (SEQ ID NO: 71), G4S (SEQ ID NO: 72) or (G4S)2 (SEQ ID NO: 73).

[0029] In certain embodiments, the N-terminus of the first Fc polypeptide is linked to an IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0030] In certain embodiments, the C-terminus of the first Fc polypeptide is linked to an IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0031] In certain embodiments, the fusion proteins described herein comprise a single IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0032] In certain embodiments, this second Fc polypeptide forms an Fc dimer with the first Fc polypeptide.

[0033] In certain embodiments, the first Fc polypeptide and the second Fc polypeptide each comprise a modification that promotes heterodimerization.

[0034] In a specific embodiment, one of the Fc polypeptides has a T366W substitution and the other Fc polypeptide has T366S, L368A, and Y407V substitutions according to EU numbering.

[0035] In a specific embodiment, the first Fc polypeptide contains a T366S, L368A, and Y407V substitution, and the second Fc polypeptide comprises a T366W substitution.

[0036] In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 9-16 and 19-22; and the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 25-32 and 35-38.

[0037] In a specific embodiment, the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A, and Y407V substitutions.

[0038] In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 17-18 and 74-75; and the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 33-34 and 97-98.

[0039] In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises a native FcRn binding site.

[0040] In certain embodiments, the first Fc polypeptide and the second Fc polypeptide have no effector function.

[0041] In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises a modification that reduces effector function.

[0042] In certain embodiments, the modification that reduces effector function is a substitution, according to EU numbering, of Ala at position 234 and Ala at position 235; Ala at position 234, Ala at position 235 and Gly at position 329; or Ala at position 234, Ala at position 235 and Ser at position 329.

[0043] In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 11-16, and 19-22.

[0044] In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:11, 12, 19, and 20.

[0045] In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 15, 16, 21, and 22.

[0046] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 50-69 and 83-92.

[0047] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:50-53.

[0048] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:54-57.

[0049] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:83-86.

[0050] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 58-61 and 91-92.

[0051] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:62-65.

[0052] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:87-90.

[0053] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:66-67.

[0054] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:68-69.

[0055] In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:27-32 and 35-38.

[0056] In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:27, 28, 35, and 36.

[0057] In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:31, 32, 37, and 38.

[0058] In a specific embodiment, the first Fc polypeptide linked to the IDUA amino acid sequence comprises any one of the amino acid sequences of SEQ ID NOs: 50-65 and 83-92; and the second Fc polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 35-38.

[0059] In a specific embodiment, the first Fc polypeptide linked to the IDUA amino acid sequence comprises any one of the amino acid sequences of SEQ ID NOs: 50-57 and 83-86; and the second Fc polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 35-36.

[0060] In a specific embodiment, the first Fc polypeptide linked to the IDUA amino acid sequence comprises any one of the amino acid sequences of SEQ ID NOs: 58-65 and 87-92; and the second Fc polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 37-38.

[0061] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises the amino acid sequence of any one of SEQ ID NOs: 66-69; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 35-38.

[0062] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises the amino acid sequence of any one of SEQ ID NOs: 66-67; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 35-36.

[0063] In certain embodiments, the first Fc polypeptide linked to the IDUA amino acid sequence comprises any one of the amino acid sequences of SEQ ID NOs: 68-69; and the second Fc polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 37-38.

[0064] In certain embodiments, uptake of the IDUA amino acid sequence into the brain is at least 5-fold greater than uptake of the IDUA amino acid sequence in the absence of the first Fc polypeptide and the second Fc polypeptide, or compared to uptake of unmodified IDUA enzyme in the presence of the second Fc polypeptide that results in TfR binding.

[0065] In certain embodiments, the first Fc polypeptide is not modified to bind to a blood-brain barrier (BBB) ​​receptor and the second Fc polypeptide is modified to specifically bind to TfR.

[0066] In certain embodiments, the protein does not include immunoglobulin heavy and / or light chain variable region sequences or antigen-binding portions thereof.

[0067] In certain embodiments, a polypeptide is provided comprising an Fc polypeptide linked to an alpha-L-iduronidase (IDUA) amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof, wherein the Fc polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 12 and comprises one or more modifications that promote heterodimerization with another Fc polypeptide.

[0068] In certain embodiments, the Fc polypeptide is linked to the IDUA enzyme, IDUA variant amino acid sequence, or catalytically active fragment thereof by a peptide bond or by a polypeptide linker.

[0069] In certain embodiments, the polypeptide comprises, from N-terminus to C-terminus: an IDUA enzyme, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; a polypeptide linker; and an Fc polypeptide.

[0070] In certain embodiments, the polypeptide comprises, from N-terminus to C-terminus: an Fc polypeptide; a polypeptide linker; and an IDUA enzyme, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0071] In certain embodiments, the Fc polypeptide comprises T366S, L368A and Y407V substitutions according to EU numbering.

[0072] In certain embodiments, the Fc polypeptide comprises the following substitutions, according to EU numbering: Ala at position 234 and Ala at position 235; Ala at position 234, Ala at position 235 and Gly at position 329; or Ala at position 234, Ala at position 235 and Ser at position 329.

[0073] In certain embodiments, the polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 50-69 and 83-92.

[0074] In certain embodiments, a protein is provided that comprises: 1) a polypeptide comprising an Fc polypeptide linked to an alpha-L-iduronidase (IDUA) amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof, wherein the Fc polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 12 and contains one or more modifications that promote heterodimerization with another Fc polypeptide; and 2) another Fc polypeptide.

[0075] Certain embodiments provide a pharmaceutical composition comprising a fusion protein described herein or a polypeptide described herein and a pharma- ceutically acceptable carrier and / or excipient.

[0076] Certain embodiments provide a polynucleotide comprising a nucleic acid sequence encoding a polypeptide described herein (eg, an IDUA-Fc fusion polypeptide described herein).

[0077] Certain embodiments provide vectors comprising the polynucleotides described herein.

[0078] Certain embodiments provide a host cell comprising a polynucleotide described herein or a vector described herein, in certain embodiments, the host cell further comprises a polynucleotide comprising a nucleic acid sequence encoding another polypeptide described herein (e.g., another Fc polypeptide, such as a TfR binding modified Fc polypeptide).

[0079] Certain embodiments provide a method for producing a polypeptide comprising an Fc polypeptide linked to an IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof, comprising culturing a host cell under conditions in which a polypeptide encoded by a polynucleotide described herein is expressed.

[0080] Certain embodiments provide pairs of polynucleotides comprising: a first nucleic acid sequence encoding a first Fc polypeptide linked to an IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; and a second nucleic acid sequence encoding a second Fc polypeptide, as described herein.

[0081] Certain embodiments also provide one or more vectors that comprise a pair of polynucleotides described herein.For example, certain embodiments provide a single vector that comprises a pair of polynucleotides.Other embodiments provide two vectors, the first vector comprises the first polynucleotide from the pair, and the second vector comprises the second polynucleotide from the pair.

[0082] Certain embodiments provide host cells comprising a pair of polynucleotides described herein, or one or more of the vectors described herein. Certain embodiments provide a method for producing a protein comprising a first Fc polypeptide and a second Fc polypeptide linked to an IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof, comprising culturing a host cell under conditions in which a pair of polynucleotides described herein are expressed.

[0083] Certain embodiments provide a method of treating MPS I, comprising administering to a patient in need thereof a protein as described herein or a polypeptide as described herein. In certain embodiments, a therapeutically effective amount of the protein or polypeptide is administered.

[0084] Certain embodiments provide a protein as described herein, or a polypeptide as described herein, for use in the treatment of MPS I in a patient in need thereof.

[0085] Certain embodiments provide for the use of a protein as described herein, or a polypeptide as described herein, in the preparation of a medicament for the treatment of MPS I in a patient in need thereof.

[0086] Certain embodiments provide a method of reducing accumulation of toxic metabolites in a patient with MPS I, comprising administering to the patient a protein described herein or a polypeptide described herein. In certain embodiments, a therapeutically effective amount (e.g., a therapeutically effective amount) of the protein or polypeptide is administered.

[0087] Certain embodiments provide a protein as described herein, or a polypeptide as described herein, for use in reducing the accumulation of toxic metabolites in a patient with MPS I.

[0088] Certain embodiments provide for the use of a protein as described herein or a polypeptide as described herein in the preparation of a medicament for reducing the accumulation of toxic metabolites in a patient with MPS I.

[0089] In certain embodiments, the toxic metabolite comprises heparan sulfate-derived oligosaccharides or dermatan sulfate-derived oligosaccharides. [Brief description of the drawings]

[0090] [Figure 1] Illustrative diagram of exemplary ETV:IDUA fusion proteins, in which the IDUA enzyme is fused to the N-terminus of an Fc polypeptide (A) or to the C-terminus of an Fc polypeptide (B). [Diagram 2]In vitro evaluation of the enzymatic activity of IDUA-Fc fusion proteins and Aldurazyme (laronidase). [Diagram 3] Assessment of the cellular activity of ETV:IDUA Fusion 1 compared to Laronidase in fibroblasts from Hurler (MPS I) patients and healthy controls using LCMS quantification of heparan sulfate and dermatan sulfate. [Figure 4A] Evaluation of ETV:IDUA Fusion 3 serum PK in TfR knock-in mice ("TfRmu / huKI"). [Figure 4B] Evaluation of ETV:IDUA Fusion 4 serum PK in TfR knock-in mice ("TfRmu / huKI"). [Figure 4C] Evaluation of ETV:IDUA Fusion 6 serum PK in TfR knock-in mice ("TfRmu / huKI"). [Figure 5A] Evaluation of ETV:IDUA Fusion 3 brain PK in TfR knock-in mice ("TfRmu / huKI"). [Figure 5B] Assessment of ETV:IDUA Fusion 4 brain PK in TfR knock-in mice ("TfRmu / huKI"). [Figure 5C] Assessment of ETV:IDUA Fusion 6 brain PK in TfR knock-in mice ("TfRmu / huKI"). [Figure 6A] Evaluation of pharmacodynamic responses (total GAG levels) in CSF in a comparative study performed in healthy and diseased mouse models of MPS I. The healthy mouse model is represented by TfR knock-in mice ("TfRmu / hu") and the diseased mouse model is represented by TfR knock-in mice in which the IDUA gene is knocked out ("IDUA KO;TfRmu / hu"). Graphs show mean ± SEM and p-values: one-way ANOVA, Dunnett's multiple comparison test; ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. [Figure 6B]Evaluation of pharmacodynamic responses (total GAG levels) in the brain in a comparative study performed in healthy and diseased mouse models of MPS I. The healthy mouse model is represented by TfR knock-in mice ("TfRmu / hu") and the diseased mouse model is represented by TfR knock-in mice in which the IDUA gene is knocked out ("IDUA KO;TfRmu / hu"). Graphs show mean ± SEM and p-values: one-way ANOVA, Dunnett's multiple comparison test; ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. [Figure 6C] Evaluation of pharmacodynamic responses (total GAG levels) in the liver in a comparative study performed in healthy and diseased mouse models of MPS I. The healthy mouse model is represented by TfR knock-in mice ("TfRmu / hu") and the diseased mouse model is represented by TfR knock-in mice in which the IDUA gene has been knocked out ("IDUA KO;TfRmu / hu"). Graphs show mean ± SEM and p-values: one-way ANOVA, Dunnett's multiple comparison test; ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. [Figure 6D] Evaluation of pharmacodynamic responses (total GAG levels) in urine in a comparative study performed in healthy and diseased mouse models of MPS I. The healthy mouse model is represented by TfR knock-in mice ("TfRmu / hu") and the diseased mouse model is represented by TfR knock-in mice in which the IDUA gene has been knocked out ("IDUA KO;TfRmu / hu"). Graphs show mean ± SEM and p-values: one-way ANOVA, Dunnett's multiple comparison test; ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0091] Currently, there is a need for new therapeutics for the treatment of MPS I, particularly those that treat severe MPS I with a neurocognitive phenotype. Described herein is a specific enzyme replacement therapy, termed ETV:IDUA, that has the ability to cross the BBB and treat both peripheral and CNS symptoms of MPS I. As used herein, the term "ETV:IDUA" refers to a protein (e.g., a dimeric protein) that can be transported across the BBB and includes a first Fc polypeptide linked (e.g., fused) to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof; and a second Fc polypeptide.

[0092] Protein molecules comprising IDUA enzyme-Fc fusion polypeptides As described herein, certain embodiments provide protein molecules comprising an IDUA enzyme-Fc fusion polypeptide. The IDUA enzyme incorporated into the protein is catalytically active, i.e., retains enzymatic activity. In some aspects, the protein described herein comprises: (a) a first Fc polypeptide, which may comprise a modification (e.g., one or more modifications that promote heterodimerization) or may be a wild-type Fc polypeptide; and an IDUA enzyme; and (b) a second Fc polypeptide, which may comprise a modification (e.g., one or more modifications that promote heterodimerization) or may be a wild-type Fc polypeptide; and optionally an IDUA enzyme, wherein the first and / or second Fc polypeptide comprises a modification that results in binding to a blood-brain barrier (BBB) ​​receptor, e.g., the transferrin receptor (TfR).

[0093] In some embodiments, the proteins described herein comprise the full-length IDUA wild-type sequence. In some embodiments, the proteins described herein comprise the mature IDUA wild-type sequence. As described herein, several polymorphisms have been reported in the wild-type IDUA protein sequence. For example, the IDUA enzyme may comprise an H or Q at position 33 and / or an A or T at position 622 according to the EU numbering. In some embodiments, the proteins described herein comprise catalytically active fragments or variants of the wild-type IDUA sequence. For example, the IDUA enzyme may comprise an E at position 27 according to the EU numbering, or the amino acid may be absent. Other IDUA enzyme truncations are also described herein. Thus, in some embodiments, the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 39-49, 78-82, and 99, or comprises the amino acid sequence of SEQ ID NOs: 39-49, 78-82, and 99. In some embodiments, the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 39, 40, 45, 78, and 99, or comprises the amino acid sequence of any one of SEQ ID NOs: 39, 40, 45, 78, and 99. In certain embodiments, X1 is H in such sequences. In certain embodiments, in such sequences, X1 is Q. In certain embodiments, in such sequences, X2 is A. In certain embodiments, in such sequences, X2 is T. In certain embodiments, in such sequences, X3 is E. In certain embodiments, in such sequences, X3 is absent.In some embodiments, the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 41-44, or comprises the amino acid sequence of any one of SEQ ID NOs: 41-44. In some embodiments, the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 46-49, or comprises the amino acid sequence of any one of SEQ ID NOs: 46-49. In some embodiments, the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 79-82, or comprises the amino acid sequence of any one of SEQ ID NOs: 79-82.

