Fusion protein containing sulfoglucosamine sulfohydrolase enzyme and method thereof

Abstract

The present invention provides a fusion protein containing a sulfoglucosamine sulfohydrolase enzyme and a method for producing such a protein. [Solution] Provided herein are proteins comprising a sulfoglucosamine sulfohydrolase (SGSH) enzyme-Fc fusion polypeptide that can be transported across the blood-brain barrier (BBB). Certain embodiments also provide a method of using such proteins to treat Sanfilippo syndrome A. Sanfilippo syndrome, or MPSIII, is a rare neurodegenerative disorder resulting from specific defects in lysosomal function.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 091,800, filed October 14, 2020. The entire contents of the above-referenced application are incorporated herein by reference. [Background technology]

[0002] Sanfilippo syndrome, or MPSIII, is a rare neurodegenerative disorder caused by specific defects in lysosomal function. The most common type of Sanfilippo syndrome is type A, which is caused by a gene mutation in the SGSH gene. Insufficient N-sulfoglucosamine sulfohydrolase (SGSH) activity leads to the accumulation of heparan sulfate-derived oligosaccharides, resulting in lysosomal dysfunction in multiple organs and tissues, particularly the brain and spinal cord. Treatment for Sanfilippo syndrome remains largely supportive; while the deficient enzyme can be administered intravenously, it has little effect on the brain because it is difficult to deliver the recombinant enzyme across the blood-brain barrier (BBB). Therefore, more effective treatments are needed to address both the peripheral and central nervous system (CNS) symptoms of Sanfilippo syndrome A. [Overview of the Initiative] [Means for solving the problem]

[0003] Accordingly, provided herein is a specific enzyme replacement therapy having the ability to cross the BBB and treat both the peripheral and CNS symptoms of San Filippo syndrome A. In particular, certain embodiments provide a protein comprising (a) a first N-sulfoglucosamine sulfohydrolase (SGSH) amino acid sequence, an SGSH variant amino acid sequence, or a first Fc polypeptide linked to a catalytically active fragment thereof; and (b) a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a second Fc polypeptide linked to a catalytically active fragment thereof, wherein the second Fc polypeptide has at least 80% identity to SEQ ID NO: 37 and comprises a sequence having Ala at position 389 according to EU numbering. In some embodiments, the second Fc polypeptide comprises Glu at position 380; Ala at position 389; and Asn at position 390, according to EU numbering. In some embodiments, the second Fc polypeptide contains, 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 certain embodiments, the second Fc polypeptide binds specifically to the transferrin receptor (TfR) or can bind specifically to the TfR. In certain embodiments, the second Fc polypeptide binds to the apical domain of the TfR. In certain embodiments, the binding of the protein to the TfR does not substantially inhibit the binding of transferrin to the TfR. In certain embodiments, the protein binds to the TfR with an affinity of about 100 nM to about 500 nM, or optionally about 150 nM to about 400 nM. In certain embodiments, proteins can be transported by crossing the target blood-brain barrier.

[0004] In a particular embodiment, the first SGSH amino acid sequence includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to any one of sequence numbers 58-60.

[0005] In a particular embodiment, the second SGSH amino acid sequence includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to any one of sequence numbers 58-60.

[0006] In certain embodiments, a first Fc polypeptide is linked to a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof by peptide bonds or a polypeptide linker. In certain embodiments, a second Fc polypeptide is linked to a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof by peptide bonds or a polypeptide linker. In certain embodiments, the polypeptide linker is a flexible polypeptide linker. In certain embodiments, the flexible polypeptide linker is a glycine-rich linker. In certain embodiments, the polypeptide linker is GS (SEQ ID NO: 7), G4S (SEQ ID NO: 8), or (G4S)2 (SEQ ID NO: 9).

[0007] In certain embodiments, the N-terminus of the first Fc polypeptide is linked to a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. In certain embodiments, the C-terminus of the first Fc polypeptide is linked to a first SGSH amino acid sequence, an SGSH 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 SGSH amino acid sequence, an SGSH 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 SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. In certain embodiments, the N-terminus of the first Fc polypeptide is linked to a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof; and the N-terminus of the second Fc polypeptide is linked to a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. In certain embodiments, the C-terminus of the first Fc polypeptide is linked to a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof; and the C-terminus of the second Fc polypeptide is linked to a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof.

[0008] In certain embodiments, the first Fc polypeptide and the second Fc polypeptide each contain a modification that promotes heterodimerization. In certain embodiments, according to EU numbering, one of the Fc polypeptides has a T366W substitution and the other Fc polypeptide has T366S, L368A, and Y407V substitutions.

[0009] In certain embodiments, the first Fc polypeptide contains the T366S, L368A, and Y407V substitutions, and the second Fc polypeptide contains the T366W substitution. In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 12-19 and 28-31; the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 34-41 and 54-57.

[0010] In certain embodiments, the first Fc polypeptide contains the T366W substitution, and the second Fc polypeptide contains the T366S, L368A, and Y407V substitutions. In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 24-27; the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 48-53.

[0011] In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises a native FcRn binding site.

[0012] In certain embodiments, the first Fc polypeptide and the second Fc polypeptide do not have effector functions. In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises a modification that reduces effector function. In certain embodiments, the modification that reduces effector function is the substitution of Ala at position 234 and Ala at position 235 according to EU numbering.

[0013] In certain embodiments, the first Fc polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 14-19 and 26-31. In certain embodiments, the first Fc polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 14, 15, 28, and 29. In certain embodiments, the first Fc polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 18, 19, 30, and 31. In certain embodiments, the first Fc polypeptide linked to the first SGSH amino acid sequence includes an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 61-88 and 117-118. In a particular embodiment, the first Fc polypeptide linked to the first SGSH amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to one of sequence numbers 61-68, 73-76, 81-84, and 117-118.

[0014] In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 36-41 and 50-57. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 36, 37, 54, and 55. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs: 40, 41, 56, and 57. In a particular embodiment, the second Fc polypeptide linked to the second SGSH amino acid sequence includes an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of sequence numbers 89-116 and 119-120.

[0015] In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide include amino acid changes to the native Fc sequence that extend the serum half-life. In certain embodiments, the amino acid changes include substitutions of Tyr at position 252, Thr at position 254, and Glu at position 256 according to EU numbering. In certain embodiments, the amino acid changes include substitutions of Leu at position 428 and Ser at position 434 according to EU numbering. In certain embodiments, the amino acid changes include substitutions of Ser or Ala at position 434 according to EU numbering.

[0016] In a particular embodiment, the first Fc polypeptide linked to the first SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 61-68, 73-76, and 81-84; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 89-96, 101-104, and 109-112.

[0017] In a particular embodiment, the first Fc polypeptide linked to the first SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 61 to 64; the second Fc polypeptide linked to the second SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 89 to 92. In a particular embodiment, the first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NOs. 63 or 64; the second Fc polypeptide linked to the second SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NOs. 91 or 92.

[0018] In a particular embodiment, the first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 75 or 76; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 103 or 104.

[0019] In a particular embodiment, the first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 83 or 84; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 111 or 112.

[0020] In a particular embodiment, the first Fc polypeptide linked to the first SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 65-68; the second Fc polypeptide linked to the second SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 93-96. In a particular embodiment, the first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NOs. 67 or 68; the second Fc polypeptide linked to the second SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NOs. 95 or 96.

[0021] In a particular embodiment, the first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 118; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 120.

[0022] In certain embodiments, the uptake of the SGSH amino acid sequence into the brain is at least 10 times greater than the uptake of the SGSH amino acid sequence in the absence of the first and second Fc polypeptides, or compared to the uptake of an unmodified SGSH enzyme that results in TfR binding to the second Fc polypeptide.

[0023] In certain embodiments, the first Fc polypeptide is not modified to bind to blood-brain barrier (BBB) ​​receptors, while the second Fc polypeptide is modified to specifically bind to TfR.

[0024] In certain embodiments, the protein does not include immunoglobulin heavy chain and / or light chain variable region sequences or their antigen-binding moieties.

[0025] In certain embodiments, a polypeptide is provided comprising an SGSH amino acid sequence, an SGSH variant amino acid sequence, or an Fc polypeptide linked to a catalytically active fragment thereof, wherein the Fc polypeptide i) comprises a sequence having at least 90% identity to SEQ ID NO: 37, ii) has one or more modifications that facilitate heterodimerization with another Fc polypeptide, and iii) has Ala at position 389 according to EU numbering. In some embodiments, the Fc polypeptide comprises, according to EU numbering, Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the Fc polypeptide comprises, 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 certain embodiments, the Fc polypeptide specifically binds to or can specifically bind to a transferrin receptor (TfR). In certain embodiments, the Fc polypeptide is linked to an SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof by a peptide bond or by a polypeptide linker. In certain embodiments, the polypeptide is a fusion polypeptide comprising: from the N-terminus to the C-terminus: an SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment; a polypeptide linker; and the Fc polypeptide. In certain embodiments, the polypeptide is a fusion polypeptide comprising: from the N-terminus to the C-terminus: the Fc polypeptide; a polypeptide linker; and an SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment.

[0026] In certain embodiments, the Fc polypeptide contains T366S, L368A and Y407V substitutions according to EU numbering. In certain embodiments, the Fc polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 97-100, 105-108, and 113-116.

[0027] In certain embodiments, the Fc polypeptide contains a T366W substitution. In certain embodiments, the Fc polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of sequence numbers 89-96, 101-104, 109-112, and 119-120.

[0028] In certain embodiments, the protein comprises an Fc polypeptide, which is dimerized into another Fc polypeptide. Thus, certain embodiments provide proteins comprising the Fc polypeptide and other Fc polypeptides described herein.

[0029] Certain embodiments provide a polynucleotide comprising a nucleic acid sequence encoding a polypeptide described herein. Certain embodiments also provide a vector comprising the polynucleotide described herein. Certain embodiments provide a host cell comprising the polynucleotide described herein or the vector described herein. In certain embodiments, such a host cell further comprises a polynucleotide comprising a nucleic acid sequence encoding another Fc polypeptide.

[0030] Provided herein are methods for producing the proteins or polypeptides described herein.

[0031] A particular embodiment provides a method for generating a polypeptide comprising an SGSH amino acid sequence, an SGSH variant amino acid sequence, or an Fc polypeptide linked to a catalytically active fragment, the method comprising culturing host cells under conditions in which a polypeptide encoded by a polynucleotide described herein is expressed.

[0032] Certain embodiments provide a pair of polynucleotides comprising: a first nucleic acid sequence encoding a first Fc polypeptide linked to a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof; and a second nucleic acid sequence encoding a second Fc polypeptide linked to a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. Certain embodiments also provide one or more vectors comprising the polynucleotide pairs described herein. Certain embodiments provide a host cell comprising the polynucleotide pairs described herein, or one or more vectors described herein.

[0033] A particular embodiment provides a method for producing a protein comprising a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a first Fc polypeptide linked to a catalytically active fragment thereof, and a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a second Fc polypeptide linked to a catalytically active fragment thereof, the method comprising culturing host cells under conditions in which the polynucleotide pairs described herein are expressed.

[0034] Certain embodiments provide a pharmaceutical composition comprising a protein or polypeptide described herein and a pharmaceutically acceptable carrier and / or excipient.

[0035] A particular embodiment provides a method for treating Sanfilippo syndrome A, comprising administering a protein or polypeptide described herein to a patient in need thereof.

[0036] Certain embodiments provide a protein or polypeptide described herein for use in the treatment of patients requiring treatment for Sanfilippo syndrome A.

[0037] Certain embodiments provide the use of the proteins or polypeptides described herein in the preparation of a medicament for the treatment of Sanfilippo syndrome A in patients requiring treatment for the same condition.

[0038] A particular embodiment provides a method for reducing the accumulation of toxic metabolites in a patient having Sanfilippo syndrome A, comprising administering a protein or polypeptide described herein to the patient.

[0039] Certain embodiments provide a protein or polypeptide described herein for use in reducing the accumulation of toxic metabolites in patients with Sanfilippo syndrome A.

[0040] Certain embodiments provide the use of the proteins or polypeptides described herein in the preparation of a pharmacopoeia for reducing the accumulation of toxic metabolites in patients with Sanfilippo syndrome A.