[0094] As discussed above, in some embodiments, the IDUA enzyme is a variant or catalytically active fragment of an IDUA protein (e.g., comprising an IDUA amino acid sequence described herein). In some embodiments, a catalytically active variant or fragment of an IDUA 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 a wild-type IDUA enzyme.

[0095] In some embodiments, the IDUA enzyme, or catalytically active variant or fragment thereof, present in the proteins described herein retains at least 25% of its activity compared to its activity when not conjugated to an Fc polypeptide or a TfR-binding Fc polypeptide. In some embodiments, the IDUA 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 conjugated to an Fc polypeptide or a TfR-binding Fc polypeptide. In some embodiments, the IDUA 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 conjugated to an Fc polypeptide or a TfR-binding Fc polypeptide. In some embodiments, fusion to an Fc polypeptide does not reduce the activity of the IDUA enzyme, or a catalytically active variant or fragment thereof. In some embodiments, fusion to a TfR-binding Fc polypeptide does not reduce the activity of the IDUA enzyme.

[0096] Fc Polypeptide Modification The Fc polypeptide incorporated in the fusion proteins described herein may include certain modifications. For example, the Fc polypeptide may include a modification that results in binding to a blood-brain barrier (BBB) ​​receptor, e.g., the transferrin receptor (TfR). In addition, the Fc polypeptide may include other modifications, such as modifications that promote heterodimerization, increase serum stability or serum half-life, modulate effector function, affect glycosylation, and / or reduce immunogenicity in humans. Thus, in certain embodiments, the fusion proteins described herein include two Fc polypeptides, one Fc being a wild-type Fc polypeptide, e.g., a human IgG1 Fc polypeptide; the other Fc being modified to bind to a blood-brain barrier (BBB) ​​receptor, e.g., the transferrin receptor (TfR), and optionally further comprising one or more additional modifications. In certain other embodiments, both Fc polypeptides each include an independently selected modification (e.g., a modification described herein). For example, in certain embodiments, the fusion proteins described herein comprise two Fc polypeptides, where one Fc is not modified to bind to a BBB receptor, but comprises one or more other modifications described herein; the other Fc is modified to bind to a blood-brain barrier (BBB) ​​receptor, e.g., the transferrin receptor (TfR), and optionally further comprises one or more additional modifications. In certain other embodiments, the fusion proteins described herein comprise two Fc polypeptides, where both Fc polypeptides are modified to bind to a blood-brain barrier (BBB) ​​receptor, e.g., the transferrin receptor (TfR), and optionally further comprise one or more additional modifications.

[0097] The amino acid residues specified in the various Fc modifications, including those introduced into modified Fc polypeptides that bind to BBB receptors, e.g., TfR, are numbered herein using EU index numbering. Any Fc polypeptide, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc polypeptide, can have a modification, e.g., an amino acid substitution, at one or more positions described herein.

[0098] The modified Fc polypeptide present in the fusion proteins described herein (e.g., that enhances heterodimerization and / or BBB receptor binding) may have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to a native Fc region sequence or a fragment thereof, e.g., a fragment at least 50 amino acids in length or at least 100 amino acids in length, or more. In some embodiments, the amino acid sequence of the native Fc is the Fc region sequence of SEQ ID NO:1. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 1-110 of SEQ ID NO:1 or amino acids 111-217 of SEQ ID NO:1, or a fragment thereof, e.g., a fragment at least 50 amino acids in length or at least 100 amino acids in length or more.

[0099] In some embodiments, the modified (e.g., heterodimerization and / or BBB receptor binding enhancing) Fc polypeptide comprises at least 50 amino acids, or at least 60, 65, 70, 75, 80, 85, 90 or 95 or more, or at least 100 or more amino acids, corresponding to the amino acid sequence of a native Fc region. In some embodiments, the modified Fc polypeptide comprises at least 25 contiguous amino acids, or at least 30, 35, 40 or 45 contiguous amino acids, or 50 contiguous amino acids, or at least 60, 65, 70, 75, 80, 85, 90 or 95 or more contiguous amino acids, or 100 or more contiguous amino acids, corresponding to the amino acid sequence of a native Fc region, such as SEQ ID NO:1.

[0100] Modifications for blood-brain barrier (BBB) ​​receptor binding In some aspects, provided herein is a fusion protein that can be transported across the blood-brain barrier (BBB). Such proteins include modified Fc polypeptides that bind to BBB receptors. BBB receptors are expressed in the BBB endothelium, as well as other cell and tissue types. In some embodiments, the BBB receptor is the transferrin receptor (TfR).

[0101] In some embodiments, the fusion proteins described herein specifically bind to the TfR. In some embodiments, the fusion proteins described herein specifically bind to the TfR with an affinity of about 50 nM to about 500 nM. In some embodiments, the protein binds (e.g., specifically binds) to TfR with an affinity of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nM. In some embodiments, the protein binds to TfR with an affinity of about 100 to about 500 nM. In some embodiments, the protein binds to TfR with an affinity of about 100 nM to about 300 nM, or about 200 nM to about 450 nM. In some embodiments, the protein binds to TfR with an affinity of about 250 nM. In certain embodiments, the protein binds to TfR with an affinity of about 150 to about 400 nM, or about 200 to about 400 nM, or about 250 nM to about 350 nM, or about 300 to about 350 nM.

[0102] In some embodiments, modified Fc polypeptides that specifically bind to TfR comprise substitutions in the CH3 domain. In some embodiments, modified Fc polypeptides comprise a human Ig CH3 domain, such as an IgG CH3 domain, that is modified for TfR binding activity. The CH3 domain can be from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH3 domain refers to the segment of amino acids from about position 341 to about position 447, numbered according to the EU numbering scheme.

[0103] In some embodiments, an engineered Fc polypeptide that specifically binds to TfR can bind to the apical domain of TfR and bind to TfR without blocking or otherwise inhibiting transferrin binding to TfR. In some embodiments, transferrin binding to TfR is not substantially inhibited. In some embodiments, transferrin binding to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, transferrin binding to TfR is inhibited by less than about 20% (e.g., less than about 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0104] In some embodiments, the modified (e.g., BBB receptor binding) Fc polypeptide present in the fusion proteins described herein comprises substitutions at amino acid positions 384, 386, 387, 388, 389, 413, 415, 416 and 421 according to the EU numbering scheme.

[0105] In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises an Ala at position 389 according to EU numbering. In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.

[0106] In additional embodiments, the modified Fc polypeptide further comprises one, two or three substitutions at positions including 414, 424 and 426 according to the EU numbering scheme. In some embodiments, position 414 is Lys, Arg, Gly or Pro, position 424 is Ser, Thr, Glu or Lys, and / or position 426 is Ser, Trp or Gly.

[0107] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 111-217 of SEQ ID NO:23; and comprises amino acids at EU index positions 380, 384-390, and / or 413-421 of SEQ ID NO:23. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 111-216 of SEQ ID NO:24; and comprises amino acids at EU index positions 380, 384-390, and / or 413-421 of SEQ ID NO:23 or 24. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:23 or 24; and comprises amino acids at EU index positions 380, 384-390, and / or 413-421 of SEQ ID NO:23 or 24.

[0108] In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:23 or 24 and has an Ala at position 389 according to EU numbering. In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:23 or 24 and comprises the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 23 or 24 and comprises the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.

[0109] In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:23 or 24.

[0110] Additional Fc Polypeptide Mutations In some embodiments, the fusion proteins described herein comprise two Fc polypeptides, where one or both Fc polypeptides each comprise an independently selected modification (e.g., a modification described herein). Non-limiting examples of other mutations that can be introduced into one or both Fc polypeptides include, for example, mutations that increase the serum stability or serum half-life of the Fc polypeptide, mutations that modulate the effector function of the Fc polypeptide, mutations that affect the glycosylation of the Fc polypeptide, mutations that reduce the immunogenicity of the Fc polypeptide in humans, and / or mutations that result in the knob and hole heterodimerization of the Fc polypeptide. Examples of various modifications that can be included in the Fc polypeptide are described in WO2019 / 070577, which is incorporated by reference in its entirety for all purposes. In some embodiments, each Fc polypeptide present in the fusion protein independently has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a corresponding wild-type Fc polypeptide (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide).

[0111] In some embodiments, the Fc polypeptide present in the fusion protein has knob and hole mutations that promote heterodimer formation and prevent homodimer formation. Generally, the modification introduces a protrusion ("knob") at the interface of one polypeptide and a corresponding cavity ("hole") at the interface of another polypeptide, such that the protrusion can be located within the cavity to promote heterodimer formation and thereby prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing a large amino acid side chain with a smaller one (e.g., alanine or threonine). In some embodiments, such additional mutations are in positions in the Fc polypeptide that do not adversely affect binding of the polypeptide to a BBB receptor, e.g., TfR.

[0112] In one exemplary embodiment of the knob and hole approach for dimerization, position 366 (numbered according to the EU numbering scheme) of one of the Fc polypeptides present in the fusion protein contains a tryptophan instead of the native threonine. The other Fc polypeptide of the dimer has a valine instead of the native tyrosine at position 407 (numbered according to the EU numbering scheme). The other Fc polypeptide may further contain substitutions, in which the native threonine at position 366 (numbered according to the EU numbering scheme) is replaced with a serine and the native leucine at position 368 (numbered according to the EU numbering scheme) is replaced with an alanine. Thus, one of the Fc polypeptides of the fusion proteins described herein has a T366W knob mutation and the other Fc polypeptide has a Y407V mutation, which is usually accompanied by T366S and L368A hole mutations. In certain embodiments, the first Fc polypeptide contains a T366S, L368A, and Y407V substitution and the second Fc polypeptide contains a T366W substitution. In certain other embodiments, the first Fc polypeptide contains a T366W substitution and the second Fc polypeptide contains a T366S, L368A, and Y407V substitution.

[0113] In some embodiments, modifications to enhance serum half-life may be introduced. For example, in some embodiments, one or both Fc polypeptides present in a fusion protein described herein may contain a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256, numbered according to the EU numbering scheme. Thus, one or both Fc polypeptides may have M252Y, S254T, and T256E substitutions. Alternatively, one or both Fc polypeptides may have M428L and N434S substitutions, numbered according to the EU numbering scheme. Alternatively, one or both Fc polypeptides may have N434S or N434A substitutions.

[0114] In some embodiments, one or both Fc polypeptides present in the fusion proteins described herein may contain a modification that reduces effector function, i.e., a modification that reduces the ability to induce a particular biological function upon binding to an Fc receptor expressed on an effector cell that mediates the effector function. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector functions may vary depending on the class of antibody. For example, native human IgG1 and IgG3 antibodies can induce ADCC and CDC activity upon binding to the appropriate Fc receptor present on immune system cells; native human IgG1, IgG2, IgG3, and IgG4 can induce ADCP function upon binding to the appropriate Fc receptor present on immune cells.

[0115] In some embodiments, one or both Fc polypeptides present in the fusion proteins described herein may be engineered to contain other modifications for heterodimerization, such as electrostatic manipulation of contact residues within the naturally charged CH3-CH3 interface or hydrophobic patch modifications.

[0116] In some embodiments, one or both Fc polypeptides present in the fusion proteins described herein may contain additional modifications that modulate effector function.

[0117] In some embodiments, one or both Fc polypeptides present in the fusion proteins described herein may comprise a modification that reduces or eliminates effector function. Exemplary Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in the CH2 domain, e.g., at positions 234 and 235 according to the EU numbering scheme. For example, in some embodiments, one or both Fc polypeptides may comprise alanine residues at positions 234 and 235. Thus, one or both Fc polypeptides may have L234A and L235A (LALA) substitutions. Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, the following: a mutation at position 329 in which proline is substituted with glycine, serine, or arginine, or with an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and tryptophan residues Trp87 and Trp110 of FcγRIII. Further exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S according to the EU numbering scheme. Multiple substitutions may also be present, for example, according to the EU numbering scheme, L234A and L235A in the human IgG1 Fc region; L234A, L235A, and P329G in the human IgG1 Fc region; L234A, L235A, and P329S in the human IgG1 Fc region; S228P and L235E in the human IgG4 Fc region; L234A and G237A in the human IgG1 Fc region; L234A, L235A, and G237A in the human IgG1 Fc region; V234A and G237A in the human IgG2 Fc region; L235A, G237A, and E318A in the human IgG4 Fc region; and S228P and L236E in the human IgG4 Fc region. In some embodiments, one or both Fc polypeptides may have one or more amino acid substitutions that modulate ADCC, for example substitutions at positions 298, 333, and / or 334 according to the EU numbering scheme.

[0118] In some embodiments, the C-terminal Lys residue is removed in the Fc polypeptides described herein (ie, the Lys residue at position 447, according to the EU numbering scheme).

[0119] Exemplary Fc Polypeptides Containing Additional Mutations As non-limiting examples described herein, one or both Fc polypeptides present in the fusion proteins described herein may contain a knob mutation (e.g., T366W as numbered according to the EU numbering scheme), a hole mutation (e.g., T366S, L368A, and Y407V as numbered according to the EU numbering scheme), a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P323C ... or a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P323C as numbered according to the EU numbering scheme). 9S (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and / or mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E, numbered with reference to EU numbering, or (ii) N434S with or without M428L, numbered according to the EU numbering scheme). By way of example, SEQ ID NOs: 9-22, 25-38, 74-77, and 97-98 provide non-limiting examples of modified Fc polypeptides that include one or more of these additional mutations.

[0120] In some embodiments, an Fc polypeptide or modified Fc polypeptide may have a knob mutation (e.g., T366W, numbered according to the EU numbering scheme) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 1, 2, 23, and 24. In some embodiments, an Fc polypeptide or modified Fv polypeptide having the sequence of any one of SEQ ID NOs: 1, 2, 23, and 24 may be modified to have a knob mutation.

[0121] In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W, numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 17 and 18. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 17 and 18.

[0122] In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W, as numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 25 and 26. In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W, as numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 25 and 26, and comprises an Ala at position 389 according to the EU numbering. In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W, numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 25 or 26, and contains Glu at position 380; Ala at position 389; and Asn at position 390 according to EU numbering. In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W, numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:25 or 26, and comprises at the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs:25 and 26.

[0123] In some embodiments, the Fc polypeptide or modified Fc polypeptide may have a knob mutation (e.g., T366W as numbered according to the EU numbering scheme), a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S as numbered according to the EU numbering scheme (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of the sequences of SEQ ID NOs: 1, 2, 23, and 24. In some embodiments, an Fc polypeptide or modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 1, 2, 23, and 24 may be modified to have knob mutations and mutations that modulate effector function.

[0124] In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W, as numbered with reference to EU numbering), as well as a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S, as numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 74-75 and 76-77. In some embodiments, the modified Fc polypeptide comprises any one of SEQ ID NOs: 74-75 and 76-77.

[0125] In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering) and a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S as numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 27-32 and 35-38. In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering) and a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S as numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 27-32 and 35-38, and comprises an Ala at position 389 according to the EU numbering.In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering), as well as a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S as numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 27-32 and 35-38, and comprises the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W, as numbered with reference to EU numbering) and a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S, as numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and has at least 85% similarity to any one of the sequences of SEQ ID NOs: 27-32 and 35-38. at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, and comprises the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 27-32 and 35-38.

[0126] In some embodiments, an Fc polypeptide or modified Fc polypeptide may have a whole mutation (e.g., T366S, L368A and Y407V, numbered according to the EU numbering scheme) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 1, 2, 23, and 24. In some embodiments, an Fc polypeptide or modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 1, 2, 23, and 24 may be modified to have a whole mutation.

[0127] In some embodiments, the modified Fc polypeptide comprises a whole mutation (e.g., T366S, L368A and Y407V, numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 9 and 10. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 9 and 10.

[0128] In some embodiments, the modified Fc polypeptide comprises whole mutations (e.g., T366S, L368A and Y407V, numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 33 and 34. In some embodiments, the modified Fc polypeptide comprises whole mutations (e.g., T366S, L368A and Y407V, numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 33 and 34, and includes an Ala at position 389 according to the EU numbering. In some embodiments, the modified Fc polypeptide comprises hole mutations (e.g., T366S, L368A and Y407V, numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33 and 34, and comprises the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the modified Fc polypeptide comprises hole mutations (e.g., T366S, L368A, and Y407V, numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 33 and 34, and comprises the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs:33 and 34.

[0129] In some embodiments, the Fc polypeptide or modified Fc polypeptide may have a whole mutation (e.g., T366S, L368A and Y407V, numbered according to the EU numbering scheme), a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S, numbered according to the EU numbering scheme (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of the sequences of SEQ ID NOs: 1, 2, 23, and 24. In some embodiments, an Fc polypeptide or modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 1, 2, 23, and 24 may be modified to have hole mutations and mutations that modulate effector function.

[0130] In some embodiments, the modified Fc polypeptide comprises a whole mutation (e.g., T366S, L368A, and Y407V, numbered with reference to EU numbering), as well as a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S, numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 11-16 and 19-22. In some embodiments, the modified Fc polypeptide comprises any one of SEQ ID NOs: 11-16 and 19-22.

[0131] In some embodiments, the modified Fc polypeptide comprises a whole mutation (e.g., T366S, L368A, and Y407V, numbered with reference to EU numbering), as well as a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S, numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 97-98. In some embodiments, the modified Fc polypeptide comprises a hole mutation (e.g., T366S, L368A, and Y407V, as numbered with reference to EU numbering), as well as a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S, as numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 97-98, and comprises an Ala at position 389 according to the EU numbering.In some embodiments, the modified Fc polypeptide comprises hole mutations (e.g., T366S, L368A, and Y407V, as numbered with reference to EU numbering), as well as mutations that modulate effector function (e.g., L234A, L235A, and / or P329G or P329S, as numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 97-98, and comprises the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the modified Fc polypeptide comprises a whole mutation (e.g., T366S, L368A, and Y407V, numbered with reference to EU numbering), as well as a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G or P329S, numbered with reference to EU numbering (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)), and comprises at least one mutation to any one of SEQ ID NOs: 97-98. or at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, and comprises the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 97-98.

[0132] FcRn binding site In certain aspects, a modified (e.g., BBB receptor-binding) Fc polypeptide, or an Fc polypeptide present in a fusion protein described herein that does not specifically bind to a BBB receptor, may comprise an FcRn binding site. In some embodiments, the FcRn binding site is present in an Fc polypeptide or a fragment thereof.

[0133] In some embodiments, the FcRn binding site comprises a native FcRn binding site. In some embodiments, the FcRn binding site does not comprise an amino acid change to the amino acid sequence of the native FcRn binding site. In some embodiments, the native FcRn binding site is an IgG binding site, for example, a human IgG binding site. In some embodiments, the FcRn binding site comprises a modification that alters FcRn binding.

[0134] In some embodiments, the FcRn binding site has one or more mutated, e.g., substituted, amino acid residues, which mutation(s) increase or do not substantially decrease serum half-life (i.e., decrease serum half-life by 25% or less when assayed under identical conditions, compared to an equivalent modified Fc polypeptide having a wild-type residue at the mutated position). In some embodiments, the FcRn binding site has one or more substituted amino acid residues at positions 250-256, 307, 380, 428, and 433-436 according to the EU numbering scheme.

[0135] In some embodiments, one or more residues at or near the FcRn binding site are mutated relative to the native human IgG sequence to increase the serum half-life of the modified polypeptide. In some embodiments, mutations are introduced at one, two or three of positions 252, 254 and 256. In some embodiments, the mutations are M252Y, S254T and T256E. In some embodiments, the modified Fc polypeptide further comprises mutations M252Y, S254T and T256E. In some embodiments, the modified Fc polypeptide comprises substitutions at one, two or all three of positions T307, E380 and N434 according to the EU numbering scheme. In some embodiments, the mutations are T307Q and N434A. In some embodiments, the modified Fc polypeptide comprises mutations T307A, E380A and N434A. In some embodiments, the modified Fc polypeptide comprises substitutions at positions T250 and M428 according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide comprises the mutations T250Q and / or M428L. In some embodiments, the modified Fc polypeptide comprises substitutions at positions M428 and N434 according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide comprises the mutations M428L and N434S. In some embodiments, the modified Fc polypeptide comprises an N434S or N434A mutation.

[0136] IDUA enzyme linked to an Fc polypeptide In some embodiments, the fusion protein described herein comprises two Fc polypeptides described herein, one or both of the Fc polypeptides may further comprise a partial or complete hinge region. The hinge region may be from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is an IgG hinge region, such as an IgG1 hinge region, for example, the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 5) of the human IgG1 hinge or a portion thereof (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the hinge region is present in the N-terminal region of the Fc polypeptide.

[0137] In certain embodiments, the N-terminus of the first Fc polypeptide is linked to an IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof. In certain embodiments, the C-terminus of the first Fc polypeptide is linked to an IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0138] In certain embodiments, the fusion proteins described herein comprise a single IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0139] In certain other embodiments, the fusion protein described herein comprises a second IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof. For example, in certain embodiments, the second Fc polypeptide is linked to an IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof. In certain embodiments, the N-terminus of the second Fc polypeptide is linked to a second IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof. In certain embodiments, the C-terminus of the second Fc polypeptide is linked to a second IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0140] In certain embodiments, the N-terminus of a first Fc polypeptide is linked to a first IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; and the N-terminus of a second Fc polypeptide is linked to a second IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0141] In certain embodiments, the C-terminus of the first Fc polypeptide is linked to a first IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; and the C-terminus of the second Fc polypeptide is linked to a second IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0142] In certain embodiments, the N-terminus of the first Fc polypeptide is linked to a first IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; and the C-terminus of the second Fc polypeptide is linked to a second IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0143] In certain embodiments, the C-terminus of the first Fc polypeptide is linked to a first IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; and the N-terminus of the second Fc polypeptide is linked to a second IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof.

[0144] In some embodiments, the Fc polypeptide is joined to the IDUA enzyme by a linker, e.g., a peptide linker. In some embodiments, the Fc polypeptide is joined to the IDUA enzyme by a peptide bond or by a peptide linker, e.g., the Fc polypeptide is a fusion polypeptide. The peptide linker may be configured to allow rotation of the IDUA enzyme relative to the joined Fc polypeptide and / or to be resistant to digestion by proteases. The peptide linker may comprise natural amino acids, unnatural amino acids, or combinations thereof. In some embodiments, the peptide linker may be a flexible linker (e.g., a glycine-rich 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 may include any number of any repeating units (e.g., repeating units of Gly and Ser residues) of any length. For example, the linker may have repeats, such as two, three, four, five or more Gly4-Ser (SEQ ID NO: 72) repeats, or a single Gly4-Ser (SEQ ID NO: 72). In other aspects, the linker may be Gly-Ser (SEQ ID NO: 71). In some embodiments, the peptide linker may include a protease cleavage site, e.g., a protease cleavage site cleavable by an enzyme present in the central nervous system.

[0145] In some embodiments, the IDUA enzyme is joined to the N-terminus of the Fc polypeptide, for example, by a Gly-Ser linker (SEQ ID NO: 71), a Gly4-Ser linker (SEQ ID NO: 72), or a (Gly4-Ser)2 linker (SEQ ID NO: 73). In some embodiments, the Fc polypeptide may include a hinge sequence or partial hinge sequence at the N-terminus that is joined to a linker or directly to the IDUA enzyme.

[0146] In some embodiments, the IDUA enzyme is joined to the C-terminus of the Fc polypeptide, for example, by a Gly-Ser linker (SEQ ID NO: 71), a Gly4-Ser linker (SEQ ID NO: 72), or a (Gly4-Ser)2 linker (SEQ ID NO: 73). In some embodiments, the C-terminus of the Fc polypeptide is joined directly to the IDUA enzyme.

[0147] In some embodiments, the IDUA enzyme is conjugated to the Fc polypeptide by a chemical crosslinker. Such conjugates can be produced using well-known chemical crosslinking reagents and protocols. For example, there are numerous chemical crosslinkers known to those skilled in the art that are useful for crosslinking polypeptides with agents of interest. For example, the crosslinker is a heterobifunctional crosslinker that can be used to link molecules in a stepwise manner. Heterobifunctional crosslinkers allow for the design of more specific coupling methods for protein conjugation, thereby reducing the occurrence of undesirable side reactions such as homoprotein polymers. A wide variety of heterobifunctional crosslinkers are known in the art, including N-hydroxysuccinimide (NHS) or its water-soluble analog N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-iodoacetyl ... These include succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). These crosslinkers with N-hydroxysuccinimide moieties can be obtained as N-hydroxysulfosuccinimide analogs, which generally have higher water solubility. Additionally, crosslinkers with disulfide bridges in the linking chain can be synthesized as alkyl derivatives instead to reduce the amount of linker cleavage in vivo. In addition to heterobifunctional crosslinkers, numerous other crosslinkers exist, including homobifunctional crosslinkers and photoreactive crosslinkers.Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH), and dimethyl pimelimidate·2HCl (DMP) are examples of useful homobifunctional crosslinkers, and bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinkers.

[0148] Exemplary Protein Molecules Comprising IDUA Enzyme-Fc Fusion Polypeptides In some aspects, the fusion proteins described herein comprise a first Fc polypeptide linked to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof, and a second Fc polypeptide, wherein the first and / or second Fc polypeptide is a modified Fc capable of binding (e.g., specifically binding) to a blood-brain barrier (BBB) ​​receptor, e.g., the transferrin receptor (TfR). In certain embodiments, the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide does not comprise an immunoglobulin heavy and / or light chain variable region sequence or an antigen-binding portion thereof. In some aspects, the fusion protein further comprises a second IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof (e.g., optionally linked to the second Fc polypeptide).

[0149] In some embodiments, the first Fc polypeptide is a modified Fc polypeptide and / or the second Fc polypeptide is a modified Fc polypeptide (e.g., comprising one or more modifications described herein). For example, in some embodiments, the modified Fc polypeptide comprises one or more modifications that promote heterodimerization with another Fc polypeptide. In some embodiments, the modified Fc polypeptide comprises one or more modifications that reduce effector function. In some embodiments, the modified Fc polypeptide comprises one or more modifications that increase serum half-life. In some embodiments, the modified Fc polypeptide comprises one or more modifications that confer binding to a BBB) receptor, e.g., the transferrin receptor (TfR). For example, in certain embodiments, the Fc polypeptide capable of binding to TfR comprises an Ala at position 389 according to EU numbering. In some embodiments, the Fc polypeptide capable of binding to the TfR receptor comprises the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, an Fc polypeptide capable of binding to the TfR receptor comprises the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, such an Fc polypeptide specifically binds to TfR.

[0150] In some embodiments, the first Fc polypeptide is a modified Fc polypeptide. In some embodiments, the second Fc polypeptide is a modified Fc polypeptide. In some embodiments, the first and second Fc polypeptides are each modified Fc polypeptides. In some embodiments, the first Fc polypeptide is a modified polypeptide but does not specifically bind to TfR; and the second Fc polypeptide is a modified polypeptide that can specifically bind to TfR, optionally further comprising one or more additional modifications described herein. In other embodiments, the first Fc polypeptide is a modified polypeptide that can specifically bind to TfR, optionally further comprising one or more additional modifications described herein; the second Fc polypeptide is a modified polypeptide that does not specifically bind to TfR. In some embodiments, the first Fc polypeptide is a modified polypeptide that can specifically bind to TfR, optionally further comprising one or more additional modifications described herein; the second Fc polypeptide is a modified polypeptide that can specifically bind to TfR, optionally further comprising one or more additional modifications described herein.

[0151] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a first Fc polypeptide comprising T366S, L368A, and Y407V (hole) substitutions linked to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof; and a second polypeptide chain comprising a second Fc polypeptide comprising a T366W (knob) substitution, wherein the first and / or second Fc polypeptide is a modified polypeptide capable of binding to TfR. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises L234A and L235A (LALA) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises L234A, L235A, and P329G (LALAPG) substitutions, or further comprises L234A, L235A, and P329S (LALAPS) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises 1) L234A and L235A (LALA) substitutions; L234A, L235A, and P329G (LALAPG) substitutions; or L234A, L235A, and P329S (LALAPS) substitutions; and 2) M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises human IgG1 wild type residues at positions 234, 235, 252, 254, 256, and 366.