[0041] In certain embodiments, the toxic metabolites include oligosaccharides derived from heparan sulfate. In embodiments of the present invention, for example, the following items are provided. (Item 1) It is a protein, a. A first N-sulfoglucosamine sulfohydrolase (SGSH) amino acid sequence, an SGSH variant amino acid sequence, or a first Fc polypeptide linked to a catalytically active fragment thereof; b. A protein comprising a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a second Fc polypeptide linked to a catalytically active fragment thereof, wherein the second Fc polypeptide is capable of specifically binding to a transferrin receptor (TfR); the second Fc polypeptide comprises a sequence having at least 80% identity to SEQ ID NO: 37 and having Ala at position 389, according to EU numbering. (Item 2) The protein according to item 1, wherein the second Fc polypeptide further comprises Glu at position 380 and Asn at position 390, according to EU numbering. (Item 3) The second Fc polypeptide is located in the following position according to the EU numbering, i.e. i. Tyr at position 384; ii. Thr at position 386; iii. Glue at position 387; iv. Trp to position 388; v. Thr in position 413; vi. Glue at position 415; vii. Glu at position 416; and viii. The protein described in item 2, containing Phe at position 421. (Item 4) A protein described in any one of items 1 to 3, which can be transported by crossing the target blood-brain barrier. (Item 5) A protein described in any one of items 1 to 4, which binds to TfR with an affinity of approximately 100 nM to approximately 500 nM, or optionally approximately 150 nM to approximately 400 nM. (Item 6) The protein according to any one of items 1 to 5, wherein the second Fc polypeptide is bound to the apical domain of the TfR. (Item 7) The protein according to any one of items 1 to 6, wherein the binding of the protein to the TfR does not substantially inhibit the binding of transferrin to the TfR. (Item 8) The protein according to any one of items 1 to 7, wherein the first SGSH amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to any one of sequence numbers 58 to 60. (Item 9) The protein described in item 8, wherein the first SGSH amino acid sequence contains one of the amino acid sequences of sequence numbers 58-60. (Item 10) The protein according to any one of items 1 to 9, wherein the second SGSH amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to any one of sequence numbers 58 to 60. (Item 11) The protein described in item 10, wherein the second SGSH amino acid sequence contains one of the amino acid sequences of sequence numbers 58-60. (Item 12) The protein according to any one of items 1 to 11, wherein the first Fc polypeptide is linked to the first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof by a peptide bond or by a polypeptide linker. (Item 13) The protein according to any one of items 1 to 12, wherein the second Fc polypeptide is linked to the second SGSH amino acid sequence, SGSH variant amino acid sequence, or a catalytically active fragment thereof by a peptide bond or by a polypeptide linker. (Item 14) The polypeptide linker is a flexible polypeptide linker, item 12 or This is the protein described in section 13. (Item 15) The protein described in item 14, wherein the flexible polypeptide linker is a glycine-rich linker. (Item 16) The protein according to any one of items 12 to 15, wherein the polypeptide linker is GS (SEQ ID NO: 7), G4S (SEQ ID NO: 8), or (G4S)2 (SEQ ID NO: 9). (Item 17) The protein according to any one of items 1 to 16, wherein the N-terminus of the first Fc polypeptide is linked to the first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. (Item 18) The protein according to any one of items 1 to 16, wherein the C-terminus of the first Fc polypeptide is linked to the first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. (Item 19) The protein according to any one of items 1 to 18, wherein the N-terminus of the second Fc polypeptide is linked to the second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. (Item 20) The protein according to any one of items 1 to 18, wherein the C-terminus of the second Fc polypeptide is linked to the second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. (Item 21) The protein according to any one of items 1 to 16, wherein the N-terminus of the first Fc polypeptide is linked to the first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof; and the N-terminus of the second Fc polypeptide is linked to the second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. (Item 22) The protein according to any one of items 1 to 16, wherein the C-terminus of the first Fc polypeptide is linked to the first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof; and the C-terminus of the second Fc polypeptide is linked to the second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. (Item 23) The protein according to any one of items 1 to 22, wherein the first Fc polypeptide and the second Fc polypeptide each contain a modification that promotes heterodimerization. (Item 24) The protein according to item 23, wherein, according to EU numbering, one of the Fc polypeptides has a T366W substitution, and the other Fc polypeptide has T366S, L368A, and Y407V substitutions. (Item 25) The protein according to item 24, wherein the first Fc polypeptide contains the T366S, L368A, and Y407V substitutions, and the second Fc polypeptide contains the T366W substitution. (Item 26) The first Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 12-19 and 28-31; the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 34-41 and 54-57 A protein as described in item 25, comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of the above. (Item 27) The protein according to item 24, wherein the first Fc polypeptide contains the T366W substitution, and the second Fc polypeptide contains the T366S, L368A, and Y407V substitutions. (Item 28) The protein according to item 27, wherein the first Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 24-27; and the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 48-53. (Item 29) The protein according to any one of items 1 to 28, wherein the first Fc polypeptide and / or the second Fc polypeptide include a native FcRn binding site. (Item 30) The protein according to any one of items 1 to 28, wherein the first Fc polypeptide and the second Fc polypeptide do not have effector function. (Item 31) The protein according to any one of items 1 to 28, wherein the first Fc polypeptide and / or the second Fc polypeptide includes modifications that reduce effector function. (Item 32) The protein according to item 31, wherein the modification that reduces the effector function is the substitution of Ala at position 234 and Ala at position 235 according to EU numbering. (Item 33) The protein according to item 32, wherein the first Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity with any one of SEQ ID NOs. 14-19 and 26-31. (Item 34) The protein according to item 33, wherein the first Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 14, 15, 28, and 29. (Item 35) The protein according to item 33, wherein the first Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 18, 19, 30, and 31. (Item 36) The protein according to item 32, wherein the first Fc polypeptide linked to the first SGSH amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 61-88 and 117-118. (Item 37) The protein according to item 36, wherein the first Fc polypeptide linked to the first SGSH amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 61-68, 73-76, 81-84, and 117-118. (Item 38) The protein according to any one of items 32 to 37, wherein the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity with any one of sequence numbers 36 to 41 and 50 to 57. (Item 39) The protein according to item 38, wherein the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 36, 37, 54, and 55. (Item 40) The protein according to item 38, wherein the second Fc polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of SEQ ID NOs. 40, 41, 56, and 57. (Item 41) The protein according to any one of items 32 to 37, wherein the second Fc polypeptide linked to the second SGSH amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of sequence numbers 89 to 116 and 119 to 120. (Item 42) The protein according to item 41, wherein the second Fc polypeptide linked to the second SGSH amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of sequence numbers 89-96, 101-104, 109-112, and 119-120. (Item 43) The protein according to any one of items 1 to 42, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises an amino acid change to the native Fc sequence that prolongs the serum half-life. (Item 44) The protein according to item 43, wherein the amino acid change includes the substitution of Tyr at position 252, Thr at position 254, and Glu at position 256 according to EU numbering. (Item 45) The protein according to item 43, wherein the amino acid change includes the substitution of Leu at position 428 and Ser at position 434 according to EU numbering. (Item 46) The protein described in item 43, wherein the aforementioned amino acid change includes a substitution of Ser or Ala at position 434 according to EU numbering. (Item 47) The protein according to item 23, wherein the first Fc polypeptide linked to the first SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 61-68, 73-76, and 81-84; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 89-96, 101-104, and 109-112. (Item 48) The protein according to item 47, wherein the first Fc polypeptide linked to the first SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 61 to 64; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 89 to 92. (Item 49) The protein according to item 48, wherein the first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 63 or 64; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 91 or 92. (Item 50) The first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 75 or 76; linked to the second SGSH amino acid sequence The protein described in item 47, wherein the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 103 or 104. (Item 51) The protein according to item 47, wherein the first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 83 or 84; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 111 or 112. (Item 52) The protein according to item 47, wherein the first Fc polypeptide linked to the first SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 65-68; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises one of the amino acid sequences of SEQ ID NOs. 93-96. (Item 53) The protein according to item 52, wherein the first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 67 or 68; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 95 or 96. (Item 54) The protein according to item 23, wherein the first Fc polypeptide linked to the first SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 118; and the second Fc polypeptide linked to the second SGSH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 120. (Item 55) The protein according to any one of items 1 to 54, wherein the uptake of the SGSH amino acid sequence into the brain is at least 10 times greater than the uptake of the SGSH amino acid sequence in the absence of the first Fc polypeptide and the second Fc polypeptide, or compared to the uptake of the unmodified SGSH enzyme that results in TfR binding to the second Fc polypeptide. (Item 56) The protein described in any one of items 1 to 55, wherein 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. (Item 57) A protein according to any one of items 1 to 56, which does not contain an immunoglobulin heavy chain and / or light chain variable region sequence or its antigen-binding moiety. (Item 58) A polypeptide comprising an SGSH amino acid sequence, an SGSH variant amino acid sequence, or an Fc polypeptide linked to a catalytically active fragment thereof, wherein the Fc polypeptide is i) capable of specifically binding to a transferrin receptor (TfR); ii) comprises a sequence having at least 90% identity to SEQ ID NO: 37; iii) contains one or more modifications that promote its heterodimerization with another Fc polypeptide; and iv) has Ala at position 389 according to EU numbering. (Item 59) The polypeptide according to item 58, wherein the Fc polypeptide further comprises Glu at position 380 and Asn at position 390, according to EU numbering. (Item 60) The Fc polypeptide is located in the following positions according to the EU numbering, i.e. i. Tyr at position 384; ii. Thr at position 386; iii. Glue at position 387; iv. Trp to position 388; v. Thr in position 413; vi. Glue at position 415; vii. Glu at position 416; and viii. The polypeptide described in item 59, containing Phe at position 421. (Item 61) The polypeptide according to any one of items 58 to 60, wherein the Fc polypeptide is linked to the SGSH amino acid sequence, the SGSH variant amino acid sequence, or a catalytically active fragment thereof by a peptide bond or by a polypeptide linker. (Item 62) The polypeptide according to item 61, which is a fusion polypeptide comprising: the SGSH amino acid sequence, the SGSH variant amino acid sequence, or catalytically active fragment from the N-terminus to the C-terminus; the polypeptide linker; and the Fc polypeptide. (Item 63) The polypeptide according to item 61, which is a fusion polypeptide comprising: the Fc polypeptide from the N-terminus to the C-terminus; the polypeptide linker; and the SGSH amino acid sequence, SGSH variant amino acid sequence, or catalytically active fragment. (Item 64) The polypeptide according to any one of items 58 to 63, wherein the Fc polypeptide contains the T366S, L368A and Y407V substitutions according to EU numbering. (Item 65) A polypeptide according to item 64, comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of sequence numbers 97-100, 105-108, and 113-116. (Item 66) The polypeptide according to any one of items 58 to 63, wherein the Fc polypeptide contains the T366W substitution. (Item 67) A polypeptide according to item 66, comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to any one of sequence numbers 89-96, 101-104, 109-112, and 119-120. (Item 68) Proteins comprising polypeptides described in any one of items 58 to 67 and other Fc polypeptides. (Item 69) A pharmaceutical composition comprising a protein as described in any one of items 1-57 and 68, or a polypeptide as described in any one of items 58-67, and a pharmaceutically acceptable excipient. (Item 70) A nucleic acid sequence containing a polypeptide described in any one of items 58-67 Renucleotide. (Item 71) A vector containing the polynucleotides described in item 70. (Item 72) A host cell containing the polynucleotide described in item 70 or the vector described in item 71. (Item 73) The host cell according to item 72, further comprising a polynucleotide containing a nucleic acid sequence encoding the other Fc polypeptides. (Item 74) Linked to an SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment. A method for producing a polypeptide comprising an Fc polypeptide, comprising culturing host cells under conditions in which the polypeptide encoded by the polynucleotide described in item 70 is expressed. (Item 75) A pair of polynucleotides comprising: a first nucleic acid sequence encoding the first Fc polypeptide linked to the first SGSH amino acid sequence, SGSH variant amino acid sequence, or catalytically active fragment thereof, as described in any one of items 1 to 57; and a second nucleic acid sequence encoding the second Fc polypeptide linked to the second SGSH amino acid sequence, SGSH variant amino acid sequence, or catalytically active fragment thereof. (Item 76) One or more vectors containing the polynucleotide pairs described in item 75. (Item 77) A host cell containing a pair of polynucleotides as described in item 75, or one or more vectors as described in item 76. (Item 78) A method for producing a protein comprising a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a first Fc polypeptide linked to a catalytically active fragment thereof, and a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a second Fc polypeptide linked to a catalytically active fragment thereof, the method comprising culturing host cells under conditions in which the polynucleotide pair described in item 75 is expressed. (Item 79) A method for treating Sanfilippo syndrome A, comprising administering a protein described in any one of items 1 to 57 and 68 or a polypeptide described in any one of items 58 to 67 to a patient in need thereof. (Item 80) A protein or polypeptide as described in any one of items 1-57 and 68, or as described in any one of items 58-67, for use in the treatment of patients requiring treatment for Sanfilippo syndrome A. (Item 81) Use of any one of the proteins described in items 1-57 and 68 or any one of the polypeptides described in items 58-67 in the preparation of a medicament for the treatment of Sanfilippo syndrome A in patients requiring treatment for the same condition. (Item 82) A method for reducing the accumulation of toxic metabolites in a patient having Sanfilippo syndrome A, comprising administering to the patient a protein described in any one of items 1 to 57 and 68 or a polypeptide described in any one of items 58 to 67. (Item 83) A protein or polypeptide as described in any one of items 1-57 and 68, or as described in any one of items 58-67, for use in reducing the accumulation of toxic metabolites in patients with Sanfilippo syndrome A. (Item 84) Use of a protein or polypeptide described in any one of items 1-57 and 68, or any one of items 58-67, in the preparation of a pharmaceutical product for reducing the accumulation of toxic metabolites in patients with Sanfilippo syndrome A. (Item 85) The toxic metabolites include heparan sulfate-derived oligosaccharides, as described in any one of items 82-84, the protein, or use described herein. [Brief explanation of the drawing]

[0042] [Figure 1]A shows an exemplary ETV:SGSH fusion protein with various linker lengths between the SGSH enzyme and the Fc polypeptide hinge region. B shows an exemplary ETV:SGSH fusion protein with various linker lengths between the SGSH enzyme and the Fc polypeptide hinge region. C shows an exemplary ETV:SGSH fusion protein with various linker lengths between the SGSH enzyme and the Fc polypeptide hinge region. [Figure 2] This shows the fGly content of SGSH-Fc and ETV:SGSH fusion proteins as measured by LCMS. [Figure 3] This paper presents in vitro evaluations of the enzymatic activity of SGSH-Fc and ETV:SGSH fusion proteins. [Figure 4] This report describes the evaluation of cellular activity of SGSH-Fc fusion protein in fibroblasts from MPSIIIA patients and healthy controls using a 35S pulse tracking assay. [Figure 5] This shows the serum concentrations of SGSH-Fc and ETV:SGSH fusion proteins. [Figure 6A] The liver concentrations of SGSH-Fc and ETV:SGSH fusion protein after 2 hours are shown. [Figure 6B] This shows the liver concentrations of SGSH-Fc and ETV:SGSH fusion protein after 8 hours. [Figure 7] This shows the brain concentrations of SGSH-Fc and ETV:SGSH fusion proteins. [Figure 8] This shows the total heparan sulfate level in the liver. [Figure 9] This indicates the total heparan sulfate level in the brain. [Figure 10] This shows the total heparan sulfate level in CSF. [Figure 11] This shows total heparan sulfate levels in the brain after administration of two different ETV:SGSH fusion proteins. [Modes for carrying out the invention]

[0043] Currently, there is a need for new therapeutic agents for the treatment of Sanfilippo syndrome A, particularly those that treat the neurocognitive phenotype. Described herein is a specific enzyme replacement therapy called ETV:SGSH, which has the ability to cross the blood-brain barrier (BBB) ​​and treat both the peripheral and CNS symptoms of Sanfilippo syndrome A. As used herein, the term "ETV:SGSH" refers to a dimeric protein containing a first Fc polypeptide and a second Fc polypeptide, each capable of crossing the BBB and being transported, with each being bound (e.g., fused) to the SGSH enzyme, an SGSH enzyme variant, or its catalytically active fragment. As discussed in the examples, a mouse model of Sanfilippo syndrome A showed a decrease of over 50% in glycosaminoglycans (GAGs) in the brain and over 80% in GAGs in the CSF after a single intravenous administration of ETV:SGSH.