[0152] In some embodiments, the second Fc polypeptide is an engineered polypeptide capable of binding to TfR. In some embodiments, the first Fc polypeptide linked to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof is not engineered to bind to TfR. In some embodiments, the second Fc polypeptide comprises a knob mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:25-32. In some embodiments, the second Fc polypeptide comprises a knob mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 25-32, and comprises an Ala at position 389 according to EU numbering. In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 25-32, and comprises an Ala at position 389 according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390.In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of the sequences in SEQ ID NOs: 25-32 and comprises at the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, or comprises any one of the sequences in SEQ ID NOs: 25-32. In some embodiments, the first Fc polypeptide comprises a hole mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 9-16, or comprises the sequence of any one of SEQ ID NOs: 9-16. In some embodiments, the second Fc polypeptide comprises any one of SEQ ID NOs: 25-32, and the first Fc polypeptide comprises any one of SEQ ID NOs: 9-16. In some embodiments, the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 35-38. In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 35-38 and comprises an Ala at position 389 according to EU numbering.In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 35-38, and contains the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 35-38 and comprises at the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, or comprises the sequence of any one of SEQ ID NOs: 35-38. In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 19-22, or comprises any one of SEQ ID NOs: 19-22.

[0153] In some embodiments, the second Fc polypeptide is not modified to bind to TfR. In some embodiments, the first Fc polypeptide linked to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof is a modified polypeptide capable of binding to TfR. In some embodiments, the second Fc polypeptide comprises a knob mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 17-18 and 74-75, or comprises any one of SEQ ID NOs: 17-18 and 74-75. In some embodiments, the first Fc polypeptide comprises a whole mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34, and 97-98. In some embodiments, the first Fc polypeptide comprises a whole mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34, and 97-98, and comprises an Ala at position 389 according to EU numbering.In some embodiments, the first polypeptide comprises a hole mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34, and 97-98, and comprises the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the first Fc polypeptide comprises a hole mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34, and 97-98, and comprises at the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, or comprises the sequence of any one of SEQ ID NOs: 33-34, and 97-98. In some embodiments, the second Fc polypeptide comprises any one of SEQ ID NOs: 17-18 and 74-75, and the first Fc polypeptide comprises any one of SEQ ID NOs: 33-34, and 97-98. In some embodiments, the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 76-77, or comprises any one of the sequences of SEQ ID NOs: 76-77.

[0154] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a first Fc polypeptide comprising a T366W (knob) substitution linked to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof; and a second polypeptide chain comprising a second Fc polypeptide comprising a T366S, L368A, and Y407V (hole) substitution, wherein the first and / or second Fc polypeptide is a modified polypeptide capable of binding to TfR. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises L234A and L235A (LALA) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises L234A, L235A, and P329G (LALAPG) substitutions, or further comprises L234A, L235A, and P329S (LALAPS) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises M252Y, S254T and T256E (YTE) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises 1) L234A and L235A (LALA) substitutions; L234A, L235A, and P329G (LALAPG) substitutions; or L234A, L235A, and P329S (LALAPS) substitutions; and 2) M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises human IgG1 wild type residues at positions 234, 235, 252, 254, 256 and 366.

[0155] In some embodiments, the second Fc polypeptide is an engineered polypeptide capable of binding to TfR. In some embodiments, the first Fc polypeptide linked to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof is not engineered to bind to TfR. In some embodiments, the second Fc polypeptide comprises a hole mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34, and 97-98. In some embodiments, the second Fc polypeptide comprises a hole mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34, and 97-98, and comprises an Ala at position 389 according to EU numbering. In some of the foregoing embodiments, the second Fc polypeptide further comprises an Ala at the following positions according to EU numbering: a Glu at position 380 and an Asn at position 390. In some of the foregoing embodiments, the second Fc polypeptide comprises the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, the second Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 33-34, and 97-98.In some embodiments, the first Fc polypeptide comprises a knob mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 17-18 and 74-75, or comprises any one of SEQ ID NOs: 17-18 and 74-75. In some embodiments, the second Fc polypeptide comprises any one of SEQ ID NOs: 33-34 and 97-98, and the first Fc polypeptide comprises any one of SEQ ID NOs: 17-18 and 74-75. In some embodiments, the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 76-77, or comprises any one of SEQ ID NOs: 76-77.

[0156] In some embodiments, the second Fc polypeptide is not modified to bind to TfR. In some embodiments, the first Fc polypeptide linked to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof is a modified polypeptide capable of binding to TfR. In some embodiments, the second Fc polypeptide comprises a hole mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:9-16, or comprises the sequence of any one of SEQ ID NOs:9-16. In some embodiments, the first Fc polypeptide comprises a knob mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 25-32. In some embodiments, the first Fc polypeptide comprises a knob mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 25-32, and comprises an Ala at position 389 according to EU numbering. In some of the foregoing embodiments, the Fc polypeptide further comprises a Glu at the following positions according to EU numbering: Glu at position 380 and Asn at position 390. In some of the foregoing embodiments, the first Fc polypeptide comprises a Glu at the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.In some embodiments, the first Fc polypeptide comprises any one of SEQ ID NOs: 25-32. In some embodiments, the second Fc polypeptide comprises any one of SEQ ID NOs: 9-16 and the first Fc polypeptide comprises any one of SEQ ID NOs: 25-32. In some embodiments, the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 19-22, or comprises any one of SEQ ID NOs: 19-22. In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 35-38, or comprises any one of SEQ ID NOs: 35-38.

[0157] In some embodiments, the IDUA enzyme present in a fusion protein described herein is linked to a first polypeptide chain comprising a first Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:9-16, or comprising the sequence of any one of SEQ ID NOs:9-16 (e.g., as a fusion polypeptide). In some embodiments, the IDUA enzyme is linked to the first Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP, SEQ ID NO:6). In some embodiments, the N-terminus of the first Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO:6). In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 19-22, or comprises the sequence of any one of SEQ ID NOs: 19-22. In some embodiments, the IDUA enzyme comprises an IDUA sequence having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-49, 78-82, and 99; or comprises the sequence of any one of SEQ ID NOs: 39-49, 78-82, and 99. In some embodiments, the IDUA sequence linked to the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 50-69, and 83-92, or comprises any one of SEQ ID NOs: 50-69, and 83-92.In some embodiments, the fusion protein comprises a second Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 25-32. In some embodiments, the fusion protein comprises a second Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 25-32 and comprises an Ala at position 389 according to EU numbering. In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 25-32, and contains the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the second polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 25-32 and comprises the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, or comprises the sequence of any one of SEQ ID NOs: 25-32. In some embodiments, the N-terminus of the second Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 6).In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 35-38. In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 35-38 and comprises an Ala at position 389 according to EU numbering. In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 35-38, and contains the following positions according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 35-38 and comprises the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, or comprises the sequence of any one of SEQ ID NOs: 35-38. In some embodiments, the second IDUA enzyme is linked to the second Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP, SEQ ID NO: 6).In some embodiments, the second IDUA enzyme comprises an IDUA sequence having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-49, 78-82, and 99, or comprises the sequence of any one of SEQ ID NOs: 39-49, 78-82, and 99. In some embodiments, the second IDUA sequence linked to the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 101-103, or comprises the sequence of any one of SEQ ID NOs: 101-103.

[0158] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising any one of the amino acid sequences of SEQ ID NOs: 50-65 and 83-92; and a second Fc polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 35-38.

[0159] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising any one of the amino acid sequences of SEQ ID NOs: 50-57 and 83-86; and a second Fc polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 35-36.

[0160] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 50-53; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 35-36.

[0161] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 50-51; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 35-36.

[0162] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:51; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO:36.

[0163] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 54-57; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 35-36.

[0164] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 83-86; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 35-36.

[0165] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 83-84; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 35-36.

[0166] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:84; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO:36.

[0167] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising any one of the amino acid sequences of SEQ ID NOs: 58-65 and 87-92; and a second Fc polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 37-38.

[0168] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 58-61; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 37-38.

[0169] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 58-59; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 37-38.

[0170] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:59; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO:38.

[0171] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 60-61; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 37-38.

[0172] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:61; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO:38.

[0173] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 62-65; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 37-38.

[0174] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 64-65; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 37-38.

[0175] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:65; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO:38.

[0176] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 87-90; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 37-38.

[0177] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 89-90; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 37-38.

[0178] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:90; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO:38.

[0179] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 91-92; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 37-38.

[0180] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:92; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO:38.

[0181] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 50-51; and a second Fc polypeptide linked to a second IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 101 or 102.

[0182] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to a first IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:51; and a second Fc polypeptide linked to a second IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs:102.

[0183] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 66-69; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 35-38.

[0184] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 66-67; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 35-36.

[0185] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO: 67; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 35-36.

[0186] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:67; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO:36.

[0187] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 68-69; and a second Fc polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 37-38.

[0188] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO: 68; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 37-38.

[0189] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to an IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:68; and a second Fc polypeptide comprising the amino acid sequence of SEQ ID NO:38.

[0190] In some embodiments, the fusion protein comprises a first Fc polypeptide linked to a first IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:67; and a second Fc polypeptide linked to a second IDUA amino acid sequence comprising the amino acid sequence of SEQ ID NO:103.

[0191] In some embodiments, the IDUA enzyme present in a fusion protein described herein is linked to a first polypeptide chain comprising a first Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 17-18 and 74-75, or comprises the sequence of any one of SEQ ID NOs: 17-18 and 74-75 (e.g., as a fusion polypeptide). In some embodiments, the first IDUA enzyme is linked to the first Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the N-terminus of the first Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 6). In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 76-77, or comprises the sequence of any one of SEQ ID NOs: 76-77. In some embodiments, the IDUA enzyme comprises an IDUA sequence having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-49, 78-82, and 99, or comprises the sequence of any one of SEQ ID NOs: 39-49, 78-82, and 99. In some embodiments, the IDUA sequence linked to the Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 100 and 104, or comprises any one of SEQ ID NOs: 100 and 104.In some embodiments, the fusion protein comprises a second Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34 and 97-98. In some embodiments, the fusion protein comprises a second Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34 and 97-98, and comprises an Ala at position 389 according to EU numbering. In some of the foregoing embodiments, the second polypeptide further comprises the following positions according to EU numbering: a Glu at position 380 and an Asn at position 390. In some of the foregoing embodiments, the second Fc polypeptide comprises at the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some of the foregoing embodiments, the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 33-34 and 97-98. In some embodiments, the N-terminus of the second Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 6). In some embodiments, the second IDUA enzyme is linked to the second Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP, SEQ ID NO: 6).

[0192] In some embodiments, the IDUA enzyme present in a fusion protein described herein is linked (e.g., as a fusion polypeptide) to a first polypeptide chain comprising a first Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 25-32. In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 25-32 and comprises an Ala at position 389 according to EU numbering. In some of the foregoing embodiments, the first polypeptide further comprises a Glu at position 380 and an Asn at position 390 according to EU numbering. In some of the foregoing embodiments, the first Fc polypeptide comprises at the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some of the foregoing embodiments, the first Fc polypeptide comprises a sequence according to any one of SEQ ID NOs: 25-32. In some embodiments, the IDUA enzyme is linked to the first Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the N-terminus of the first Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 6). In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 35-38.In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 35-38 and comprises an Ala at position 389 according to EU numbering. In some of the foregoing embodiments, the first polypeptide further comprises a Glu at position 380 and an Asn at position 390 according to EU numbering. In some of the foregoing embodiments, the first Fc polypeptide comprises a Glu at position 380; a Tyr at position 384; a Thr at position 386; a Glu at position 387; a Trp at position 388; an Ala at position 389; an Asn at position 390; a Thr at position 413; a Glu at position 415; a Glu at position 416; and a Phe at position 421 according to EU numbering. In some of the foregoing embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 35-38. In some embodiments, the IDUA enzyme comprises an IDUA sequence having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-49, 78-82, and 99, or comprises the sequence of any one of SEQ ID NOs: 39-49, 78-82, and 99. In some embodiments, the IDUA sequence linked to the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 101-103, or comprises the sequence of any one of SEQ ID NOs: 101-103. In some embodiments, the fusion protein has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:9-16, or comprises any one of SEQ ID NOs:9-16.In some embodiments, the N-terminus of the second Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 6). In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 19-22, or comprises the sequence of any one of SEQ ID NOs: 19-22. In some embodiments, the second IDUA enzyme is linked to the second Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the second IDUA enzyme comprises an IDUA sequence having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-49, 78-82, and 99, or comprises the sequence of any one of SEQ ID NOs: 39-49, 78-82, and 99. In some embodiments, the second IDUA sequence linked to the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 50-69 and 83-92, or comprises the sequence of any one of SEQ ID NOs: 50-69 and 83-92.

[0193] In some embodiments, the IDUA enzyme present in a fusion protein described herein is linked (e.g., as a fusion polypeptide) to a first polypeptide chain comprising a first Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34 and 97-98. In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 33-34 and 97-98 and comprises an Ala at position 389 according to EU numbering. In some of the foregoing embodiments, the first polypeptide further comprises a Glu at position 380 and an Asn at position 390 according to EU numbering. In some of the foregoing embodiments, the first Fc polypeptide comprises at the following positions according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some of the foregoing embodiments, the first Fc polypeptide comprises a sequence of any one of SEQ ID NOs: 33-34 and 97-98. In some embodiments, the IDUA enzyme is linked to the first Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the N-terminus of the first Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 6).In some embodiments, the IDUA enzyme comprises an IDUA sequence having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-49, 78-82, and 99, or the sequence of any one of SEQ ID NOs: 39-49, 78-82, and 99. In some embodiments, the fusion protein comprises a second Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 17-18 and 74-75, or the sequence of any one of SEQ ID NOs: 17-18 and 74-75. In some embodiments, the N-terminus of the second Fc polypeptide comprises a portion of an IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 6). In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 76-77, or comprises the sequence of any one of SEQ ID NOs: 76-77. In some embodiments, the second IDUA enzyme is linked to the second Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the second IDUA enzyme comprises an IDUA sequence having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-49, 78-82, and 99, or comprises any one of SEQ ID NOs: 39-49, 78-82, and 99.In some embodiments, the second IDUA sequence linked to the Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 100 and 104, or comprises any one of SEQ ID NOs: 100 and 104.

[0194] In some embodiments, the fusion proteins described herein comprise 1) a first Fc polypeptide linked to an IDUA amino acid sequence, and 2) a second Fc polypeptide; wherein each polypeptide consists of an amino acid sequence described in the previous embodiment.