[0044] Protein molecules containing SGSH enzyme-FC fusion polypeptide As described herein, certain embodiments provide protein molecules comprising an SGSH enzyme-Fc fusion polypeptide. The SGSH enzyme incorporated into the protein is catalytically active, i.e., retains enzymatic activity. In some embodiments, the proteins described herein comprise (i) an Fc polypeptide that may contain a modification (e.g., one or more modifications that promote heterodimerization) or may be a wild-type Fc polypeptide; and an SGSH enzyme; and (ii) an Fc polypeptide containing a modification that results in binding to a blood-brain barrier (BBB) ​​receptor, e.g., a transferrin receptor (TfR), and optionally one or more additional modifications (e.g., one or more modifications that promote heterodimerization); and an SGSH enzyme.

[0045] In some embodiments, the proteins described herein comprise catalytically active fragments or variants of wild-type SGSH. In some embodiments, the SGSH enzyme is a variant or catalytically active fragment of the SGSH protein comprising any one of the amino acid sequences of SEQ ID NOs. 58, 59, and 60. In some embodiments, the catalytically active variant or fragment of the SGSH enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the activity of the wild-type SGSH enzyme.

[0046] In some embodiments, the SGSH enzyme, or its catalytic variant or fragment present in the proteins described herein, retains at least 25% of its activity compared to its activity when not fused to an Fc polypeptide or TfR-linked Fc polypeptide. In some embodiments, the SGSH enzyme, or its catalytic variant or fragment, 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 fused to an Fc polypeptide or TfR-linked Fc polypeptide. In some embodiments, the SGSH enzyme, or its catalytic variant or fragment, retains at least 80%, 85%, 90%, or 95% of its activity compared to its activity when not fused to an Fc polypeptide or TfR-linked Fc polypeptide. In some embodiments, fusion to an Fc polypeptide does not reduce the activity of the SGSH enzyme, or its catalytic variant or fragment. In some embodiments, fusion to TfR-binding Fc polypeptide does not reduce the activity of the SGSH enzyme.

[0047] Fc polypeptide modification The Fc polypeptide incorporated into the fusion protein described herein may include certain modifications. For example, the Fc polypeptide may include modifications that result in binding to a blood-brain barrier (BBB) ​​receptor, such as a transferrin receptor (TfR). Furthermore, 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 protein described herein comprises two Fc polypeptides, one of which is a wild-type Fc polypeptide, such as a human IgG1 Fc polypeptide; and the other Fc is modified to bind to a blood-brain barrier (BBB) ​​receptor, such as a transferrin receptor (TfR), and optionally further includes one or more additional modifications. In certain other embodiments, both Fc polypeptides each include independently selected modifications (e.g., modifications described herein).

[0048] The amino acid residues specified in various Fc modifications, including those introduced into modified Fc polypeptides that bind to BBB receptors, such as TfR, are numbered herein using EU index numbering. Any Fc polypeptide, such as IgG1, IgG2, IgG3, or IgG4, may have modifications, such as amino acid substitutions, at one or more of the positions described herein.

[0049] The modified Fc polypeptide present in the fusion proteins described herein (e.g., heterodimerization and / or enhanced BBB receptor binding) may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with respect to the native Fc region sequence or a fragment thereof, for example, a fragment of at least 50 amino acids or at least 100 amino acids 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 with respect to amino acids 1 to 110 or amino acids 111 to 217 of SEQ ID NO: 1, or fragments thereof, for example, fragments of at least 50 amino acids or at least 100 amino acids or more.

[0050] In some embodiments, the modified (e.g., heterodimerizing and / or enhancing BBB receptor binding) Fc polypeptide contains 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 the native Fc region. In some embodiments, the modified Fc polypeptide contains at least 25 consecutive amino acids, or at least 30, 35, 40 or 45 consecutive amino acids, or 50 consecutive amino acids, or at least 60, 65, 70, 75, 80, 85, 90 or 95 or more consecutive amino acids, or 100 or more consecutive amino acids, corresponding to the amino acid sequence of the native Fc region such as SEQ ID NO: 1.

[0051] Modification for blood-brain barrier (BBB) ​​receptor binding In some embodiments, what is provided herein is a fusion protein that can be transported across the blood-brain barrier (BBB). Such a protein comprises a modified Fc polypeptide that binds to a BBB receptor. The BBB receptor is expressed in the BBB endothelium, as well as in other cell and tissue types. In some embodiments, the BBB receptor is a transferrin receptor (TfR).

[0052] In some embodiments, the fusion proteins described herein bind specifically to TfR. In some embodiments, the fusion proteins described herein bind specifically to TfR with an affinity of about 50 nM to about 500 nM. In some embodiments, the protein binds to TfR with an affinity of approximately 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 (e.g., specifically). In some embodiments, the protein binds to TfR with an affinity of approximately 100 to approximately 500 nM. In some embodiments, the protein binds to TfR with an affinity of about 100 nM to about 300 nM, or about 150 nM to about 250 nM, or about 200 nM to about 250 nM. In some embodiments, the protein binds to TfR with an affinity of about 230 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.

[0053] In some embodiments, the modified Fc polypeptide that specifically binds to TfR includes a substitution in the CH3 domain. In some embodiments, the modified Fc polypeptide includes a human Ig CH3 domain, such as an IgG CH3 domain, which is modified for TfR binding activity. The CH3 domain may be from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH3 domain refers to the amino acid segment at approximately positions 341–447, numbered according to the EU numbering scheme.

[0054] In some embodiments, a modified Fc polypeptide that specifically binds to TfR can bind to the apical domain of TfR and to TfR without blocking or inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is substantially uninhibited. In some embodiments, the binding of transferrin 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, the binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%).

[0055] In some embodiments, the modified (e.g., BBB receptor-binding) Fc polypeptide present in the fusion proteins described herein includes substitutions at amino acid positions 384, 386, 387, 388, 389, 413, 415, 416, and 421, according to the EU numbering scheme.

[0056] In some embodiments, the modified Fc polypeptide that specifically binds to TfR contains Ala at position 389, according to EU numbering. In some embodiments, the modified Fc polypeptide that specifically binds to TfR 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 that specifically binds to TfR contains 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.

[0057] In additional embodiments, the modified Fc polypeptide further includes one, two, or three substitutions at positions 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.

[0058] 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: 32; and includes amino acids at EU index positions 380, 384-390 and / or 413-421 of SEQ ID NO: 32. 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: 33; and includes amino acids at EU index positions 380, 384-390 and / or 413-421 of SEQ ID NO: 32 or 33. 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: 32 or 33; and contains amino acids at EU index positions 380, 384-390 and / or 413-421 of SEQ ID NO: 32 or 33.

[0059] 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: 32 or 33, and has 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: 32 or 33, and has Ala at position 380, at position 389, and Asn at position 390, 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: 32 or 33, and includes, 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.

[0060] In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 33.

[0061] Additional Fc polypeptide mutations In some embodiments, the fusion proteins described herein comprise two Fc polypeptides, one or both of which comprise independently selected modifications (e.g., modifications described herein). Non-limiting examples of other mutations that may 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 effector function, mutations that affect glycosylation, mutations that reduce immunogenicity in humans, and / or mutations that result in heterodimerization by knobs and holes.

[0062] In some embodiments, the 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 the corresponding wild-type Fc polypeptide (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide).

[0063] In some embodiments, the Fc polypeptide present in the fusion protein includes knob and hole mutations that promote heterodimerization and inhibit homodimerization. Generally, the modification introduces a protrusion ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") at the interface of the second polypeptide, so that the protrusion can be positioned within a cavity to promote heterodimerization and, consequently, inhibit homodimerization. The protrusion is constructed by replacing a smaller amino acid side chain (e.g., tyrosine or tryptophan) at the interface of the first polypeptide. A cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing a larger amino acid side chain (e.g., alanine or threonine). In some embodiments, such additional mutations are located at the Fc polypeptide site where they do not adversely affect polypeptide binding to BBB receptors, e.g., TfR.

[0064] In one exemplary embodiment of the knob and whole approach for dimerization, one of the Fc polypeptides present in the fusion protein has tryptophan at position 366 (numbered according to the EU numbering scheme) instead of native threonine. The other Fc polypeptide of the dimer has valine at position 407 (numbered according to the EU numbering scheme) instead of native tyrosine. The other Fc polypeptide may further include substitutions, in which the native threonine at position 366 (numbered according to the EU numbering scheme) is substituted with serine, and the native leucine at position 368 (numbered according to the EU numbering scheme) is substituted with alanine. Thus, one of the Fc polypeptides of the fusion protein described herein has the T366W knob mutation, and the other Fc polypeptide has the Y407V mutation, which is typically accompanied by the T366S and L368A whole mutations. In certain embodiments, the first Fc polypeptide contains the T366S, L368A, and Y407V substitutions, and the second Fc polypeptide contains the T366W substitution. In certain other embodiments, the first Fc polypeptide contains the T366W substitution, and the second Fc polypeptide contains the T366S, L368A, and Y407V substitutions.

[0065] In some embodiments, modifications can be introduced to enhance the serum half-life. For example, in some embodiments, one or both Fc polypeptides present in the fusion protein described herein may contain tyrosine at position 252, threonine at position 254, and 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.

[0066] In some embodiments, one or both Fc polypeptides present in the fusion proteins described herein may include modifications that reduce effector function, i.e., modifications that reduce their ability to induce certain biological functions when bound to Fc receptors expressed on effector cells that mediate effector function. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cell-mediated 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 differ depending on the class of antibody. For example, native human IgG1 and IgG3 antibodies can induce ADCC and CDC activity when bound to appropriate Fc receptors present on immune system cells, while native human IgG1, IgG2, IgG3, and IgG4 can induce ADCP function when bound to appropriate Fc receptors present on immune cells.

[0067] In some embodiments, one or both Fc polypeptides present in the fusion protein described herein may also be manipulated to include other modifications for heterodimerization, such as electrostatic manipulation of contact residues within the naturally charged CH3-CH3 interface or hydrophobic patch modifications.

[0068] In some embodiments, one or both of the Fc polypeptides present in the fusion protein described herein may include additional modifications that modulate effector function.

[0069] In some embodiments, one or both Fc polypeptides present in the fusion protein described herein may include modifications that reduce or eliminate effector function. Exemplary Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in the CH2 domain at positions 234 and 235 according to the EU numbering scheme. For example, in some embodiments, one or both Fc polypeptides may contain alanine residues at positions 234 and 235. Therefore, one or both Fc polypeptides may include L234A and L235A (LALA) substitutions.

[0070] Additional Fc polypeptide mutations that modulate effector function may include, but are not limited to, mutations in which proline at position 329 is replaced with glycine or arginine, or with an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and the 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 exist, for example, according to the EU numbering scheme, L234A and L235A of the Fc region of human IgG1; L234A, L235A, and P329G of the Fc region of human IgG1; L234A, L235A, and P329S of the Fc region of human IgG1; S228P and L235E of the Fc region of human IgG4, L234A and G237A of the Fc region of human IgG1, L234A, L235A, and G237A of the Fc region of human IgG1, V234A and G237A of the Fc region of human IgG2, L235A, G237A, and E318A of the Fc region of human IgG4, and S228P and L236E of the Fc region of human IgG4. 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.

[0071] In some embodiments, the C-terminal Lys residue is removed in the Fc polypeptide described herein (i.e., the Lys residue at position 447 according to the EU numbering scheme).

[0072] Exemplary Fc polypeptides including additional mutations As non-limiting examples described herein, one or both Fc polypeptides present in the fusion proteins described herein may be knob mutations (e.g., T366W numbered according to the EU numbering scheme), hole mutations (e.g., T366S, L368A, and Y407V numbered according to the EU numbering scheme), or mutations that modulate effector function (e.g., L234A, L235A, and / or P329G or P32 Additional mutations may include 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). For example, Sequence IDs 12-19, 24-31, 34-41, and 48-57 provide non-limiting examples of modified Fc polypeptides containing one or more of these additional mutations.

[0073] In some embodiments, the Fc polypeptide may have a knob mutation (e.g., T366W numbered according to the EU numbering scheme) and at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 1, 2, 32, and 33. In some embodiments, the Fc polypeptide having any one of sequence numbers 1, 2, 32, and 33 may be modified to have a knob mutation.

[0074] 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to either sequence 24 or 25. In some embodiments, the modified Fc polypeptide comprises either sequence 24 or 25.

[0075] In some embodiments, the modified Fc polypeptide includes a knob mutation (e.g., T366W numbered with reference to EU numbering) and contains Ala at position 389, having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to either sequence SEQ ID NO: 34 or 35, and according to EU numbering. In some embodiments, the modified Fc polypeptide includes Glu at position 380; Ala at position 389; and Asn at position 390, having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to sequence SEQ ID NO: 34 or 35, and according to EU numbering. In some embodiments, the modified 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 SEQ ID NO: 34 or 35, and includes, 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 includes either the sequence of SEQ ID NO: 34 or 35.

[0076] In some embodiments, the Fc polypeptide may have a knob mutation (e.g., T366W 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 1, 2, 32, and 33. In some embodiments, the Fc polypeptide having any one of sequence numbers 1, 2, 32, and 33 can be modified to have a knob mutation and a mutation that modulates effector function.

[0077] In some embodiments, the modified Fc polypeptide includes knob mutations (e.g., T366W numbered with reference to EU numbering), as well as mutations that modulate 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 either sequence 26 or 27. In some embodiments, the modified Fc polypeptide includes either sequence 26 or 27.

[0078] In some embodiments, the modified Fc polypeptide includes knob mutations (e.g., T366W numbered with reference to EU numbering), as well as mutations that modulate 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)), having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one sequence of sequence numbers 36-41 and 54-57, and containing Ala at position 389 according to EU numbering. In some embodiments, the modified 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 of SEQ ID NOs. 36-41 and 54-57, and includes, 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 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the sequences of SEQ ID NOs. 36-41 and 54-57, and contains, according to EU numbering, at the following positions: 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 contains at least one of the sequences of SEQ ID NOs. 36-41 and 54-57.

[0079] In some embodiments, the Fc polypeptide may have a hole mutation (e.g., T366S, L368A, and Y407V numbered according to the EU numbering scheme), as well as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 1, 2, 32, and 33. In some embodiments, the Fc polypeptide having any one of sequence numbers 1, 2, 32, and 33 may be modified to have a hole mutation.

[0080] In some embodiments, the modified Fc polypeptide includes whole mutations (e.g., T366S, L368A, and Y407V numbered with reference to EU numbering) and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 12 and 13. In some embodiments, the modified Fc polypeptide includes any one of sequence numbers 12 and 13.

[0081] In some embodiments, the modified Fc polypeptide includes a whole mutation (e.g., T366S, L368A, and Y407V numbered with reference to EU numbering) and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 48 and 49, and includes Ala at position 389, according to EU numbering. In some embodiments, the modified Fc polypeptide includes at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 48 and 49, and includes Glu at position 380; Ala at position 389; and Asn at position 390, according to EU numbering. In some embodiments, the modified Fc polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the sequences of SEQ ID NOs. 48 and 49, and includes, according to EU numbering, at the following positions: 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 includes any one of the sequences of SEQ ID NOs. 48 and 49.

[0082] In some embodiments, the 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the sequences of SEQ ID NOs: 1, 2, 32, and 33. In some embodiments, an Fc polypeptide having any one of sequence numbers 1, 2, 32, and 33 can be modified to have a hole mutation and a mutation that modulates effector function.

[0083] In some embodiments, the modified Fc polypeptide includes whole mutations (e.g., T366S, L368A, and Y407V numbered with reference to EU numbering), as well as mutations that modulate 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 sequence of SEQ ID NOs. 14-19 and 28-31.

[0084] In some embodiments, the modified Fc polypeptide includes hole mutations (e.g., T366S, L368A, and Y407V numbered with reference to EU numbering), as well as mutations that modulate 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)), having 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 sequence numbers 50-53, and containing Ala at position 389 according to EU numbering. In some embodiments, the modified 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 of SEQ ID NOs. 50-53, and includes, 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 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the sequences of SEQ ID NOs. 50-53, and contains, according to EU numbering, at the following positions: 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 contains at least one of the sequences of SEQ ID NOs. 50-53.

[0085] FcRn binding site In certain embodiments, a modified (e.g., BBB receptor-binding) Fc polypeptide, or an Fc polypeptide present in the fusion protein described herein that does not specifically bind to the BBB receptor, may contain an FcRn binding site. In some embodiments, the FcRn binding site is located within the Fc polypeptide or a fragment thereof.

[0086] In some embodiments, the FcRn binding site includes a native FcRn binding site. In some embodiments, the FcRn binding site does not involve amino acid changes 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 includes modifications that alter the FcRn binding.

[0087] In some embodiments, the FcRn binding site has one or more mutated, e.g., substituted amino acid residues, which increase or substantially decrease the serum half-life (i.e., reduce the serum half-life by 25% or less when assayed under the same conditions compared to a modified Fc polypeptide equivalent with 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.

[0088] In some embodiments, one or more residues at or near the FcRn binding site are mutated relative to the native human IgG sequence to extend the serum half-life of the modified polypeptide. In some embodiments, the mutation is 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 the mutations M252Y, S254T, and T256E. In some embodiments, the modified Fc polypeptide includes 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 includes mutations T307A, E380A, and N434A. In some embodiments, the modified Fc polypeptide includes substitutions at positions T250 and M428 according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide includes the T250Q and / or M428L mutation. In some embodiments, the modified Fc polypeptide includes substitutions at positions M428 and N434 according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide includes the M428L and N434S mutations. In some embodiments, the modified Fc polypeptide includes the N434S or N434A mutation.

[0089] SGSH enzyme bound to Fc polypeptide In some embodiments, the fusion protein described herein comprises two Fc polypeptides described herein, one or both of which 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 the IgG1 hinge region, e.g., the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 5) or a portion thereof of the human IgG1 hinge (e.g., DKTHTCPPCP, SEQ ID NO: 6). In some embodiments, the hinge region is located in the N-terminal region of the Fc polypeptide.

[0090] In certain embodiments, the N-terminus of the first Fc polypeptide is linked to a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof. In certain embodiments, the C-terminus of the first Fc polypeptide is linked to a first SGSH amino acid sequence, an SGSH 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 SGSH amino acid sequence, an SGSH 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 SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof.

[0091] In a particular embodiment, the N-terminus of the first Fc polypeptide is linked to a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof; and the N-terminus of the second Fc polypeptide is linked to a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof.

[0092] In a particular embodiment, the C-terminus of the first Fc polypeptide is linked to a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof; and the C-terminus of the second Fc polypeptide is linked to a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof.

[0093] In some embodiments, the Fc polypeptide is linked to the SGSH enzyme by a linker, such as a peptide linker. In some embodiments, the Fc polypeptide is linked to the SGSH enzyme by a peptide bond or by a peptide linker, for example, the Fc polypeptide is a fusion polypeptide. The peptide linker can be configured such that the SGSH enzyme is rotatable with respect to the linked Fc polypeptide, and / or the peptide linker is resistant to digestion by proteases. The peptide linker may contain native amino acids, non-native 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 Gly, Asn, Ser, Thr, Ala, etc. Such a linker may be designed using known parameters, may be of arbitrary length, and may contain any number of repeating units of arbitrary length (e.g., repeating units of Gly and Ser residues). For example, the linker may have two, three, four, five or more Gly4-Ser (SEQ ID NO: 8) repeats, or a single Gly4-Ser (SEQ ID NO: 8). In other embodiments, the linker may be Gly-Ser (SEQ ID NO: 7). In some embodiments, the peptide linker may include a protease cleavage site, for example, a protease cleavage site that can be cleaved by an enzyme present in the central nervous system.

[0094] In some embodiments, the SGSH enzyme is conjugated to the N-terminus of the Fc polypeptide by, for example, a Gly-Ser linker (SEQ ID NO: 7), a Gly4-Ser linker (SEQ ID NO: 8), or a (Gly4-Ser)2 linker (SEQ ID NO: 9). In some embodiments, the Fc polypeptide may contain a hinge sequence or partial hinge sequence at its N-terminus that is conjugated to the linker or directly conjugated to the SGSH enzyme.

[0095] In some embodiments, the SGSH enzyme is conjugated to the C-terminus of the Fc polypeptide by, for example, a Gly-Ser linker (SEQ ID NO: 7), a Gly4-Ser linker (SEQ ID NO: 8), or a (Gly4-Ser)2 linker (SEQ ID NO: 9). In some embodiments, the C-terminus of the Fc polypeptide is conjugated directly to the SGSH enzyme.

[0096] In some embodiments, the SGSH enzyme is conjugated to an Fc polypeptide by a chemical crosslinking agent. Such conjugates can be prepared using well-known chemical crosslinking reagents and protocols. For example, there are numerous chemical crosslinking agents known to those skilled in the art that are useful for crosslinking polypeptides with the desired active ingredient. For example, crosslinking agents are heterobifunctional crosslinking agents that can be used to link molecules stepwise. Heterobifunctional crosslinking agents allow for the design of more specific coupling methods for conjugating proteins, thereby reducing the occurrence of undesirable side reactions such as homoprotein polymers. A wide variety of heterobifunctional crosslinking agents are known in the art, including N-hydroxysuccinimide (NHS) or its water-soluble analogs N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), and Examples 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 crosslinking agents having an N-hydroxysuccinimidide moiety can generally be obtained as N-hydroxysulfosuccinimidide analogs with higher water solubility. Furthermore, crosslinking agents having disulfide crosslinks in the linking chain can be synthesized as alkyl derivatives instead to reduce the amount of linker cleavage in vivo. In addition to heterobifunctional crosslinking agents, there are numerous other crosslinking agents, including homobifunctional crosslinking agents and photoreactive crosslinking agents.Disuccinimidyl subcrate (DSS), bismaleimide hexane (BMH), and dimethylpimelimidate·2HCl (DMP) are examples of useful homobifunctional crosslinking agents, while bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinking agents.

[0097] Exemplary protein molecules including SGSH enzyme-FC fusion polypeptide In some embodiments, the fusion protein described herein comprises a first Fc polypeptide linked to a first SGSH enzyme, an SGSH enzyme variant, or a catalytically active fragment thereof; and a second Fc polypeptide linked to a second SGSH enzyme, an SGSH enzyme variant, or a catalytically active fragment thereof, wherein the second Fc polypeptide contains Ala at position 389 according to EU numbering; and the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide. In some embodiments, the second Fc polypeptide contains Glu at position 380, i.e., Ala at position 389, and Asn at position 390, according to EU numbering. In some embodiments, the second Fc polypeptide contains, 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 binds specifically to TfR. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide do not contain immunoglobulin heavy chain and / or light chain variable region sequences or their antigen-binding moieties. In some embodiments, the first Fc polypeptide is a modified Fc polypeptide. In certain embodiments, the second Fc polypeptide (i.e., the modified Fc polypeptide) includes one or more additional modifications. In some embodiments, the modified Fc polypeptide described herein includes one or more modifications that facilitate heterodimerization with the other Fc polypeptide. In some embodiments, the modified Fc polypeptides described herein contain one or more modifications that reduce effector function. In some embodiments, the modified Fc polypeptides described herein contain one or more modifications that extend serum half-life.

[0098] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a first Fc polypeptide containing a first Fc polypeptide containing T366S, L368A, and Y407V (hole) substitutions; and a second polypeptide chain comprising a second Fc polypeptide bound to TfR and containing a T366W (knob) substitution. 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 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 include 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 contains human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0099] In some embodiments, the second Fc polypeptide comprises a knob mutation, namely the LALA / LALAPG / LALAPS mutation and / or the YTE mutation, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 34-41, and contains Ala at position 389, according to EU numbering. In some embodiments, the second Fc polypeptide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 34-41, and contains Ala at the following positions, namely Glu at position 380; Ala at position 389; and Asn at position 390, 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 the sequences of SEQ ID NOs. 34-41, and includes, 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 includes any one of the sequences of SEQ ID NOs. 34-41. 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 sequence numbers 12-19; ​​or comprises any one of sequence numbers 12-19.In some embodiments, the second Fc polypeptide comprises one of SEQ ID NOs: 34-41, and the first Fc polypeptide comprises one of SEQ ID NOs: 12-19. In some embodiments, the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide comprises a portion of the 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%, at least 96%, at least 97%, at least 98%, or at least 99% identity to one of SEQ ID NOs: 54-57, and contains 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 the sequences of SEQ ID NOs. 54-57, and includes, 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 of SEQ ID NOs. 54-57, and includes, according to EU numbering, at the following positions: 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 includes any one of the sequences of SEQ ID NOs. 54-57. In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 28-31; or it contains any one of sequence numbers 28-31.