[0195] In some embodiments, the fusion proteins described herein comprise 1) a first Fc polypeptide linked to an IDUA amino acid sequence; and 2) a second Fc polypeptide linked to a second IDUA amino acid sequence, each polypeptide consisting of an amino acid sequence described in the previous embodiments.

[0196] The fusion proteins and other compositions described herein may have a wide range of binding affinities. For example, in some embodiments, the protein has an affinity for the transferrin receptor (TfR) in the range of about 50 mM to about 500 nM, or about 100 nM to about 500 nM. In some embodiments, the affinity for TfR is in the range of about 50 nM to about 300 nM. In some embodiments, the affinity for TfR is in the range of about 100 nM to about 350 nM. In some embodiments, the affinity for TfR is in the range of about 150 nM to about 400 nM. In some embodiments, the affinity for TfR is in the range of about 200 nM to about 400 nM. In some embodiments, the affinity for TfR is in the range of about 200 nM to about 450 nM. In some embodiments, the affinity for TfR is a monovalent affinity.

[0197] Assessment of protein activity The activity of the fusion proteins described herein that contain the IDUA enzyme can be assessed using a variety of assays, including assays that measure activity in vitro using artificial substrates, such as those described in the Examples section.

[0198] In some embodiments, tissue samples are evaluated using the assays described above, but typically include multiple freeze-thaw cycles, such as 2, 3, 4 or 5 or more, prior to the sonication step to ensure rupture of the microvesicles.

[0199] Samples that may be evaluated by the assays described herein include brain, liver, kidney, lung, spleen, plasma, serum, cerebrospinal fluid (CSF) and urine. In some embodiments, CSF samples from patients who have received an enzyme-Fc fusion protein (e.g., IDUA-Fc fusion protein) described herein may be evaluated.

[0200] Nucleic Acids, Vectors, and Host Cells The polypeptide chains contained in the fusion proteins described herein are usually prepared using recombinant methods. Thus, in some aspects, the present disclosure provides an isolated nucleic acid comprising a nucleic acid sequence encoding any of the polypeptide chains comprising the Fc polypeptides described herein, as well as a host cell into which a nucleic acid used to replicate the nucleic acid encoding the polypeptide and / or to express the polypeptide has been introduced. In some embodiments, the host cell is a eukaryotic organism, e.g., a human cell.

[0201] In another aspect, a polynucleotide is provided that comprises a nucleotide sequence encoding one or more of the polypeptide chains described herein. In some embodiments, the polynucleotide encodes one of the polypeptide sequences described herein. In some embodiments, the polynucleotide encodes two of the polypeptide sequences described herein. The polynucleotide may be single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA. In certain embodiments, the polynucleotide is cDNA. In some embodiments, the polynucleotide is RNA.

[0202] Some embodiments also provide pairs of nucleic acid sequences, each nucleic acid sequence encoding a polypeptide described herein. For example, certain embodiments provide pairs of nucleic acid sequences, where a first nucleic acid sequence of the pair encodes a first Fc polypeptide linked to a first IDUA amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; and a second nucleic acid sequence of the pair encodes a second Fc polypeptide, where the first and / or second Fc polypeptide is a modified Fc capable of binding (e.g., capable of specifically binding) to a blood-brain barrier (BBB) ​​receptor, such as the transferrin receptor (TfR).

[0203] In some embodiments, the polynucleotide is contained within a nucleic acid construct, or the pair of polynucleotides is contained within one or more nucleic acid constructs. In some embodiments, the construct is a replicable vector. In some embodiments, the vector is selected from a plasmid, a viral vector, a phagemid, a yeast chromosomal vector, and a non-episomal mammalian vector.

[0204] In some embodiments, the polynucleotide is operably linked to one or more regulatory nucleotide sequences in an expression construct. In one set of embodiments, the nucleic acid expression construct is adapted for use as a surface expression library. In some embodiments, the library is adapted for surface expression in yeast. In some embodiments, the library is adapted for surface expression in phage. In another set of embodiments, the nucleic acid expression construct is adapted for expression of a polypeptide in a system capable of isolating the polypeptide in milligram or gram amounts. In some embodiments, the system is a mammalian cell expression system. In some embodiments, the system is a yeast cell expression system.

[0205] Expression vehicles for recombinant polypeptide production include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived, pEMBL-derived, pEX-derived, pBTac-derived, and pUC-derived plasmids for expression in prokaryotic cells such as E. coli. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) may be used for transient expression of polypeptides in eukaryotic cells. In some embodiments, it may be desirable to express recombinant polypeptides by use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors. Additional expression systems include adenovirus, adeno-associated virus, and other viral expression systems.

[0206] The vector may be transformed into any suitable host cell. In some embodiments, the host cell, for example, a bacterial or yeast cell, may be adapted for use as a surface expression library. In certain cells, the vector is expressed in the host cell and expresses relatively large amounts of the polypeptide. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, an NS0 cell, a Y0 cell, a HEK293 cell, a COS cell, a Vero cell, or a HeLa cell.

[0207] Host cells transfected with an expression vector(s) encoding one or more Fc polypeptide chains described herein can be cultured under appropriate conditions to result in expression of one or more polypeptides. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide may be retained in the cytoplasm or in a membrane fraction, and the cells harvested and lysed, and the polypeptide isolated using a desired method.

[0208] Treatment method The fusion proteins described herein may be used therapeutically to treat MPS I.

[0209] Accordingly, certain embodiments provide a method of reducing accumulation of toxic metabolites (e.g., heparan sulfate-derived oligosaccharides or dermatan sulfate-derived oligosaccharides) in a subject with MPS I, comprising administering to the subject a protein described herein.

[0210] Certain embodiments provide a protein as described herein for use in reducing the accumulation of toxic metabolites (e.g., heparan sulfate-derived oligosaccharides or dermatan sulfate-derived oligosaccharides) in a subject with MPS I.

[0211] Certain embodiments provide for the use of a protein as described herein in the preparation of a medicament for reducing accumulation of toxic metabolites (e.g., heparan sulfate-derived oligosaccharides or dermatan sulfate-derived oligosaccharides) in a subject with MPS I.

[0212] Certain embodiments also provide a method of treating MPS I, comprising administering a protein described herein to a subject in need thereof.

[0213] Certain embodiments provide a protein as described herein for use in the treatment of MPS I in a subject in need thereof.

[0214] Certain embodiments provide for the use of a protein as described herein in the preparation of a medicament for treating MPS I in a subject in need thereof.

[0215] In some embodiments, administration of a protein (e.g., linked to an IDUA enzyme) increases the C of IDUA in the brain compared to uptake of IDUA when not linked to a fusion protein described herein, or compared to uptake of IDUA linked to a reference protein (e.g., a fusion protein described herein that does not have a modification to the second Fc polypeptide that results in TfR binding). max is improved (e.g., increased).

[0216] In some embodiments, the C of IDUA in the brain max is improved (e.g., increased) by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.2-fold, 2.4-fold, 2.6-fold, 2.8-fold, 3-fold, 4-fold, 5-fold, 6-fold, or more, compared to uptake of IDUA when not linked to a fusion protein described herein, or compared to uptake of IDUA linked to a reference protein (e.g., a fusion protein described herein that does not have a modification to the second Fc polypeptide that results in TfR binding).

[0217] The fusion proteins described herein are administered to a subject in a therapeutically effective amount or dose.

[0218] In various embodiments, the fusion proteins described herein are administered parenterally, hi some embodiments, the proteins are administered intravenously.

[0219] In some parenteral embodiments, the fusion proteins described herein are administered intraperitoneally, intradermally, or intramuscularly, hi some embodiments, the fusion proteins described herein are administered intrathecally, such as by epidural administration, or intraventricularly.

[0220] Pharmaceutical Compositions and Kits In other aspects, pharmaceutical compositions and kits are provided that include the fusion proteins described herein.

[0221] Pharmaceutical Compositions Guidance for preparing formulations for use in the present disclosure can be found in many handbooks on pharmaceuticals and formulations known to those of skill in the art.

[0222] In some embodiments, a pharmaceutical composition comprises a fusion protein described herein and further comprises one or more pharma- ceutically acceptable carriers and / or excipients. A pharma- ceutically acceptable carrier includes any solvent, dispersion medium, or coating that is physiologically compatible with the active agent and does not interfere with or otherwise inhibit the activity of the active agent.

[0223] Dosages and desired drug concentrations of the pharmaceutical compositions described herein may vary depending on the particular use envisioned.

[0224] kit In some embodiments, a kit for use in treating MPS I is provided that comprises a fusion protein as described herein.

[0225] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a fusion protein described herein and further comprises one or more additional therapeutic agents for use in treating the neurological symptoms of MPS I. In some embodiments, the kit further comprises instructional materials containing directions (i.e., protocols) for carrying out the methods described herein (e.g., instructions for using the kit to administer a fusion protein comprising an IDUA enzyme across the blood-brain barrier). The instructional materials typically include, but are not limited to, written or printed materials. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), and the like. Such media may include the address of an internet site providing such instructional materials.

[0226] Specific Definitions As used herein, unless the content clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a polypeptide" can include two or more such molecules, and so forth.

[0227] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate that reasonable deviations from that numerical value and values ​​known to one of ordinary skill in the art, for example, ±20%, ±10% or ±5%, are within the intended meaning of the stated value.

[0228] As used interchangeably herein, the terms "subject," "individual," and "patient" 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 one embodiment, the patient is a human. In some embodiments, the human is a patient in need of treatment for MPS I. In some embodiments, the patient has one or more signs or symptoms of MPS I.

[0229] The term "pharmaceutical 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.

[0230] The term "administering" refers to a method of delivering an agent (e.g., an MPS I therapeutic agent such as ETV:IDUA therapy 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, parenteral, intravenous, intradermal, intramuscular, intrathecal, or intraperitoneal delivery. In one embodiment, a polypeptide described herein is administered intravenously.

[0231] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention to alter the natural course of the individual being treated, and may be performed 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.

[0232] The phrase "effective amount" refers to an amount of a compound described herein 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.

[0233] 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 an individual, and the ability of the substance / molecule to induce a desired response in an individual. A therapeutically effective amount encompasses an amount in which the toxic or harmful effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective to achieve a desired prophylactic result at the required dosage and duration of administration. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is used in subjects before or at an early stage of disease.

[0234] As used herein, "alpha-L-iduronidase", "iduronidase alpha-L", "L-iduronidase", "iduronidase", or "IDUA" refers to alpha-L-iduronidase (EC 3.2.1.76), an enzyme involved in the lysosomal degradation of glycosaminoglycans such as dermatan sulfate and heparan sulfate. Mutations in the IDUA gene are associated with MSP I and result from impaired degradation of heparan sulfate and dermatan sulfate. The term "IDUA" or "IDUA enzyme" as used herein as a component of a protein, optionally including an Fc polypeptide, is catalytically active and encompasses functional variants, including allelic and splice variants, as well as catalytically active fragments thereof. The sequence of human IDUA is available at UniProt entry P35475 and is encoded by the human IDUA gene at 4p16.3. The full length sequence is provided as SEQ ID NO:39 and may have an H or Q at position 33 and / or an A or T at position 622, according to EU numbering. As used herein, a "mature" IDUA sequence refers to a form of the polypeptide chain lacking the signal sequence of the full-length naturally occurring polypeptide chain. An embodiment of the amino acid sequence of a mature human IDUA polypeptide is provided as SEQ ID NO:40, which corresponds to amino acids 27-653 of the full-length human sequence. As used herein, a "shortened, truncated" IDUA sequence refers to a catalytically active fragment of the full-length naturally occurring polypeptide chain (e.g., SEQ ID NOs:45-49, 78-82, and 99 (wherein X3 is absent)). The structure of human IDUA has been well characterized. IDUA sequences for non-human primates have also been described, including chimpanzees (e.g., UniProt entry A0A2R9ALZ1 for Pan paniscus (bonobo)). The mouse IDUA sequence is available in Uniprot entry P48441.An IDUA 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 IDUA or a fragment thereof when assayed under the same conditions. A catalytically active IDUA 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 IDUA or a variant thereof when assayed under the same conditions. The commercially available recombinant form of IDUA is called Aldurazyme or Laronidase, both terms referring to the same recombinant form.

[0235] "Transferrin receptor" or "TfR" as used herein refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 7. 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. The 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:8.

[0236] As used herein, a "fusion protein" or "[IDUA enzyme]-Fc fusion protein" refers to a dimeric protein comprising a first Fc polypeptide linked (e.g., fused) to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof (i.e., an "[IDUA]-Fc fusion polypeptide"); and a second Fc polypeptide (e.g., forming an Fc dimer with the first Fc polypeptide). The second Fc polypeptide may also be linked (e.g., fused) to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof. The first Fc polypeptide and / or the second Fc polypeptide may be linked to the IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof by a peptide bond or a polypeptide linker. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that facilitate its heterodimerization with other Fc polypeptides. The first Fc polypeptide and / or the second Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that confer binding to the transferrin receptor. The first Fc polypeptide and / or the second Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that reduce effector function. In certain embodiments, the first Fc polypeptide and the second Fc polypeptide have no effector function. The first Fc polypeptide and / or the second Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that extend serum half-life. In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide do not comprise an immunoglobulin heavy and / or light chain variable region sequence or an antigen-binding portion thereof. In certain embodiments, the first Fc polypeptide and the second Fc polypeptide do not comprise an immunoglobulin heavy and / or light chain variable region sequence or an antigen-binding portion thereof.

[0237] As used herein, "fusion polypeptide" or "[IDUA enzyme]-Fc fusion polypeptide" refers to an Fc polypeptide linked (e.g., fused) to an IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof. The Fc polypeptide may be linked to the IDUA enzyme, an IDUA enzyme variant, or a catalytically active fragment thereof by a peptide bond or a polypeptide linker. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that promote its heterodimerization with another Fc polypeptide. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that confer binding to the transferrin receptor. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that reduce effector function. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that extend serum half-life.

[0238] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. An Fc polypeptide contains a constant region sequence including at least a CH2 domain and / or a CH3 domain, and may contain at least a portion of the hinge region. Generally, an Fc polypeptide does not contain a variable region.

[0239] "Modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, e.g., a substitution, deletion, or insertion, compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of a native Fc polypeptide.

[0240] 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 composed 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.

[0241] 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.

[0242] 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 naturally occurring Fc polypeptide that binds to FcRn.

[0243] As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. For 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 the EU numbering scheme, 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 hinge region sequence. Polypeptides of CH2 and CH3 domains may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where the CH2 domain is numbered from 1 to 110 and the CH3 domain is numbered from 1 to 107 according to the IMGT Scientific chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. The Fc region refers to the segment of amino acids from about position 231 to about position 447 as numbered according to the EU numbering scheme, but as used herein, 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:5).

[0244] "Naturally occurring," "native," or "wild type" are used to describe an entity that can be found in nature as distinct from that which has been artificially produced. For example, a nucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified in a laboratory is naturally occurring. Additionally, "wild type" refers to a normal gene, or an organism found in nature with no known mutations.

[0245] For example, the terms "wild-type," "native," and "naturally occurring" with respect to a CH3 or CH2 domain are used herein to refer to a domain having a sequence that occurs in nature.

[0246] As used herein, the term "mutant" with respect to mutant polypeptides or mutant polynucleotides is used interchangeably with "variant". Variants with respect to a given wild-type CH3 or CH2 domain reference sequence can include naturally occurring allelic variants. A "non-naturally occurring" CH3 or CH2 domain refers to a variant or mutant domain that does not naturally occur in a cell and is generated by genetic modification of a native CH3 domain or CH2 domain polynucleotide or polypeptide, for example, using genetic engineering or mutagenesis techniques. A "variant" includes any domain that contains at least one amino acid mutation relative to the wild type. Mutations can include substitutions, insertions, and deletions.

[0247] 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.

[0248] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

[0249] 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.

[0250] 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.

[0251] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics 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.

[0252] As used herein, the term "protein" refers to either a dimer (i.e., two) or multimer (i.e., three or more) of a polypeptide or a single-chain polypeptide. The single-chain polypeptides of a protein can be joined by covalent bonds, e.g., disulfide bonds, or by non-covalent interactions.

[0253] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to modifications 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 can also be identified. For example, the group of charged or polar amino acids may be subdivided into subgroups including a "positively charged subgroup" consisting of Lys, Arg, and His, a "negatively charged subgroup" consisting of Glu and Asp, and a "polar subgroup" consisting of Asn and Gln. In another example, the aromatic or cyclic group may be divided into subgroups including a "nitrogen ring subgroup" consisting of Pro, His, and Trp, and a "phenyl subgroup" consisting of Phe and Tyr. In another further example, the aliphatic group may be divided into subgroups, such as an "aliphatic non-polar subgroup" consisting of Val, Leu, Gly, and Ala, and an "aliphatic slightly polar subgroup" consisting of Met, Ser, Thr, and Cys. Examples of conservative mutation categories include amino acid substitutions of amino acids within the above subgroups, including, for example: Lys to Arg, or vice versa, to maintain a positive charge; Glu to Asp, or vice versa, to maintain a negative charge; Ser to Thr, or vice versa, to maintain a free -OH; and Gln to Asn, or vice versa, to maintain a free -NH2.In some embodiments, a hydrophobic amino acid is substituted for a naturally occurring hydrophobic amino acid, e.g., in the active site, to preserve the hydrophobicity.

[0254] The term "identical" or percent "identity" in the context of two or more polypeptide sequences refers to two or more sequences or subsequences that have the same or a certain percentage of amino acid residues that are identical, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% 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. In some embodiments, a sequence having a specified percent identity to a reference sequence differs from the reference sequence by one or more conservative substitutions.

[0255] In the case of polypeptide sequence comparison, one amino acid sequence usually serves as a reference sequence and is 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 public software with known algorithms to achieve maximum alignment. Such programs include 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 the sequence comparison of polypeptide sequences, the BLASTP algorithm standard protein BLAST is used to align two protein sequences with default parameters.

[0256] The phrases "corresponding to," "determined with reference to," or "numbered with reference 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 a particular reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a modified Fc polypeptide "corresponds to" an amino acid in SEQ ID NO:1 if that residue matches the amino acid in SEQ ID NO:1 when optimally aligned to that sequence. A polypeptide that is aligned to a reference sequence need not be the same length as the reference sequence.

[0257] 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.

[0258] As used herein, "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 Dcan be determined by measuring the kinetics of complex formation and dissociation, for example, using surface plasmon resonance (SPR) methods, such as the Biacore™ system; kinetic 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 reflecting a 1:1 interaction between a polypeptide and its target, but also to K values, which may reflect strong binding. D The apparent affinity is also included, from which the

[0259] As used herein, the terms "specifically bind" or "selectively bind" to a target, e.g., TfR, when referring to an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding fusion protein described herein, refer to a binding reaction whereby the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding fusion protein binds to a target with higher affinity, higher avidity, and / or longer duration than it binds to a structurally different target. In typical embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding fusion protein has at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more greater affinity for a specific target, e.g., TfR, compared to an unrelated target, when assayed under the same affinity assay conditions. As used herein, the terms "specific binding to," "specifically binds to," or "is specific for" a particular target (e.g., TfR) refer to, for example, binding to a target that is more selective than, e.g., 10-fold, 100-fold, 1,000-fold, 10,000-fold, or more. -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 Equilibrium dissociation constant K DIn some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding 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 engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding fusion protein may bind exclusively to the human TfR.

[0260] The term "variable region" or "variable domain" refers to the domain in an antibody heavy or light chain that is derived from germline variable (V), diversity (D), or joining (J) genes (and not from constant (Cμ and Cδ) gene segments) and confers to the antibody its antigen-binding specificity. Typically, antibody variable regions contain four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions".

[0261] 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 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).

[0262] The following examples are intended to be non-limiting. EXAMPLES

[0263] Example 1: Construction of a fusion protein containing alpha-L-iduronidase (IDUA). Design and Cloning An IDUA-Fc fusion protein was designed that includes (i) a first fusion polypeptide in which the human IDUA enzyme is fused to a human IgG1 fragment comprising an Fc region ("IDUA-Fc fusion polypeptide"), and (ii) a modified human IgG1 fragment comprising a mutation in the Fc region that confers transferrin receptor (TfR) binding ("modified Fc polypeptide"). Fusion proteins were also designed that include (i) a first fusion polypeptide in which the human IDUA enzyme sequence is fused to a human IgG1 fragment comprising an Fc region ("IDUA-Fc fusion polypeptide"), and (ii) a second fusion polypeptide in which the human IDUA enzyme sequence is fused to a modified human IgG1 fragment comprising a mutation in the Fc region that confers TfR binding ("IDUA-Fc fusion polypeptide that binds TfR"). IDUA-Fc fusion polypeptides were generated in which the IDUA sequence was fused to the N-terminus and C-terminus of the human IgG1 Fc region. In all constructs, the signal peptide MGWSCIILFLVATATGAYA (SEQ ID NO: 70) was inserted upstream of the fusion to facilitate secretion. The fragment of the human IgG1 Fc region used corresponds to amino acids D104-K330 (EU numbering, positions 221-447, including the 10 amino acids of the hinge (positions 221-230)) of the sequence of UniProtKB ID P01857. Expression vectors encoding (i) the IDUA-Fc fusion polypeptide and (ii) the modified Fc polypeptide separately were generated and co-transfected into Chinese Hamster Ovary (CHO) cells to generate a heterodimeric fusion protein containing the IDUA enzyme ("monozyme"). Expression vectors separately encoding (i) an IDUA-Fc fusion polypeptide and (ii) an IDUA-Fc fusion polypeptide that binds TfR were also generated and co-transfected into Chinese hamster ovary (CHO) cells to generate a heterodimeric fusion protein containing the IDUA enzyme ("bizyme"). In some constructs, the IgG1 fragment contains an additional mutation that facilitates heterodimerization of the two Fc regions.

[0264] The IDUA-Fc fusion polypeptide comprising the mature human IDUA sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence having hole and LALA mutations has the sequence of SEQ ID NO: 50 or 51. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0265] The IDUA-Fc fusion polypeptide comprising the mature human IDUA sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence having hole and LALAPS mutations has the sequence of SEQ ID NO: 58 or 59. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0266] The IDUA-Fc fusion polypeptide comprising the mature human IDUA sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence having hole and LALAPS mutations has the sequence of SEQ ID NO: 60 or 61. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0267] The IDUA-Fc fusion polypeptide comprising a mature, truncated human IDUA sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence with hole and LALAPS mutations has the sequence of SEQ ID NO: 64 or 65. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0268] The IDUA-Fc fusion polypeptide comprising the mature human IDUA sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence having hole and LALAPS mutations has the sequence of SEQ ID NO: 91 or 92. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0269] The IDUA-Fc fusion polypeptide comprising the mature human IDUA sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence having knob and LALA mutations has the sequence of SEQ ID NO: 104 or 100. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0270] The IDUA-Fc fusion polypeptide, comprising a mature human IDUA sequence fused to the N-terminus of a TfR-binding modified IgG1 Fc polypeptide sequence with knob and LALA mutations, has the sequence of SEQ ID NO: 101 or 102. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0271] The Fc-IDUA fusion polypeptide, comprising a mature human IDUA sequence fused to the C-terminus of a TfR-binding modified IgG1 Fc polypeptide sequence with knob and LALA mutations, has the sequence of SEQ ID NO: 103. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0272] The Fc-IDUA fusion polypeptide comprising the mature human IDUA sequence fused to the C-terminus of an IgG1 Fc polypeptide sequence with hole and LALAPS mutations has the sequence of SEQ ID NO: 68. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0273] The Fc-IDUA fusion polypeptide comprising the mature human IDUA sequence fused to the C-terminus of an IgG1 Fc polypeptide sequence with hole and LALAPS mutations has the sequence of SEQ ID NO: 69. The IDUA enzyme was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 72), and the N-terminus of the Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0274] The TfR binding modified Fc polypeptide with the knob and LALA mutations has the sequence of SEQ ID NO: 35 or 36. The N-terminus of the modified Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0275] The TfR-binding modified Fc polypeptide with knob and LALAPS mutations has the sequence of SEQ ID NO: 37 or 38. The N-terminus of the modified Fc polypeptide included a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0276] An initial "N-terminal monozyme" IDUA-Fc fusion protein ("ETV:IDUA Fusion 1") was generated, which comprises a TfR-binding modified Fc polypeptide having the sequence of SEQ ID NO:35 and an IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO:50. The IDUA-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc polypeptide has the sequence of SEQ ID NO:36 and / or the IDUA-Fc fusion polypeptide has the sequence of SEQ ID NO:51. Thus, as used herein, the term ETV:IDUA Fusion 1 may be used to refer to a protein molecule having an unprocessed sequence (i.e., SEQ ID NOs:35 and 50); a protein molecule that includes one or more processed sequences (i.e., selected from SEQ ID NOs:36 and 51); or a mixture including processed and unprocessed protein molecules.

[0277] A second "N-terminal monozyme" IDUA-Fc fusion protein ("ETV:IDUA Fusion 2") was generated, which comprises a TfR-binding modified Fc polypeptide having the sequence of SEQ ID NO:37 and an IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO:58. The IDUA-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc polypeptide has the sequence of SEQ ID NO:38 and / or the IDUA-Fc fusion polypeptide has the sequence of SEQ ID NO:59. Thus, as used herein, the term ETV:IDUA Fusion 2 may be used to refer to a protein molecule having an unprocessed sequence (i.e., SEQ ID NOs:37 and 58); a protein molecule that includes one or more processed sequences (i.e., selected from SEQ ID NOs:38 and 59); or a mixture including processed and unprocessed protein molecules.

[0278] A third "N-terminal monozyme" IDUA-Fc fusion protein ("ETV:IDUA Fusion 3") was generated, which comprises a TfR-binding modified Fc polypeptide having the sequence of SEQ ID NO:37 and an IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO:60. The IDUA-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc polypeptide has the sequence of SEQ ID NO:38 and / or the IDUA-Fc fusion polypeptide has the sequence of SEQ ID NO:61. Thus, as used herein, the term ETV:IDUA Fusion 3 may be used to refer to a protein molecule having an unprocessed sequence (i.e., SEQ ID NOs:37 and 60); a protein molecule that includes one or more processed sequences (i.e., selected from SEQ ID NOs:38 and 61); or a mixture including processed and unprocessed protein molecules.

[0279] A fourth "N-terminal monozyme" IDUA-Fc fusion protein ("ETV:IDUA Fusion 4") was produced, which comprises a TfR-binding modified Fc polypeptide having the sequence of SEQ ID NO:37 and an IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO:64. The IDUA-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc polypeptide has the sequence of SEQ ID NO:38 and / or the IDUA-Fc fusion polypeptide has the sequence of SEQ ID NO:65. Thus, as used herein, the term ETV:IDUA Fusion 4 may be used to refer to a protein molecule having an unprocessed sequence (i.e., SEQ ID NOs:37 and 64); a protein molecule that includes one or more processed sequences (i.e., selected from SEQ ID NOs:38 and 65); or a mixture including processed and unprocessed protein molecules.

[0280] A fifth "N-terminal monozyme" IDUA-Fc fusion protein ("ETV:IDUA Fusion 5") was generated, which comprises a TfR-binding modified Fc polypeptide having the sequence of SEQ ID NO:37 and an IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO:91. The IDUA-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc fusion polypeptide has the sequence of SEQ ID NO:38 and / or the IDUA-Fc fusion polypeptide has the sequence of SEQ ID NO:92. Thus, as used herein, the term ETV:IDUA Fusion 5 may be used to refer to a protein molecule having an unprocessed sequence (i.e., SEQ ID NOs:37 and 91); a protein molecule that includes one or more processed sequences (i.e., selected from SEQ ID NOs:38 and 92); or a mixture including processed and unprocessed protein molecules.

[0281] A "C-terminal monozyme" IDUA-Fc fusion protein ("ETV:IDUA Fusion 6") was generated, which comprises a TfR-binding modified Fc polypeptide having the sequence of SEQ ID NO:37 and an IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO:68. The IDUA-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc polypeptide has the sequence of SEQ ID NO:38. Thus, as used herein, the term ETV:IDUA Fusion 6 may be used to refer to a protein molecule comprising SEQ ID NO:37 and 68, a protein molecule comprising SEQ ID NO:38 and 68, or a mixture of protein molecules comprising SEQ ID NO:37 and 68 and protein molecules comprising SEQ ID NO:38 and 68. Table 1 shows sequences of additional exemplary IDUA-Fc fusion proteins and recombinant proteins. [Table 1]

[0282] A composition comprising ETV:IDUA (e.g., any of the fusion proteins described above) may be used to refer to a composition comprising protein molecules having unprocessed sequences; to refer to a composition comprising protein molecules that include one or more processed sequences; or to refer to a mixture comprising processed and unprocessed protein molecules.