[0100] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a first Fc polypeptide comprising a T366W (knob) substitution; and a second polypeptide chain comprising a second Fc polypeptide bound to TfR and comprising T366S, L368A, and Y407V (hole) substitutions. 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 include 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 contains human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0101] In some embodiments, the second Fc polypeptide comprises a whole mutation, a LALA / LALAPG / LALAPS mutation, and / or a YTE mutation, and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 48-53, and contains Ala at position 389 according to EU numbering. In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 48-53, and contains Ala at the following positions, i.e., Glu at position 380; Ala at position 389; and Asn at position 390, 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 the sequences of SEQ ID NOs. 48-53, and includes, according to EU numbering, at the following positions: 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 includes any one of the sequences of SEQ ID NOs. 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 the sequences of SEQ ID NOs. 24-27; or comprises any one of the sequences of SEQ ID NOs. 24-27.In some embodiments, the second Fc polypeptide comprises one of SEQ ID NOs: 48-53, and the first Fc polypeptide comprises one of SEQ ID NOs: 24-27. In some embodiments, the N-terminus of the modified Fc polypeptide and / or Fc polypeptide comprises a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 6).

[0102] In some embodiments, the first SGSH enzyme present in the 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%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs. 12-19, or comprises any one of SEQ ID NOs. 12-19 (e.g., as a fusion polypeptide). In some embodiments, the first SGSH enzyme is linked to the first Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or part thereof (e.g., DKTHTCPPCP, SEQ ID NO. 6). In some embodiments, the N-terminus of the first Fc polypeptide comprises a part of the 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%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs. 28-31; or it contains a sequence of any one of SEQ ID NOs. 28-31. In some embodiments, the first SGSH enzyme contains an SGSH sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs. 58-60; or it contains a sequence of any one of SEQ ID NOs. 58-60. In some embodiments, the first SGSH sequence linked to the Fc polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 61-68, 73-76, 81-84, and 117-118; or it contains any one of sequence numbers 61-68, 73-76, 81-84, and 117-118.In some embodiments, the fusion protein has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 34-41, and contains Ala at position 389, according to EU numbering. In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 34-41, and contains Glu at position 380; Ala at position 389; and Asn at position 390, according to EU numbering. In some embodiments, the second polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the sequences of SEQ ID NOs. 34-41, and includes, 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 includes any one of the sequences of SEQ ID NOs. 34-41. In some embodiments, the second SGSH enzyme is linked to the second Fc polypeptide by a linker such as a flexible linker and / or a hinge region or part thereof (e.g., DKTHTCPPCP, SEQ ID NO. 6). In some embodiments, the N-terminus of the second Fc polypeptide includes a portion of the 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%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 54-57, and includes 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 the sequences of SEQ ID NOs. 54-57, and includes, 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 of SEQ ID NOs. 54-57, and includes, according to EU numbering, at the following positions: 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 includes any one of the sequences of SEQ ID NOs. 54-57. In some embodiments, the second SGSH enzyme comprises an SGSH sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 58-60; or comprises any one of sequence numbers 58-60. In some embodiments, the second SGSH sequence linked to the second Fc polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 89-96, 101-104, 109-112, and 119-120, and contains 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 the sequences of SEQ ID NOs. 89-96, 101-104, 109-112, and 119-120, and includes, 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 of SEQ ID NOs. 89-96, 101-104, 109-112, and 119-120, and includes, according to EU numbering, at the following positions: 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 includes any one of the sequences of SEQ ID NOs. 89-96, 101-104, 109-112, and 119-120.

[0103] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing one of the sequences of SEQ ID NOs. 61-64, and a second SGSH-Fc polypeptide containing one of the sequences of SEQ ID NOs. 89-92.

[0104] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing one of the sequences of SEQ ID NOs. 65-68, and a second SGSH-Fc polypeptide containing one of the sequences of SEQ ID NOs. 93-96.

[0105] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing one of the sequences of SEQ ID NOs. 73-76, and a second SGSH-Fc polypeptide containing one of the sequences of SEQ ID NOs. 101-104.

[0106] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing one of the sequences of SEQ ID NOs: 81-84, and a second SGSH-Fc polypeptide containing one of the sequences of SEQ ID NOs: 109-112.

[0107] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing one of the sequences of SEQ ID NOs: 117-118, and a second SGSH-Fc polypeptide containing one of the sequences of SEQ ID NOs: 119-120.

[0108] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 61 or 62, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 89 or 90.

[0109] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 65 or 66, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 93 or 94.

[0110] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 63 or 64, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 91 or 92.

[0111] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 64, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 92.

[0112] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 67 or 68, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 95 or 96.

[0113] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 68, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 96.

[0114] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 73 or 74, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 101 or 102.

[0115] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 75 or 76, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 103 or 104.

[0116] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 76 and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 104.

[0117] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 81 or 82, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 109 or 110.

[0118] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 83 or 84, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 111 or 112.

[0119] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 84 and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 112.

[0120] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 117 and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 119.

[0121] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 118, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 120.

[0122] In some embodiments, the first SGSH enzyme present in the 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%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs. 24-27, or comprises any one of SEQ ID NOs. 24-27 (e.g., as a fusion polypeptide). In some embodiments, the first SGSH enzyme is linked to the first Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or part thereof (e.g., DKTHTCPPCP, SEQ ID NO. 6). In some embodiments, the N-terminus of the first Fc polypeptide comprises a part of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO. 6). In some embodiments, the first SGSH enzyme comprises an SGSH sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 58-60; or comprises any one of sequence numbers 58-60. In some embodiments, the first SGSH sequence linked to the Fc polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 69-72, 77-80, and 85-88; or comprises any one of sequence numbers 69-72, 77-80, and 85-88. In some embodiments, the fusion protein has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs. 48-53, and includes Ala at position 389 according to EU numbering.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 any one of the sequences of SEQ ID NOs. 48-53, and includes, 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the sequences of SEQ ID NOs. 48-53, and includes, 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 includes any one of the sequences of SEQ ID NOs. 48-53. In some embodiments, the second SGSH enzyme is linked to the second Fc polypeptide by a linker such as a flexible linker and / or a hinge region or part thereof (e.g., DKTHTCPPCP, SEQ ID NO. 6). In some embodiments, the N-terminus of the second Fc polypeptide includes a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 6). In some embodiments, the second SGSH enzyme includes an SGSH sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the sequences of SEQ ID NOs: 58-60; or includes any one of the sequences of SEQ ID NOs: 58-60.In some embodiments, the second SGSH sequence linked to the second Fc polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of sequence numbers 97-100, 105-108, and 113-116, and contains 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 the sequences 97-100, 105-108, and 113-116, and includes, 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 of SEQ ID NOs. 97-100, 105-108, and 113-116, and includes at the following positions according to EU numbering, namely 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 includes any one of the sequences of SEQ ID NOs.

[0123] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 69 or 70, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 97 or 98.

[0124] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 71 or 72, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 99 or 100.

[0125] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 77 or 78, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 105 or 106.

[0126] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 79 or 80, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NO: 107 or 108.

[0127] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 85 or 86, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NOs: 113-114.

[0128] In some embodiments, the fusion protein comprises a first SGSH-Fc fusion polypeptide containing the sequence of SEQ ID NO: 87 or 88, and a second SGSH-Fc polypeptide containing the sequence of SEQ ID NOs: 115-116.

[0129] 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) ranging from 1 pM to 10 μM. In some embodiments, the affinity for TfR is in the range of 1 nM to 5 μM or 10 nM to 1 μM. In some embodiments, the affinity for TfR is 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 450 nM. In some embodiments, the affinity for TfR is monovalent.

[0130] Evaluation of protein activity The activity of the fusion proteins described herein, including the SGSH enzyme, can be evaluated using a variety of assays, including those described in the Examples section, which measure activity in vitro using artificial substrates. Other exemplary protocols for measuring SGSH activity in vitro are provided, for example, in WO2019 / 070577.

[0131] In some embodiments, tissue samples are evaluated. Tissue samples can be evaluated using the assay described above, however, multiple free-thaw cycles, e.g., two, three, four, or five or more, are typically included before the sonication step to ensure that microvesicles are ruptured.

[0132] Samples that can 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 the enzyme-Fc fusion protein described herein (e.g., SGSH-Fc fusion protein) can be evaluated.

[0133] Nucleic acids, vectors, and host cells The polypeptide chains contained in the fusion proteins described herein are typically prepared using recombinant methods. Therefore, in some embodiments, this disclosure provides isolated nucleic acids comprising nucleic acid sequences encoding any of the polypeptide chains containing the Fc polypeptides described herein, as well as host cells into which nucleic acids used for replicating and / or expressing the polypeptides are introduced. In some embodiments, the host cells are eukaryotes, such as human cells.

[0134] In another embodiment, a polynucleotide is provided comprising a nucleotide sequence encoding one or more 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.

[0135] Some embodiments also provide pairs of nucleic acid sequences in which each nucleic acid sequence encodes a polypeptide described herein. For example, a particular embodiment provides a pair of nucleic acid sequences in which the first nucleic acid sequence of the pair encodes a first Fc polypeptide linked to a first SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof; and the second nucleic acid sequence of the pair encodes a second Fc polypeptide linked to a second SGSH amino acid sequence, an SGSH variant amino acid sequence, or a catalytically active fragment thereof.

[0136] In some embodiments, polynucleotides are contained within the nucleic acid construct, or pairs of polynucleotides are 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 plasmids, viral vectors, phagemids, yeast chromosome vectors, and non-episomal mammalian vectors.

[0137] In some embodiments, polynucleotides are operably linked to one or more regulatory nucleotide sequences in the expression construct. In a certain set of embodiments, the nucleic acid expression construct is suitable for use as a surface expression library. In some embodiments, the library is suitable for surface expression in yeast. In some embodiments, the library is suitable for surface expression in phages. In another set of embodiments, the nucleic acid expression construct is suitable for polypeptide expression in a system in which polypeptides can be isolated in milligram or gram quantities. In some embodiments, the system is a mammalian cell expression system. In some embodiments, the system is a yeast cell expression system.

[0138] Expression vehicles for recombinant polypeptide generation include plasmids and other vectors. For example, suitable vectors include the following types of plasmids for expression in prokaryotic cells such as E. coli: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids. Vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg are examples of mammalian expression vectors suitable for eukaryotic cell transfection. Alternatively, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of polypeptides in eukaryotic cells. In some embodiments, it may be desirable to express recombinant polypeptides using 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 those for adenoviruses, adeno-associated viruses, and other viruses.

[0139] The vector can be transformed into any suitable host cell. In some embodiments, host cells, such as bacteria or yeast cells, may be suitable for use as a surface expression library. In certain cells, the vector is expressed in the host cell, expressing relatively large amounts of polypeptides. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cells are mammalian cells such as Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, NS0 cells, Y0 cells, HEK293 cells, COS cells, Vero cells, or HeLa cells.

[0140] Host cells transfected with one or more expression vectors encoding one or more Fc polypeptide chains described herein can be cultured under appropriate conditions to produce expression of one or more polypeptides. The polypeptides can be secreted from a mixture of the polypeptide-containing cells and culture medium and isolated. Alternatively, the polypeptides may be retained in the cytoplasm or membrane fraction, the cells may be recovered and lysed, and the polypeptides isolated using a desired method.

[0141] Treatment method The fusion proteins described herein can be used therapeutically to treat Sanfilippo syndrome A.

[0142] Accordingly, certain embodiments provide a method for reducing the accumulation of toxic metabolites (e.g., oligosaccharides derived from heparan sulfate) in subjects having Sanfilippo syndrome A, the method comprising administering the proteins described herein to the subjects.

[0143] Certain embodiments provide the proteins described herein for use in reducing the accumulation of toxic metabolites (e.g., oligosaccharides derived from heparan sulfate) in subjects having Sanfilippo syndrome A.

[0144] Certain embodiments provide the use of the proteins described herein in the preparation of a pharmaceutical for reducing the accumulation of toxic metabolites (e.g., oligosaccharides derived from heparan sulfate) in subjects having Sanfilippo syndrome A.

[0145] Certain embodiments also provide a method for treating Sanfilippo syndrome A, which comprises administering the protein described herein to a subject in need thereof.

[0146] Certain embodiments provide the proteins described herein for use in the treatment of subjects requiring treatment for Sanfilippo syndrome A.

[0147] Certain embodiments provide the use of the protein described herein in the preparation of a pharmacopoeci for the treatment of Sanfilippo syndrome A in subjects requiring treatment of the syndrome.

[0148] In some embodiments, administration of a protein (e.g., linked to the SGSH enzyme) improves the uptake of SGSH in the brain compared to SGSH not linked to the fusion protein described herein, or compared to SGSH linked to a reference protein (e.g., the fusion protein described herein, which does not have modification to a second Fc polypeptide resulting in TfR binding). max It improves (for example, increases).

[0149] In some embodiments, C of SGSH in the brain max Compared to the uptake of SGSH when not linked to the fusion protein described herein, or compared to the uptake of SGSH linked to a reference protein (e.g., the fusion protein described herein that does not have modification to a second Fc polypeptide resulting in TfR binding), the uptake is improved by at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.4, 2.6, 2.8, 3, 4, 5, 6, or more (e.g., increased).

[0150] The fusion proteins described herein are administered to the subject in a therapeutically effective dose or amount.

[0151] In various embodiments, the fusion protein described herein is administered parenterally. In some embodiments, the protein is administered intravenously.

[0152] In some parenteral embodiments, the fusion protein described herein is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, the fusion protein described herein is administered intradermally or intramuscularly. In some embodiments, the fusion protein described herein is administered intrathecally, such as by epidural administration, or intraventricularly.

[0153] In other embodiments, the fusion proteins described herein may be administered orally, transpulmonaryly, intranasally, intraocularly, or topically. Transpulmonary administration may be employed, for example, by using a formulation comprising an inhaler or nebulizer and an aerosolizing agent.

[0154] Pharmaceutical compositions and kits In other embodiments, pharmaceutical compositions and kits comprising the fusion protein described herein are provided.

[0155] Pharmaceutical composition Guidelines for preparing formulations for use in this disclosure can be found in many handbooks on pharmaceuticals and formulations known to those skilled in the art.

[0156] In some embodiments, the pharmaceutical composition comprises a fusion protein described herein and further comprises one or more pharmaceutically acceptable carriers and / or excipients. Examples of pharmaceutically acceptable carriers include any solvent, dispersion medium, or coating, which are physiologically compatible with the active agent and do not interfere with or otherwise inhibit the activity of the active agent.

[0157] The dosages and desired drug concentrations of the pharmaceutical compositions described herein may vary depending on the specific intended use. Exemplary dosages are provided herein.

[0158] kit In some embodiments, a kit is provided for use in the treatment of Sanfilippo syndrome A, comprising the fusion protein described herein.

[0159] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit further comprises one or more additional therapeutic agents for use in treating the neurological symptoms of Sanfilippo syndrome A, comprising the fusion protein described herein. In some embodiments, the kit further comprises explanatory materials containing instructions (i.e., protocols) for carrying out the methods described herein (e.g., instructions for use for using the kit to administer the fusion protein containing the SGSH enzyme across the blood-brain barrier). The explanatory materials typically include, but are not limited to, written or printed materials. Any medium capable of storing and communicating such instructions to end users is contemplated by this 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), etc. Such media may include addresses of internet sites providing such explanatory materials.

[0160] A certain definition As used herein, unless otherwise clearly indicated, the singular forms “a,” “an,” and “the” include multiple references. Therefore, for example, a reference to “a polypeptide” may include two or more such molecules.

[0161] As used herein, the terms “about” and “approximately” indicate, when used to modify a quantity specified by a number or range, that a reasonable deviation from that number and a value known to those skilled in the art, e.g., ±20%, ±10%, or ±5%, is within the intended meaning of the stated value.

[0162] Where 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 human. In some embodiments, the human is a patient requiring treatment for Sanfilippo syndrome A. In some embodiments, the patient has one or more signs or symptoms of Sanfilippo syndrome A.

[0163] The term "pharmaceutically acceptable excipient" refers to a non-active pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, buffers, carriers, or preservatives.

[0164] The term "administer" refers to a method of delivering a drug (e.g., a Sanfilippo syndrome A treatment such as the ETV:SGSH 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, oral, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, or intraperitoneal delivery. In one embodiment, the polypeptide described herein is administered intravenously.

[0165] As used herein, “treatment” (and its grammatical variations such as “to treat” or “to treat”) refers to a clinical intervention to alter the natural processes of the individual being treated, which may be carried out for preventive purposes or during a clinicopathological process. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, slowing of disease progression, recovery or remission of the condition, and remission or improvement of prognosis.

[0166] The term "effective dose" means the amount of a compound described herein that (i) treats or prevents a particular disease, condition or disorder, (ii) reduces, improves 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.

[0167] The “therapeutic dose” of a substance / molecule disclosed herein may vary depending on factors such as the individual’s medical condition, age, sex, and weight, as well as the substance / molecule’s ability to induce a desired response in the individual. The therapeutic dose encompasses the amount in which the therapeutically beneficial effects of the substance / molecule outweigh the toxic or harmful effects. The “preventive dose” refers to the amount effective in achieving the desired preventive outcome at the required dosage and duration of administration. Typically, but not always, the preventive dose will be less than the therapeutic dose because the preventive dose is used in subjects before or at an early stage of the disease.

[0168] As used herein, “sulfoglucosamine sulfohydrolase,” “N-sulfoglucosamine sulfohydrolase,” or “SGSH” refers to N-sulfoglucosamine sulfohydrolase (EC3.10.1.1), an enzyme involved in the lysosomal degradation of heparan sulfate. Mutations in this gene are associated with Sanfilippo syndrome A, a type of mucopolysaccharidosis III, which is a lysosomal storage disorder resulting from impaired degradation of heparan sulfate. As used herein, the term “SGSH” refers to a component of a protein containing an Fc polypeptide, and encompasses catalytically active functional variants, including alleles and splice variants of wild-type SGSH or its fragments. The sequence of human SGSH is available in UniProt entry P51688 and is encoded by the human SGSH gene at 17q25.3. The full-length sequence is provided as Sequence ID 58. As used herein, the “mature” SGSH sequence refers to the naturally occurring form of the polypeptide chain lacking the signal sequence of the full-length polypeptide chain. The amino acid sequence of the mature human SGSH polypeptide is provided as Sequence ID No. 59, corresponding to amino acids 21-502 of the full-length human sequence. As used herein, the “shortened” SGSH sequence refers to a catalytically active fragment of a naturally occurring full-length polypeptide chain. The structure of human SGSH is well-characterized. An exemplary structure is available under PDB accession code 4MHX. SGSH sequences from non-human primates, including chimpanzees, have also been described (UniProt entry K7C218). Mouse SGSH sequences are available under Uniprot entry Q9EQ08. SGSH variants, for example, have 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 SGSH or fragment when assayed under identical conditions.A catalytically active SGSH fragment, for example, when assayed under identical conditions, exhibits 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 SGSH or its variant.

[0169] As used herein, “transferrin receptor” or “TfR” refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is defined by Sequence ID No. 10. Sequences of transferrin receptor protein 1 from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus macaque, 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 exemplary reference sequences, e.g., allelic variants of the human sequence encoded by the gene at the transferrin receptor protein 1 locus. The full-length transferrin receptor protein includes a short N-terminal intracellular domain, a transmembrane domain, 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 defined in Sequence ID No. 11.

[0170] As used herein, “fusion protein” or “[SGSH enzyme]-Fc fusion protein” refers to a dimer protein comprising a first Fc polypeptide (i.e., “[SGSH]-Fc fusion polypeptide”) linked (e.g., fused) to an SGSH enzyme, an SGSH enzyme variant, or its catalytically active fragment; and a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide. The second Fc polypeptide may also be linked (e.g., fused) to an SGSH enzyme, an SGSH enzyme variant, or its catalytically active fragment. The first Fc polypeptide and / or the second Fc polypeptide may be linked to an SGSH enzyme, an SGSH enzyme variant, or their catalytically active fragment by a peptide bond or polypeptide linker. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that promote its heterodimerization with other Fc polypeptides. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that confer binding to a transferrin receptor. The first Fc polypeptide and / or the second Fc polypeptide may 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 may be a modified Fc polypeptide containing one or more modifications that extend the serum half-life. In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide do not contain immunoglobulin heavy chain and / or light chain variable region sequences or their antigen-binding moieties. In some embodiments, the first Fc polypeptide and the second Fc polypeptide do not contain immunoglobulin heavy chain and / or light chain variable region sequences or their antigen-binding moieties.

[0171] As used herein, “fusion polypeptide” or “[SGSH enzyme]-Fc fusion polypeptide” refers to an Fc polypeptide linked (e.g., fused) to an SGSH enzyme, an SGSH enzyme variant, or a catalytically active fragment thereof. An Fc polypeptide can be linked to an SGSH enzyme, an SGSH enzyme variant, or a catalytically active fragment thereof by a peptide bond or polypeptide linker. An Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that promote its heterodimerization with another Fc polypeptide. An Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that confer binding to a transferrin receptor. An Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that reduce effector function. An Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that extend its serum half-life.

[0172] 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. Fc polypeptides contain a constant region sequence comprising at least a CH2 domain and / or a CH3 domain, and may contain at least a portion of a hinge region. Generally, Fc polypeptides do not contain a variable region.

[0173] A "modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, such as a substitution, deletion, or insertion, compared to the wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the native Fc polypeptide.

[0174] The term "FcRn" refers to the fetal Fc receptor. Binding of Fc polypeptides to FcRn reduces the clearance of Fc polypeptides and prolongs their serum half-life. The human FcRn protein is a heterodimer composed of a protein approximately 50 kDa in size, similar to major histocompatibility (MHC) class I proteins, and β2-microglobulin approximately 15 kDa in size.

[0175] As used herein, “FcRn binding site” refers to the region of an Fc polypeptide that binds to FcRn. In human IgG, FcRn binding sites numbered using the EU index include 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 in SEQ ID NO: 1.

[0176] As used herein, "native FcRn binding site" refers to a region of an Fc polypeptide that binds to an FcRn and has the same amino acid sequence as a naturally occurring Fc polypeptide region that binds to an FcRn.

[0177] As used herein, the terms “CH3 domain” and “CH2 domain” refer to immunoglobulin constant region domain polypeptides. For the purposes of this application, a CH3 domain polypeptide refers to the amino acid segment from approximately 341 to approximately 447, numbered according to the EU, and a CH2 domain polypeptide refers to the amino acid segment from approximately 231 to approximately 340, numbered according to the EU numbering scheme, and does not include the hinge region sequence. CH2 and CH3 domain polypeptides may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, in which case, according to the IMGT Scientific chart numbering (IMGT website), the numbering for CH2 domains is 1 to 110 and the numbering for CH3 domains is 1 to 107. The CH2 and CH3 domains are part of the Fc region of immunoglobulins. The Fc region refers to the amino acid segment from approximately 231 to approximately 447, numbered according to the EU numbering scheme, but as used herein, may 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).

[0178] The terms "naturally occurring," "native," or "wild-type" are used to describe objects found in nature that are distinct from those that have been artificially produced. For example, a nucleotide sequence found in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified in a laboratory is considered naturally occurring. Furthermore, "wild-type" refers to an organism found in nature with normal genes or no known mutations.

[0179] For example, the terms “wild-type,” “native,” and “naturally occurring” with respect to CH3 or CH2 domains are used herein to refer to domains having sequences that are naturally present.

[0180] As used herein, the term “variant” with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with “variant.” A variant with respect to a given wild-type CH3 or CH2 domain reference sequence may include naturally occurring allelic variants. A “non-natural” CH3 or CH2 domain refers to a variant or mutant domain that does not naturally exist in cells and is produced by genetic modification of a native CH3 domain or CH2 domain polynucleotide or polypeptide, for example, by using genetic engineering or mutagenesis techniques. A “variant” includes any domain that contains at least one amino acid mutation with respect to the wild type. Mutations may include substitutions, insertions, and deletions.

[0181] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and amino acid mimes that function in a manner similar to naturally occurring amino acids.

[0182] Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been later 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., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimes" refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0183] 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. Stereoimers of naturally occurring 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.

[0184] In this specification, amino acids are referred to by their commonly known three-letter abbreviations or IUPAC-IUB symbols. It may be referred to using one of the single-letter symbols recommended by the Biochemical Nomenclature Commission.

[0185] The terms “polypeptide” and “peptide” are used interchangeably herein and refer to polymers of amino acid residues in a single chain. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring and unnaturally occurring amino acid polymers. Amino acid polymers may consist entirely of L-amino acids, entirely of D-amino acids, or mixtures of L-amino acids and D-amino acids.

[0186] As used herein, the term "protein" refers to either a polypeptide or a dimer (i.e., two) or polymer (i.e., three or more) of a single-chain polypeptide. Single-chain polypeptides of a protein can be joined by covalent bonds, such as disulfide bonds, or by non-covalent interactions.

[0187] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to modifications that result in the substitution of an amino acid in another amino acid that can be classified as having similar characteristics. Examples of classifications of the conserved amino acid groups defined in this way include the "charged / polar group" which includes 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" which includes Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and the "aliphatic group" which includes Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids can be divided into subgroups including a "positively charged subgroup" containing Lys, Arg, and His, a "negatively charged subgroup" containing Glu and Asp, and a "polar subgroup" containing Asn and Gln. In another example, the aromatic or cyclic group can be divided into subgroups including a "nitrogen ring subgroup" containing Pro, His, and Trp, and a "phenyl subgroup" containing Phe and Tyr. In yet another example, the aliphatic group can be divided into subgroups, for example, an "aliphatic nonpolar subgroup" containing Val, Leu, Gly, and Ala, and an "aliphatic micropolar subgroup" containing Met, Ser, Thr, and Cys. Examples of the classification of conservative mutations include amino acid substitutions within the above subgroups, such as, but not limited to, substitutions of Lys with Arg or vice versa so that a positive charge can be maintained, Glu with Asp or vice versa so that a negative charge can be maintained, Ser with Thr or vice versa so that a free -OH can be maintained, and Gln with Asn or vice versa so that a free -NH2 can be maintained.In some embodiments, hydrophobic amino acids are substituted with naturally occurring hydrophobic amino acids, for example, at the active site, to preserve their hydrophobicity.

[0188] In relation to two or more polypeptide sequences, the terms “identical” or “percent “identical” refer to two or more sequences or subsequences that, when compared and aligned to the greatest extent possible across a comparison window or specified region using a sequence comparison algorithm or by manual alignment and visual inspection, have amino acid residues that are identical or identical to a certain percentage, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, across a specific region. In some embodiments, a sequence having a specified percentage identity with respect to a reference sequence differs from the reference sequence by one or more conservative substitutions.

[0189] In polypeptide sequence comparison, typically a single amino acid sequence functions as a reference sequence, which is then compared to the candidate sequence. Alignment can be performed using various methods available to those skilled in the art, such as visual alignment or using publicly available software with known algorithms, to achieve the best possible alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to achieve the best possible alignment can be determined by those skilled in the art. For the purposes of this application, the BLASTP algorithm standard protein BLAST is used to align two protein sequences with default parameters for polypeptide sequence comparison.