[0283] An IDUA-Fc fusion protein lacking the mutations that confer TfR binding was similarly designed and constructed.

[0284] A first non-TfR binding "N-terminal monozyme" IDUA-Fc fusion protein ("IDUA-Fc Fusion 12") was generated, which comprises an Fc polypeptide having the sequence of SEQ ID NO: 76 and an IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO: 83. The IDUA-Fc fusion protein may also be further processed during cell culture production such that the Fc polypeptide has the sequence of SEQ ID NO: 77 and / or the IDUA-Fc fusion polypeptide has the sequence of SEQ ID NO: 84. Thus, as used herein, the term IDUA-Fc fusion protein may be used to refer to protein molecules having unprocessed sequences (i.e., SEQ ID NOs: 76 and 83); protein molecules that include one or more processed sequences (i.e., selected from SEQ ID NOs: 77 and 84); or a mixture including processed and unprocessed protein molecules.

[0285] A second non-TfR binding "N-terminal monozyme" IDUA-Fc fusion protein ("IDUA-Fc Fusion 13") was generated, which comprises an Fc polypeptide having the sequence of SEQ ID NO: 76 and a second IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO: 50. The IDUA-Fc fusion protein may also be further processed during cell culture production such that the Fc polypeptide has the sequence of SEQ ID NO: 77 and / or the IDUA-Fc fusion polypeptide has the sequence of SEQ ID NO: 51. Thus, as used herein, the term IDUA-Fc fusion protein may be used to refer to protein molecules having unprocessed sequences (i.e., SEQ ID NOs: 76 and 50); to protein molecules that include one or more processed sequences (i.e., selected from SEQ ID NOs: 77 and 51); or to a mixture including processed and unprocessed protein molecules.

[0286] A non-TfR binding "N-terminal bizyme" IDUA-Fc fusion protein ("IDUA-Fc Fusion 14") was generated, which comprises a first IDUA-Fc polypeptide having the sequence of SEQ ID NO: 104 and a second IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO: 50. The IDUA-Fc fusion protein may also be further processed during cell culture production such that the first IDUA-Fc polypeptide has the sequence of SEQ ID NO: 100 and / or the second IDUA-Fc fusion polypeptide has the sequence of SEQ ID NO: 51. Thus, as used herein, the term IDUA-Fc Fusion 14 may be used to refer to protein molecules having unprocessed sequences (i.e., SEQ ID NOs: 104 and 50); protein molecules that include one or more processed sequences (i.e., selected from SEQ ID NOs: 100 and 51); or a mixture including processed and unprocessed protein molecules. Table 2 shows the sequences of exemplary IDUA-Fc fusion proteins and recombinant proteins that do not bind to TfR. [Table 2]

[0287] A composition comprising ETV:IDUA (e.g., any of the fusion proteins described above) may be used to refer to a composition that comprises a protein molecule having an unprocessed sequence; to refer to a composition that comprises a protein molecule that comprises one or more processed sequences; or to refer to a mixture that comprises processed and unprocessed protein molecules.

[0288] Recombinant protein expression and purification To express recombinant IDUA-Fc fusion proteins, ExpiCHO cells (Thermo Fisher Scientific) were transfected with the relevant DNA constructs using the Expifectamine™ CHO transfection kit according to the manufacturer's instructions (Thermo Fisher Scientific). Cells were grown in ExpiCHO™ Expression medium supplemented with feed as described in the manufacturer's protocol at 37°C, 5% CO2 and 125 rpm in an orbital shaker (Infors HT Multitron). Briefly, logarithmically growing ExpiCHO cells were transfected with 0.8 μg of total DNA plasmid per mL of culture volume at 6 × 10 6 Cells were transfected at a density of 10000 cells / ml. After transfection, cells were returned to 37°C. 18-22 hours after transfection, the transfected cultures were supplemented with nutrient feeds and the cell culture temperature was reduced to 32°C for the duration of the production run. Transfected cell culture supernatants were harvested 120 hours after transfection by centrifugation at 4000 rpm for 15 min. The clarified supernatants were filtered (0.22 μM membrane) and stored at 4°C.

[0289] IDUA-Fc fusion proteins with or without engineered Fc regions that confer TfR binding were purified from cell culture supernatants using Protein A affinity chromatography. The supernatant was loaded onto a HiTrap MabSelect Prisma Protein A affinity column (GE Healthcare Life Sciences, using Akta Pure System). The column was then washed with 10 column volumes (CV) of PBS. Bound proteins were eluted using 50 mM citrate buffer pH 3.6. Immediately after elution, fractions were neutralized using 1 M Tris, pH 8 (1:8 dilution). The Protein A pool was further purified using cation exchange chromatography (CEX). The Protein A pool was diluted 10-fold with sodium acetate buffer, pH 5.5, to facilitate binding to a HiTrap® SP High Performance column (Cytiva, SKU 17-1152-01). The column was eluted with a linear salt gradient of 20 mM sodium acetate, 0.5 M NaCl, pH 5.5 over 30 CV. Fractions were further analyzed by HPLC-SEC. Fractions with purity greater than 95% were pooled and dialyzed against 1XPBS, pH 7.4. The homogeneity of the final bulk of the IDUA-Fc fusion was assessed by a number of techniques including reducing and non-reducing calipers (microcapillary electrophoresis-SDS) and HPLC-SEC.

[0290] Recombinant IDUA with a C-terminal hexahistidine tag (SEQ ID NOs: 93-95) was expressed in ExpiCHO cells as described above. To purify the hexahistidine-tagged IDUA enzyme, the transfected supernatant was dialyzed overnight against 15 L of 20 mM HEPES pH 7.4 containing 100 mM NaCl. The dialyzed supernatant was bound to a HiTrap column (GE Healthcare Life Sciences (using Akta Pure System)). After binding, the column was washed with 20 CV of PBS. Bound proteins were eluted using PBS containing 500 mM imidazole. Pooled fractions containing the IDUA enzyme were diluted 1:10 with 50 mM Tris pH 7.5 and further purified using Q Sepharose High Performance (GE Healthcare). After binding, the column was washed with 10 CV of 50 mM Tris pH 7.5. Bound proteins were eluted using a linear gradient to 50 mM Tris pH 7.5 and 0.5 M NaCl and collected in 1 CV fractions. Purity of the fractions was assessed by non-reducing SDS-PAGE.

[0291] Example 2: Characterization of IDUA-Fc fusion proteins. The ETV:IDUA fusion protein, constructed and prepared as described in Example 1, was evaluated for TfR binding, enzymatic activity, and cellular potency.

[0292] IDUA-Fc fusion proteins with engineered TfR binding sites bind to human TfR To determine whether the IDUA-Fc fusion proteins with engineered TfR binding affect the ability of the modified Fc domain to interact with human TfR, the affinity of ETV:IDUA Fusion 3, 4, and 6 (Example 1) for human TfR was measured by surface plasmon resonance (SPR) using a Biacore 8K instrument (Cytiva). 5 μg / mL of IDUA-Fc fusion protein was captured on a Protein A-coated Biacore™ Series S CM5 sensor chip for 1 min, and serial 3-fold dilutions of human apical domain TfR were injected at a flow rate of 30 μL / min. Each sample was analyzed using five consecutive 60-second injections of increasing concentrations of human TfR, followed by a 60-second dissociation at the end of the injection cycle. After each injection, the chip was regenerated using 10 mM glycine-HCl (pH 1.5). Binding responses were corrected by subtracting the RU from a flow cell capturing irrelevant IgG at a similar density A. Kinetic analysis using Biacore™ Insight Evaluation Software included k on and k off A 1:1 Languir model was used to simultaneously fit the following: The monovalent binding affinities of ETV:IDUA Fusion 3, 4, and 6 to human TfR ranged from approximately 200 nM to 400 nM.

[0293] IDUA-Fc fusion proteins with engineered TfR binding sites are active in vitro The in vitro activity of engineered TfR-binding IDUA-Fc fusion proteins was evaluated to demonstrate that IDUA maintains its enzymatic activity when fused to a human IgG fragment. The in vitro activity of recombinant IDUA was measured using a one-step fluorescent enzyme assay using an artificial substrate. An aliquot of 70.96 mM 4-methylumbelliferyl-α-L-iduronide (free acid) (Cayman Chemical #19543) was diluted in assay buffer (50 mM sodium acetate, 0.5 M NaCl, 0.025% Triton® X-100, pH 4.5) to a final concentration of 2.5 mM. IDUA-Fc fusion protein was serially diluted starting at a concentration of 50 mM. 5 μL of substrate was then mixed with 5 μL of serially diluted IDUA-Fc fusion protein in a 384-well black flat-bottom microplate (NUNC#262260). The reaction was incubated at 22°C for 60 min and stopped with 10 μL of stop buffer (0.5 M sodium carbonate buffer, pH 10.3). The fluorescence of the reaction solution was then measured (excitation wavelength 365 nm, and emission wavelength 450 nm). A standard curve of 4-methylumbelliferone was fitted by nonlinear regression to calculate the amount of product, which was confirmed to be less than 10% of the total substrate cleavage. The specific activity (pmol product / min / pmol IDUA) was calculated by dividing the amount of product by the reaction time and the molar amount of IDUA.

[0294] In vitro enzyme activity assays demonstrated that the IDUA-Fc fusion proteins were active and similar between ETV:IDUA Fusion 3, 4, and 6 (Figure 2). All ETV:IDUA fusion proteins exhibited specific activities ranging from about 75% to about 90% of the activity of Aldurazyme (laronidase).

[0295] IDUA-Fc fusion proteins with engineered TfR binding sites modify substrate accumulation in vitro The cellular activity of the IDUA-Fc fusion protein was also examined in fibroblasts from Hurler (MPS I) patients and healthy controls using LCMS quantification of heparan sulfate and dermatan sulfate to assess substrate compensation. Briefly, Hurler and healthy fibroblasts were plated at a density of 75,000 cells per well in 24-well culture plates in DMEM medium supplemented with 10% FBS. Cells were cultured in this format for approximately 96 hours until confluence was reached. ETV:IDUA Fusion 1 was then added to each well in a 12-point, 5-fold serial dilution dose curve from a starting concentration of 62.5 nM. 72 hours after protein addition, cells were lysed by hypotonic shock. Lysates were transferred to a 96-well assay plate, the buffer composition was adjusted to contain 111 mM ammonium acetate and 11 mM calcium acetate, and sonication was performed to complete lysis. The protein concentration of the lysates was determined by BCA assay, and all lysates were adjusted to equal protein concentrations. Lysates were supplemented with 2 mM DTT and GAGs were digested for 3 h at 30° C. with an enzyme mixture containing heparinase I (1.25 mIU / reaction), heparinase II (1.25 mIU / reaction), heparinase III (1.25 mIU / reaction), and chondroitinase B (6.25 mIU / reaction). Digestion reactions were quenched with 10 mM EDTA and 20 ng of 4UA-2S-GlcNCOEt-6S internal standard was added to each reaction followed by denaturation at 95° C. for 10 min. Insoluble material was removed from the samples by centrifugal filtration and the clarified samples were mixed 1:1 with acetonitrile in glass LC-MS vials. Disaccharide species derived from heparan sulfate and dermatan sulfate GAGs were quantified by LC-MS / MS on a Xevo TQ-S Micro instrument equipped with an ACQUITY UPLC BEH Amide 1.7 mm, 2.1 x 150 mm column.

[0296] Fibroblasts from MPS I patients lack IDUA activity, leading to the accumulation of two glycosaminoglycan (GAG) species, heparan sulfate and dermatan sulfate. ETV:IDUA Fusion 1 exhibits similar potency to laronidase in MPS I patient-derived cells, with a low picomolar cell EC2 for reducing heparan sulfate accumulation. 50 (approximately 7 to 10 pM) (Figure 3).

[0297] IDUA-Fc fusion protein with engineered TfR binding site shows increased brain uptake in TfR KI mice TfR knock-in (hereinafter referred to as "TfR mu / hu KI" or "TfR mu / hu The peripheral (serum) and brain PK of the IDUA-Fc fusion protein was evaluated in mice. ms / hu KI mice were generated to express the human Tfrc apical domain within the mouse Tfrc gene using CRISPR / Cas9 technology as described in International Patent Application No. WO2018 / 152285. The resulting chimeric TfR was expressed in vivo under the control of the endogenous promoter. Briefly, 6-8 week old male TfR mice were mu / huKI mice (n=12 per cohort) were dosed with 40 mg / kg ETV:IDUA fusion protein and the concentrations of IDUA enzyme and ETV:IDUA intact molecules in serum and brain tissue were measured. Total IDUA enzyme levels were measured at t=0.25, 0.5, 1, 4, 8, 10, 24, and 48 hours post-dose for serum and t=1, 8, 24, and 48 hours post-dose for brain PK using a sandwich ELISA-based assay. The IDUA-Fc fusion proteins used in the analysis were as described above and prepared according to Example 1. For the measurement of total IDUA enzyme levels, polyclonal sheep anti-human IDUA antibody (Bio-Techne AF4119) was coated overnight onto MULTI-ARRAY® 96-well plates (Meso Scale Diagnostics L15XA-3). The plates were blocked with Blocker™ Casein in PBS (ThermoFisher 37528), BS, and then incubated with diluted serum or brain lysate. Ruthenium-conjugated polyclonal sheep anti-human IDUA antibody (Bio-Techne AF4119) was then added for detection. 1× Read Buffer T with detergent (Meso Scale Diagnostics R92TC-1) was added to each well, plated, and loaded into the MSD reader. Standard curves were based on individual constructs and fitted using a 4-parameter logistic curve. Results are shown in Figures 4A-4C and 5A-5C and Table 3.

[0298] ETV:IDUA Fusion 3, 4, and 6 demonstrated good stability in the circulation. High brain Cmax values ​​(>15 nM) were observed for all molecules, and brain uptake of all molecules was consistent with peripheral exposure. [Table 3]

[0299] Example 3: Product quality attributes of ETV:IDUA Fusions. Various ETV:IDUA fusion proteins were evaluated in terms of product quality. In this study, ETV:IDUA Fusion 3, 4, and 6 (Example 1) were evaluated. All constructs were prepared as described in Example 1. The homogeneity of the ETV:IDUA fusion proteins in the eluted fractions was evaluated by different techniques, such as reducing and non-reducing calipers (microcapillary electrophoresis-SDS) and HPLC-SEC. The affinity for human TfR was measured as described in Example 2.