[0190] When used in connection with the identification of a given amino acid residue in a polypeptide sequence, the phrases "corresponding to," "determined with reference to," or "numbered with reference to" refer to the position of a residue in a particular reference sequence when the given amino acid sequence is optimally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a modified Fc polypeptide "corresponds to" an amino acid of SEQ ID NO:1 when the residue aligns optimally with the amino acid of SEQ ID NO:1. A polypeptide aligned to a reference sequence need not be of the same length as the reference sequence.

[0191] The terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to nucleotide chains of any length, including DNA and RNA. 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-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA.

[0192] As used herein, the term "binding affinity" refers to the strength of non-covalent interaction between two molecules, e.g., between a single binding site on a polypeptide and its target to which it binds, e.g., a transferrin receptor. Thus, for example, unless otherwise indicated or clear from the context, this term may refer to a 1:1 interaction between a polypeptide and its target. Binding affinity can be quantified by measuring the equilibrium dissociation constant (K D ), which is the dissociation rate constant (k a , time -1 M -1 ) divided by the association rate constant (k d , time -1 ). K DThis can be determined by measuring the dynamics of complex formation and dissociation, for example, by 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” can reflect not only formal binding affinity, such as a 1:1 interaction between a polypeptide and its target, but also K, which can reflect strong binding. D This includes the apparent affinity that is calculated.

[0193] As used herein, the terms “specifically bind” or “selectively bind” to a target, e.g., TfR, refer to a binding reaction in which the manipulated TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody described herein binds to the target with higher affinity, higher affinity, and / or longer duration than when binding to structurally different targets. In a typical embodiment, the manipulated TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody, when assayed under the same affinity assay conditions, has at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold or more affinity to a specific target, e.g., TfR, compared to an unrelated target. As used herein, the terms “specifically bind to,” “specifically bind to,” or “specific to” a particular target (e.g., TfR) refer to, for example, a binding reaction in which the manipulated TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody binds to the target, e.g., 10 -4 M or less (for example, 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 of M DIt can be represented by a molecule having. In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody binds specifically to an epitope on TfR that is conserved across species (e.g., structurally conserved across species), e.g., conserved between non-human primates and the human species (e.g., structurally conserved between non-human primates and the human species). In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody can bind exclusively to human TfR.

[0194] The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is derived from germline variable (V) genes, diversity (D) genes, or joining (J) genes (and not from constant (Cμ and Cδ) gene segments) and confers the specificity of the antibody to bind an antigen. Typically, the variable region of an antibody contains four conserved "framework" regions interspersed with three hypervariable "complementary determining regions".

[0195] 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 an antigen through 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 composed of two Fab fragments linked by disulfide bridges in the hinge region), single-chain Fv (scFv), disulfide-bonded Fv (dsFv), complementary determining regions (CDRs), VL (light chain variable region), and VH (heavy chain variable region).

[0196] The following examples are intended to be non-limiting.

Example

[0197] Example 1: Construction of a fusion protein containing N-sulfo-glucosamine sulfohydrolase (SGSH). Design and cloning We designed SGSH-Fc fusion proteins containing (i) a first fusion polypeptide ("SGSH-Fc fusion polypeptide") in which a mature human SGSH enzyme is fused to a human IgG1 fragment containing an Fc region, and (ii) a second fusion polypeptide ("modified Fc polypeptide") in which a mature human SGSH enzyme is fused to a modified human IgG1 fragment containing a mutation in the Fc region that confers transferrin receptor (TfR) binding. In particular, the SGSH fragment is human IgG1 SGSH-Fc fusion polypeptides were created by fusing SGSH to the N-terminus of the Fc region. In some cases, a linker was placed between SGSH and the IgG1 fragment to reduce steric hindrance between the two fragments. In all constructs, the signal peptide MGWSCIILFLVATATGAYA (SEQ ID: 121) was inserted upstream of the fusion to promote secretion, and SGSH was shortened to consist of amino acids R21-L502 (UniProtKB ID-P51688). The human IgG1 Fc region fragment used corresponds to amino acids D104-K330 (EU numbering, positions 221-447, including 10 hinge amino acids (positions 221-230)) of the sequence UniProtKB ID P01857. A second fusion polypeptide containing SGSH fused to the modified Fc polypeptide was co-transfected with the SGSH-Fc fusion polypeptide to generate a heterodimer fusion protein ("bizyme") containing two SGSH enzymes. Some constructs include additional mutations in the IgG1 fragment, which facilitate heterodimerization of the two Fc regions. Thus, the SGSH-Fc fusion protein containing a TfR linkage used in the examples is a dimer ("bizyme") formed by a TfR-binding SGSH-Fc fusion polypeptide comprising i) an SGSH-Fc fusion polypeptide; and ii) a modified Fc polypeptide fused to a second SGSH molecule.

[0198] A control SGSH-Fc fusion protein lacking mutations conferring TfR binding was similarly designed and constructed. An exemplary control SGSH-Fc fusion protein was generated that included a first SGSH-Fc fusion polypeptide having either one of the sequences of SEQ ID NO: 61 and 63 and a second SGSH-Fc fusion polypeptide having either one of the sequences of SEQ ID NO: 69 and 71. The SGSH-Fc fusion protein may also be further processed during cell culture production such that the first SGSH-Fc fusion polypeptide has the sequence of SEQ ID NO: 62 or 64 and / or the second SGSH-Fc fusion polypeptide has the sequence of SEQ ID NO: 70 or 72. Thus, as used herein, the term SGSH-Fc fusion protein refers to a protein molecule having non-processed sequences (i.e., SEQ ID NO: 61, 63, 69, and 71); a protein molecule containing one or more processed sequences (i.e., selected from SEQ ID NO: 62, 64, 70, and 72); or a mixture containing processed and non-processed protein molecules.

[0199] The SGSH-Fc fusion polypeptide, which contains the mature human SGSH sequence fused to the N-terminus of the IgG1 Fc polypeptide sequence having the hole and LALA mutations, has one of the sequences of SEQ ID NO: 61-64. The SGSH enzyme was conjugated to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 8), and the N-terminus of the Fc polypeptide included a part of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0200] The SGSH-Fc fusion polypeptide, which contains the mature human SGSH sequence fused to the N-terminus of the IgG1 Fc polypeptide sequence having the hole and LALA mutations, has one of the sequences of SEQ ID NO: 73-76. The SGSH enzyme was conjugated to the Fc polypeptide by a GS linker (SEQ ID NO: 7), and the N-terminus of the Fc polypeptide included a part of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0201] SGSH-Fc fusion polypeptides, which contain a mature human SGSH sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence having a whole mutation and an LALA mutation, have one of the sequences from SEQ ID NOs. 81 to 84. The SGSH enzyme is linked to the Fc polypeptide by a (GGGGSGGGGS) linker (SEQ ID NO. 9), and the N-terminus of the Fc polypeptide contained a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO. 6).

[0202] SGSH-Fc fusion polypeptides containing a mature human SGSH sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence having a whole mutation and a LALAPS mutation have one of the sequences from SEQ ID NOs. 65 to 68. The SGSH enzyme conjugates to the Fc polypeptide via a GGGGS linker (SEQ ID NO. 8), and the N-terminus of the Fc polypeptide contains a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO. 6).

[0203] Fc-SGSH fusion polypeptides, which contain a mature human SGSH sequence fused to the C-terminus of an IgG1 Fc polypeptide sequence having a whole mutation and an LALA mutation, have one of the sequences SEQ ID NOs. 117-118. The SGSH enzyme is linked to the Fc polypeptide by a GGGGS linker (SEQ ID NO. 8), and the N-terminus of the Fc polypeptide contains a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO. 6).

[0204] The TfR-binding SGSH-Fc fusion polypeptide, which contains a mature human SGSH sequence fused to the N-terminus of a TfR-binding modified Fc polypeptide sequence having knob mutations and LALA mutations, has one of the sequences from SEQ ID NOs. 89 to 92. The SGSH enzyme was linked to the modified Fc polypeptide by the GGGGS linker (SEQ ID NO. 8), and the N-terminus of the modified Fc polypeptide contained a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO. 6).

[0205] The TfR-binding SGSH-Fc fusion polypeptide, which contains a mature human SGSH sequence fused to the N-terminus of a TfR-binding modified Fc polypeptide sequence having knob mutations and LALA mutations, has one of the sequences from SEQ ID NOs. 101 to 104. The SGSH enzyme is linked to the Fc polypeptide by a GS linker (SEQ ID NO: 7), and the N-terminus of the Fc polypeptide contained a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 6).

[0206] The TfR-binding SGSH-Fc fusion polypeptide, which contains a mature human SGSH sequence fused to the N-terminus of a TfR-binding modified Fc polypeptide sequence having knob mutations and LALA mutations, has one of the sequences from SEQ ID NOs. 109 to 112. The SGSH enzyme was linked to the modified Fc polypeptide by the GGGGSGGGGS linker (SEQ ID NO. 9), and the N-terminus of the modified Fc polypeptide contained a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO. 6).

[0207] The TfR-binding SGSH-Fc fusion polypeptide, which contains a mature human SGSH sequence fused to the N-terminus of a TfR-binding modified Fc polypeptide sequence having a knob mutation and a LALAPS mutation, has one of the sequences from SEQ ID NOs. 93 to 96. The SGSH enzyme was linked to the modified Fc polypeptide by a GGGGS linker (SEQ ID NO. 8), and the N-terminus of the modified Fc polypeptide contained a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO. 6).

[0208] The TfR-binding SGSH-Fc fusion polypeptide, which contains a mature human SGSH sequence fused to the C-terminus of a TfR-binding modified Fc polypeptide sequence having knob mutations and LALA mutations, has one of the sequences of SEQ ID NOs. 119-120. The SGSH enzyme was joined to the modified Fc polypeptide by the GGGGS linker (SEQ ID NO. 8), and the N-terminus of the modified Fc polypeptide contained a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO. 6).

[0209] A first “N-terminal bizyme” SGSH-Fc fusion protein (“ETV:SGSH Bizyme Structure 1”) was generated, which contained a first SGSH-Fc fusion polypeptide having the sequence of either SEQ ID NOs. 61 or 63, and a second TfR-binding SGSH-Fc fusion polypeptide having the sequence of either SEQ ID NOs. 89 or 91. The SGSH-Fc fusion protein may also be further processed during cell culture generation so that the first SGSH-Fc fusion polypeptide has the sequence of SEQ ID NOs. 62 or 64, and / or the second TfR-binding SGSH-Fc fusion polypeptide has the sequence of SEQ ID NOs. 90 or 92. Therefore, as used herein, the term ETV:SGSH Bizyme Structure 1 can be used to refer to a protein molecule having a non-processing sequence (i.e., SEQ ID NOs: 61, 63, 89, and 91); a protein molecule containing one or more processing sequences (i.e., selected from SEQ ID NOs: 62, 64, 90, and 92); or a mixture containing processing and non-processing protein molecules.

[0210] A second “N-terminal bizyme” SGSH-Fc fusion protein (“ETV:SGSH Bizyme Structure 2”) was generated, which contained a first SGSH-Fc fusion polypeptide having the sequence of either SEQ ID NOs. 73 or 75, and a second SGSH-Fc fusion polypeptide that binds to TfR, having the sequence of either SEQ ID NOs. 101 or 103. The SGSH-Fc fusion protein may also be further processed during cell culture generation so that the first SGSH-Fc fusion polypeptide has the sequence of SEQ ID NOs. 74 or 76, and / or the second SGSH-Fc fusion polypeptide that binds to TfR has the sequence of SEQ ID NOs. 102 or 104. Therefore, as used herein, the term ETV:SGSH Bizyme Structure 2 can be used to refer to a protein molecule having a non-processing sequence (i.e., SEQ ID NOs: 73, 75, 101, and 103); a protein molecule containing one or more processing sequences (i.e., selected from SEQ ID NOs: 74, 76, 102, and 104); or a mixture containing processing and non-processing protein molecules.

[0211] A third “N-terminal bizyme” SGSH-Fc fusion protein (“ETV:SGSH Bizyme Structure 3”) was generated, which contained a first SGSH-Fc fusion polypeptide having the sequence of either SEQ ID NOs. 81 or 83, and a second SGSH-Fc fusion polypeptide that binds to TfR, having the sequence of either SEQ ID NOs. 109 or 111. The SGSH-Fc fusion protein may also be further processed during cell culture generation so that the first SGSH-Fc fusion polypeptide has the sequence of SEQ ID NOs. 82 or 84, and / or the second SGSH-Fc fusion polypeptide that binds to TfR has the sequence of SEQ ID NOs. 110 or 112. Therefore, as used herein, the term ETV:SGSH Bizyme Structure 3 can be used to refer to a protein molecule having a non-processing sequence (i.e., SEQ ID NOs: 81, 83, 109, and 111); a protein molecule containing one or more processing sequences (i.e., selected from SEQ ID NOs: 82, 84, 110, and 112); or a mixture containing processing and non-processing protein molecules.