[0300] result As described in Example 2, the measured human TfR affinities for ETV:IDUA Fusion 3 and 6 were comparable (K D is in the range of approximately 200-400 nM). Expression titers of ETV:IDUA Fusion 3, 4, and 6 exceeded approximately 100 mg / L.

[0301] Recovery rates following Protein A chromatographic purification of ETV:IDUA Fusion 3, 4, and 6 were assessed. Analysis of post-Protein A pools of all structures showed a purity of at least 85% (measured by HPLC-SEC) with at least about 90% intact ETV structure (maintenance of the modified Fc dimer containing knob and hole pairs). Post-Protein A pools of all structures were subjected to cation exchange chromatography (CEX) for further refinement. Post-CEX pools of all structures achieved purity levels of >99% (measured by HPLC-SEC) with >95% intact ETV structure.

[0302] A summary of the product quality characteristics of the ETV:IDUA fusion protein is shown in Table 4. [Table 4]

[0303] Example 4: Comparative study of ETV:IDUA fusion protein and laronidase in a disease model of MPS I. A representative ETV:IDUA fusion protein (ETV:IDUA Fusion 3) was compared to laronidase, the standard of care enzyme replacement therapy, in a mouse model of MPS I.

[0304] result IDUA KO;TfR after a single intravenous (IV) administration of ETV:IDUA and laronidase mu / hu Total GAG levels in the brain, CSF, and liver of KI mice were compared for the extent to which the protein reduced them. ETV:IDUA Fusion 3 was administered to mice at equimolar amounts to laronidase (0.85 mg / kg), approximately 10 times the equimolar amount (8.8 mg / kg), and at a high dose level (40 mg / kg), with laronidase administered at a dose consistent with a clinically relevant dose for treatment (0.58 mg / kg). Total GAG was determined as the sum of the major heparan sulfate (HS) (D0A0, D0S0) and dermatan sulfate (DS) (D0a4) derived disaccharides. As shown in Figures 6A-6D and Table 5, 7 days after a single dose, ETV:IDUA protein was able to reduce total GAG levels in both cerebrospinal fluid (CSF) and brain tissue, and this reduction was superior to the effect achieved with laronidase. Reductions in total GAG levels were seen at all three doses of ETV:IDUA, including equimolar doses of laronidase (CSF: 79% reduction vs. 36% reduction; brain: 38% reduction vs. N / S reduction). In the liver, total GAG reductions were similar between laronidase and ETV:IDUA protein at all doses administered. In urine, ETV:IDUA reduced total GAG levels equivalently to laronidase (0.85 mg / kg) at equimolar doses. However, at higher doses of ETV:IDUA (e.g., 8.8 mg / kg and 40 mg / kg), reductions in urinary total GAG levels were better than laronidase. [Table 5]

[0305] Data in Figures 6A-6D represent approximate mean amounts + / - standard error of the mean. The results show that ETV:IDUA robustly reduced substrate accumulation in the CSF, brain, and urine, and that this reduction represented an improvement over standard of care ERT treatment.

[0306] Experimental Method ETV:IDUA Fusion 3 was expressed and purified as described in Example 1. Laronidase was obtained commercially (NDC 58468-0070-1, lot number 0Y0432).

[0307] The MPS I mouse model used in this study is a mouse model in which the gene encoding IDUA has been knocked out, but which also has the human TfR apical domain knocked out into the mouse TfR (referred to herein as "IDUA KO; TfR mu / hu KI" or "IDUA KO;TfR mu / hu IDUA KO mice were obtained from The Jackson Laboratories (JAX stock number 004068). Briefly, TfR mu / hu KI male mice (see Example 2; herein referred to as "TfR mu / hu ") were crossed with female trout heterozygous for the IDUA mutation to produce TfR mu / hu Homozygous IDUA KO mice on a KI homozygous background were generated. Mice used in this study were mixed sex and housed under a 12-h light-dark cycle with food (#25502, irradiated; LabDiet) and water available ad libitum.

[0308] In this study, IDUA KO;TfR mu / hu KI mice were administered a single intravenous injection of ETV:IDUA Fusion 3 or laronidase and the pharmacodynamic response was evaluated. In particular, peripheral administration of the proteins significantly reduced the IDUA KO;TfR mu / hu The effects of KI on total GAG levels in the brain, CSF, liver, and urine of IDUA KO;TfR mice at approximately 2 months of age were examined. mu / huKI were injected intravenously (i.v.) with saline, ETV:IDUA fusion protein (0.85, 8.8, or 40 mg / kg body weight), or laronidase (0.58 mg / kg body weight) (n=4–5 / group). Approximately 2-month-old littermates TfRs injected i.v. with saline were mu / hu KI mice (non-MPS I mice) were used as controls. Urine samples were collected on days 6 and 7 after the single dose and pooled for analysis. On day 7 after the single dose, all animals were sacrificed and brain, CSF, and liver tissues were harvested. All collected tissues and fluids were flash frozen on dry ice and stored at -80°C until analysis.

[0309] Heparan sulfate and dermatan sulfate species were measured in vivo using an LC-MS / MS-based method described below. Tissue aliquots (50 mg) were homogenized in water (500–750 μL) using a Qiagen TissueLyzer II at 30 Hz for 3 min twice. The homogenates were transferred to a 96-well deep plate and sonicated with 20 × 1 s pulses using a 96-tip sonicator (Q Sonica). The sonicated homogenates were centrifuged at 17,000 × g for 20 min at 4 °C to pellet cellular debris. The resulting lysates were transferred to a clear 96-well deep plate and a BCA was performed to quantify total protein. Heparan sulfate (HS) and dermatan sulfate (DS) in the samples were digested to the corresponding disaccharides prior to LC-MS / MS analysis. Protein lysates (from brain and liver tissues) and CSF and urine were mixed with heparinase I, II, III and the internal standard D4UA-2S-GlcNCOEt-6S (HD009, Iduron Ltd, Manchester, UK) in a digestion buffer consisting of ammonium acetate, calcium acetate, and DTT, and shaken (700 rpm) in a PCR plate at 30°C for 3 h. For urine, stable labeled creatinine (internal standard, N-methyl-D3, 98%) was also added to the mixture. After 3 h of incubation, the reaction was stopped by adding 0.0025 M EDTA to each sample, and the mixture was boiled at 95°C for 10 min to inactivate the enzymes. The digested samples from brain, liver, CSF, and urine were centrifuged, and the supernatant was transferred to a cellulose acetate filter plate (Millipore, MSUN03010) and centrifuged again. For brain, CSF and urine samples, the resulting eluates were mixed with an equal volume of acetonitrile in a 96-well glass vial plate and analyzed by mass spectrometry as follows: For liver, the resulting eluates were mixed with two volumes of acetonitrile.

[0310] Quantification of HS- and DS-derived disaccharides in biological fluids and tissues was performed using liquid chromatography (Exion LC, Sciex, Framingham, MA, USA; Agilent 1290 Infinity II HPLC, Agilent Technologies Inc., Santa Clara, CA, USA) coupled to electrospray mass spectrometry (Sciex Triple Quad 7500, Sciex, Framingham, MA, USA; Sciex Triple Quad 6500+, Sciex, Framingham, MA, USA). For both liquid chromatography analyses, samples were injected onto an ACQUITY UPLC BEH Amide 1.7 mm, 2.1 × 150 mm column (Waters Corporation, Milford, MA, USA) at a flow rate of 0.6 mL / min and a column temperature of 55 °C. Mobile phase A consisted of water with 10 mM ammonium formate and 0.1% formic acid, and mobile phase B consisted of acetonitrile with 0.1% formic acid. The gradient was programmed as follows: 0.0-6.0 min at 80% B, 6.0-6.01 min at 80% B-20% B, 6.01-8.0 min at 20% B-20% B, 8.0-8.1 min at 20% B-80% B, and hold 8.1-10 min at 80% B. For the Sciex Triple Quad 7500, electrospray ionization was performed in negative ionization mode applying the following settings: curtain gas 40; ion spray voltage -4500; temperature 450 °C; ion source gas 1 at 50; and ion source gas 2 at 60. Data acquisition was performed using Sciex OS 2.1.6.59781 in multiple reaction monitoring mode (MRM) with the following settings: dwell time 100 msec; collision energy -30; entrance potential -10; collision cell exit potential -10. For the Sciex Triple Quad 6500+, electrospray ionization was performed in negative ionization mode applying the following settings: curtain gas 20, ion spray voltage -4500, temperature 450°C; ion source gas 1 at 50, and ion source gas 2 at 60.Data acquisition was performed using Analyst 1.7.1 with MRM using the following settings: dwell time 100 msec; collision energy -30; declustering potential -80; entrance potential -10; collision cell exit potential -10. Individual disaccharide species were identified based on retention time and MRM transitions using commercially available reference standards (Iduron Ltd). On the Sciex Triple Quad 7500 and Sciex Triple Quad 6500+, the following disaccharide transitions were monitored: D0A0 (HS), m / z 378>87; D0S0 (HS), m / z 416>138, and D0a4 (m / z 458>300); D4UA-2S-GlcNCOEt-6S (internal standard) m / z 472>97. Disaccharide amounts were normalized to total protein levels measured by BCA assay or to the amount of body fluid used per sample. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10] [Table 6-11]

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 6-28

[0311] 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 and 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. A protein, a. a fusion polypeptide comprising a first Fc polypeptide linked to an alpha-L-iduronidase (IDUA) amino acid sequence, an IDUA variant amino acid sequence, or a catalytically active fragment thereof; b. a second Fc polypeptide comprising a sequence having at least 90% identity to SEQ ID NO: 28 and capable of specifically binding to transferrin receptor (TfR).

2. wherein said second Fc polypeptide is located at the following position according to EU numbering: i. Glu at position 380; ii. Tyr at position 384; iii. Thr at position 386; iv. Glu at position 387; v. Trp at position 388; vi. Ala at position 389; vii. Asn at position 390; viii. Thr at position 413; ix. Glu at position 415; x. Glu at position 416; and xi. Phe at position 421; The protein of claim 1 further comprising:

3. The protein of claim 1 , wherein the IDUA amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to any one of SEQ ID NOs: 39, 40, 45, 78, and 99.

4. The protein of claim 1, wherein the IDUA amino acid sequence comprises any one of the amino acid sequences of SEQ ID NOs: 41-44, 46-49, and 79-82.

5. The protein of claim 1 , wherein the first Fc polypeptide is linked to the IDUA amino acid sequence, the IDUA variant amino acid sequence, or a catalytically active fragment thereof by a polypeptide linker.

6. The protein of claim 1 , wherein the N-terminus or C-terminus of the first Fc polypeptide is linked to the IDUA amino acid sequence, the IDUA variant amino acid sequence, or a catalytically active fragment thereof.

7. The protein of claim 1, wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide.

8. 2. The protein of claim 1, wherein one of the Fc polypeptides has a T366W substitution and the other Fc polypeptide has T366S, L368A, and Y407V substitutions according to EU numbering.

9. 9. The protein of claim 8, wherein the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 9-16 and 19-22; and the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 25-32 and 35-38.

10. 2. The protein of claim 1, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises a modification that reduces effector function, wherein the modification that reduces effector function is a substitution of Ala at position 234 and Ala at position 235; Ala at position 234, Ala at position 235 and Gly at position 329; or Ala at position 234, Ala at position 235 and Ser at position 329, according to EU numbering.

11. 11. The protein of claim 10, wherein the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 11-16 and 19-22.

12. The protein of claim 10, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 50-69 and 83-92.

13. 11. The protein of claim 10, wherein the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 27-32 and 35-38.

14. The protein of claim 1, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 50-65 and 83-92; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 35-38. 15) the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 50-57 and 83-86; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 35-36; or 2) the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 58-65 and 87-92; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 37-38; The protein of claim 14.

16. The protein described in claim 15, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 60 to 61; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 37 to 38.

17. The protein of claim 1, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 66-69; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 35-38. 18) the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 66-67; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 35-36; or 2) the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 68-69; and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 37-38; The protein of claim 17.

19. The protein of claim 1 , wherein the protein does not contain an immunoglobulin heavy and / or light chain variable region sequence or an antigen-binding portion thereof.

20. A pharmaceutical composition comprising the protein according to any one of claims 1 to 19 and a pharmaceutically acceptable excipient.

21. A polynucleotide comprising a nucleic acid sequence encoding the fusion polypeptide of any one of claims 1 to 19 and a second Fc polypeptide.

22. A vector comprising the polynucleotide of claim 21.

23. A host cell comprising the polynucleotide of claim 21.

24. A method for producing a protein comprising a fusion polypeptide and a second Fc polypeptide, the method comprising culturing a host cell comprising the polynucleotide of claim 21 under conditions in which the polypeptide encoded by the polynucleotide is expressed.

25. A pair of polynucleotides comprising: a first nucleic acid sequence encoding the fusion polypeptide of any one of claims 1 to 19; and a second nucleic acid sequence encoding the second Fc polypeptide.

26. 26. One or more vectors comprising the pair of polynucleotides of claim 25.

27. A host cell comprising the pair of polynucleotides of claim 25.

28. A method for producing a protein comprising a fusion polypeptide and a second Fc polypeptide, the method comprising culturing a host cell comprising a pair of polynucleotides described in claim 25 under conditions in which the polypeptide encoded by the pair of polynucleotides is expressed.

29. A composition for treating MPS I in a patient in need thereof, comprising a protein according to any one of claims 1 to 19.

30. 20. Use of a protein according to any one of claims 1 to 19 in the preparation of a medicament for the treatment of MPS I in a patient in need thereof.

31. A composition for reducing the accumulation of toxic metabolites in patients with MPS I, comprising a protein according to any one of claims 1 to 19.

32. Use of a protein according to any one of claims 1 to 19 in the preparation of a medicament for reducing the accumulation of toxic metabolites in patients with MPS I.

33. 32. The composition of claim 31, wherein the toxic metabolite comprises a heparan sulfate-derived oligosaccharide or a dermatan sulfate-derived oligosaccharide.

34. The use of claim 32, wherein the toxic metabolite comprises a heparan sulfate-derived oligosaccharide or a dermatan sulfate-derived oligosaccharide.

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