[0212] A fourth “N-terminal bizyme” SGSH-Fc fusion protein (“ETV:SGSH Bizyme Structure 4”) was generated, which contained a first SGSH-Fc fusion polypeptide having the sequence of either SEQ ID NOs. 65 or 67, and a second TfR-binding SGSH-Fc fusion polypeptide having the sequence of either SEQ ID NOs. 93 or 95. The SGSH-Fc fusion protein may also be further processed during cell culture generation so that the first SGSH-Fc fusion polypeptide has the sequence of SEQ ID NOs. 66 or 68, and / or the second TfR-binding SGSH-Fc fusion polypeptide has the sequence of SEQ ID NOs. 94 or 96. Therefore, as used herein, the term ETV:SGSH Bizyme Structure 4 can be used to refer to a protein molecule having a non-processing sequence (i.e., SEQ ID NOs. 65, 67, 93, and 95); a protein molecule containing one or more processing sequences (i.e., selected from SEQ ID NOs. 66, 68, 94, and 96); or a mixture containing processing and non-processing protein molecules.

[0213] A “C-terminal bizyme” SGSH-Fc fusion protein (“ETV:SGSH Bizyme Structure 5”) was generated, which contained a first SGSH-Fc fusion polypeptide having the sequence of either SEQ ID NOs. 117 or 118, and a second SGSH-Fc fusion polypeptide that binds to TfR, having the sequence of either SEQ ID NOs. 119 or 120. Therefore, as used herein, the term ETV:SGSH Bizyme Structure 5 can be used to refer to protein molecules containing SEQ ID NOs. 117 and 119; protein molecules containing SEQ ID NOs. 118 and 120; or mixtures containing SEQ ID NOs. 117 and / or 118 in combination with SEQ ID NOs. 119 and / or 120.

[0214] Compositions comprising ETV:SGSH (for example, the structure described above) may be used to refer to compositions comprising protein molecules having a non-processing sequence; compositions comprising protein molecules comprising one or more processing sequences; or mixtures comprising processing protein molecules and non-processing protein molecules.

[0215] Recombinant protein expression and purification To express recombinant SGSH enzyme fused to the Fc region, ExpiCHO cells (Thermo Fisher Scientific) were transfected with the relevant DNA construct using the Expifectamine®CHO transfection kit according to the manufacturer's instructions for use (Thermo Fisher Scientific). The cells were grown in an orbital shaker (Infors HT Multitron) at 37°C, 5% CO2, and 125 rpm in ExpiCHO® expression medium supplemented with feed, as described in the manufacturer's protocol. Briefly, logarithmically growing ExpiCHO® cells were transfected with 0.8 μg of total DNA plasmid per 1 mL of culture volume, at a rate of 6 × 10⁶ 6 Transfection was performed at a cell / ml density. Cultures expressing the SGSH fusion were co-transfected with a plasmid expressing the cofactor SUMF1 in a 5:1 plasmid ratio (SGSH:SUMF1). The encoded SUMF1 sequence is described in Genbank NM_182760. After transfection, cells were returned to 37°C, and 18–22 hours after transfection, the transfected cultures were refilled with feed as indicated. The transfected cell culture supernatant was collected 120 hours after transfection by centrifugation at 3,500 rpm for 20 minutes. The clarified supernatant was filtered (on a 0.22 μM membrane) and stored at 4°C.

[0216] SGSH-Fc fusion proteins containing (or not containing) an engineered Fc region conferring TfR binding were purified from cell culture supernatant using protein A affinity chromatography. The supernatant was loaded onto a HiTrap MabSelect SuRe protein A affinity column (GE Healthcare Life Sciences, using the Akta Pure System). The column was then washed with 10 column volumes (CV) of PBS. The bound proteins were eluted using 50 mM citrate / NaOH buffer pH 3.6 containing 150 mM NaCl. Immediately after elution, the fraction was neutralized with 1 M Tris pH 8 (1:7 dilution). Homogeneity of the SGSH-Fc fusion in the eluted fraction was evaluated using multiple techniques, including reduced SDS-PAGE and HPLC-SEC, and unreduced SDS-PAGE and HPLC-SEC.

[0217] Example 2: Characterization of SGSH fusion protein. Formylglycine and M6P content of the fusion protein To characterize certain properties of SGSH-Fc fusion proteins that affect the enzymatic activity of SGSH and the transport of the fusion protein, the formylglycine (fGly) and mannose-6-phosphate (M6P) content of SGSH-Fc fusion proteins were evaluated. The ETV:SGSH N-terminal bizyme (Bizyme structure 1) and control SGSH-Fc fusion protein (lacking TfR binding) described in Example 1 were used for the analysis.

[0218] Measurement of fGly content. The identity and quantity of Cys and FGly-containing peptides were simultaneously evaluated by LC-MS / MS. Briefly, approximately 20 μg of SGSH fusion protein was reduced with tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl), alkylated with iodoacetamide, and proteolytically digested with trypsin (at 70°C for 2 hours). The reaction product quenched with formic acid was analyzed by LC-MS / MS. Quantitative analysis of the peptides was performed by liquid chromatography on a UHPLC Vanquish (Thermo Scientific, CA, USA) coupled to a UV / Vis and Q Exactive Orbitrap electrospray ionization mass spectrometer (Thermo Scientific, CA, USA). For analysis, the sample was injected into a CSH C18 column (Waters Corporation, Milford, Massachusetts, USA) at 40°C with a mobile phase of water containing 0.1% formic acid. Next, the samples were subjected to a 45-minute linear gradient from 1%B to 70%B, each containing water (A) with 0.1% formic acid and acetonitrile (B) with 0.1% formic acid, respectively. The mass spectrometer was operated in full mass scan mode in positive mode. Peak areas, or so-called area under the curve (AUC), were integrated using Thermo Scientific Freestyle software. The following SGSH cysteine ​​(three major trypsin peptides containing the CXPXR motif (SEQ ID NO: 126)) were accumulated at position 70: (1) free Cys, NAFTSVSSCSPSR (SEQ ID NO: 127) (2+, m / z 671.806); (2) alkylated carbamide methyl Cys: NAFTSVSSC(CAM)SPSR (SEQ ID NO: 128) (2+, m / z 700.317); and (3) FGly peptide: NAFTSVSS(Fgly)SPSR (SEQ ID NO: 129) (2+, m / z 663.810). The calculated percentage of FGly is based on dividing the AUC of the three FGly peptides by the sum of the AUCs of the FGly peptide, free Cys peptide, and alkylated Cys peptide, and multiplying by 100. The fGly content of SGSH-Fc and ETV was found to be similar to that of SGSH (Figure 2).

[0219] Measurement of mannose-6-phosphate (M6P) content. The M6P content of the SGSH-Fc fusion protein was measured by liquid chromatography-mass spectrometry analysis. The recombinant purified protein (20 μg) was buffer-exchanged into 50 mM ammonium acetate, pH 7.0. 5 μg of the protein was taken and spiked with stable isotope labeling (SIL) 13 C6 mannose-6-phosphate (M6P-IS, Omicronbio Inc, catalog #, MAN-05, 125 ng per sample) as an internal standard. 120 μL of 6.6 M trifluoroacetic acid solution was added to the protein sample, and this was hydrolyzed with shaking at 95 °C for 105 minutes using a heater block. The sample dried under a nitrogen stream was washed with acetonitrile (ACN) and dried again. The final pellet resuspended in 50 μL of ACN:water (20:80, v:v) was analyzed by LC-MS / MS. M6P analysis was performed by liquid chromatography in a UHPLC Vanquish (Thermo Scientific, CA, USA) coupled to a UV / Vis and Q Exactive Orbitrap electrospray ionization mass spectrometer (Thermo Scientific, CA, USA). The sample was injected at 60 °C in negative ionization mode into a BEH Amide column (Waters) 1.9 μm, 2.1×150 mm with a mobile phase of water containing 0.1% formic acid and eluted with a gradient of acetonitrile containing 0.1% formic acid. The data included M6P and M6P internal standard (IS), and were collected using parallel reaction monitoring (PRM) acquisition in negative mode with an inclusion time of 1.6 - 2.2 minutes, with precursors of 259.0224 (M6P) and 265.0426 (M6P-IS). The molecular weight of M6P released from the protein was calculated using the AUC ratio of M6P / M6P-IS, and the number of moles of M6P per mole of protein was obtained. The M6P contents of SGSH-Fc and ETV:SGSH are shown in Table 1.

Table 1

[0220] SGSH-Fc fusion proteins with a modified TfR binding site bind to human TfR. To determine whether the SGSH-Fc fusion protein with manipulated TfR binding affects the ability of the modified Fc domain to interact with human TfR, the affinity of ETV:SGSH Bizyme structure 1 (Example 1) to human TfR was evaluated using a Biacore® surface plasmon resonance assay. Biacore® Series S CM5 sensor chips were immobilized with anti-human Fab (Human Fab Capture Kit from GE Healthcare). 5 μg / mL of SGSH-Fc fusion protein was captured in each flow cell for 1 minute, and a 3-fold serial dilution of human apical domain TfR was injected at a flow rate of 30 μL / min. Each sample was analyzed after 3 minutes of association and 3 minutes of dissociation. After each injection, the chip was regenerated using 10 mM glycine-HCl (pH 2.1). Binding reactions were corrected by subtracting RU from flow cells that captured unrelated IgG at similar densities. Steady-state affinity was obtained by fitting the equilibrium response to concentration using Biacore® T200 Evaluation Software v3.1. Biacore® analysis revealed that the SGSH-Fc fusion protein, which has a modified TfR binding site in the Fc region, binds to human TfR. In particular, the binding affinity of ETV:SGSH Bizyme structure 1 to human TfR was determined to be approximately 230 nM.

[0221] SGSH-Fc fusion proteins with modified TfR binding sites are active both in vitro and intracellularly. The in vitro and cellular activity of the manipulated TfR-binding SGSH-Fc fusion protein was evaluated to demonstrate that SGSH maintains its enzymatic activity when fused to a human IgG fragment. The in vitro activity of recombinant SGSH was measured using a two-step fluorescence enzyme assay with an artificial substrate. Specifically, 20 μL of 1 mM 4-methylumbelliferyl 2-deoxy-2-sulfamino-aD-glucopyranoside sodium basic (Carbosynth Limited, #EM06602), diluted in assay buffer (0.03 M sodium acetate, 0.12 M NaCl, pH 6.5), was mixed with 10–20 μL of 140 nM SGSH. The first reaction mixture was incubated at 37°C for 17 hours, followed by termination with 10 μL of 0.2 M phosphate-citrate buffer, pH 6.7. Next, 10 μL (0.5 U) of yeast α-glucosidase (Sigma, #G0660-750UN) was added to initiate the second reaction, which was incubated at 37°C for 24 hours. The reaction was then stopped by adding 100 μL of 0.5 M sodium carbonate buffer, pH 10.3. The fluorescence of the reaction solution was then measured (excitation at 365 nm and emission at 450 nm). The amount of product was calculated by fitting a standard curve for 4-methylumbelliferone using linear regression and confirmed to be less than 10% of the total substrate cleavage. The specific activity (fmol product / min / pmol SGSH) was calculated by dividing the amount of product by the reaction time and the molar amount of SGSH.

[0222] In vitro enzyme activity assays demonstrated that the SGSH-Fc fusion protein was active and similar to Fc-SGSH (control; Example 1) and ETV:SGSH (Bizyme structure 1; Example 1) (Figure 3).

[0223] The cellular activity of the SGSH-Fc fusion protein was also observed in fibroblasts derived from MPS IIIA patients and healthy controls. 35 The investigation was conducted using the S pulse chase assay, and in this assay, 35S is incorporated into the newly synthesized GAG as previously described (Boado et al., Mol. Pharm. 11(8): 2928-2934

[2014] ). Since fibroblasts from MPS IIIA patients lack SGSH activity, this allows for the following: 35 S signal accumulation increases. ETV:SGSH-Fc fusion proteins containing SGSH (Bizyme structure 1) are S 35 Low picomolecular EC to reduce the accumulation of labeling substances 50 This was shown (Figure 4), and it was highly effective in cells derived from MPS IIIA patients.

[0224] SGSH-Fc fusion proteins with a modified TfR binding site show improved brain delivery of MPSIII in a mouse model. To determine whether the TfR-binding SGSH-Fc fusion protein showed improved brain delivery compared to the control SGSH-Fc fusion protein, a human TfR knock-in (TfR mu / huKI) mice were administered 40 mg / kg of the TfR-binding SGSH-Fc fusion protein ETV:SGSH (Bizyme structure 1) or a control SGSH-Fc fusion protein lacking the mutation conferring TfR binding ("SGSH:Fc") (see Example 1). The concentrations of SGSH-Fc fusion protein in the liver and brain were measured at 2 and 8 hours post-administration using a sandwich ELISA-based assay. The SGSH-Fc fusion proteins used for analysis were those described above and prepared according to Example 1 (referred to herein as ETV:SGSH (Bizyme structure 1) and control SGSH-Fc). Polyclonal donkey anti-human IgG capture antibody specific to the Fc fragment (Jackson ImmunoResearch, #709-006-098) was coated overnight on 384-well MaxiSorp® plates (Thermo Scientific #464718). After blocking plates with 5% BSA, they were incubated with diluted serum, brain, and liver lysates. Next, HRP-conjugated polyclonal goat anti-human IgG (Jackson ImmunoResearch, #109-036-098), specific to the Fc fragment, was added for detection. The plates were colorimetrically treated with a TMB substrate, stopped with sulfuric acid, and absorbance at 450 nm was measured using a BioTek plate reader. Standard curves were constructed individually in a 3-fold dilution series from 2000 to 2.74 pM and fitted using a 5-parameter logistic curve. TfR mu / hu KI mice were created by expressing the human Tfrc apical domain within the mouse Tfrc gene using CRISPR / Cas9 technology, as described in International Patent Publication WO2018 / 152285. The resulting chimeric TfRs were expressed in vivo under the control of an endogenous promoter. The results are shown in Figures 5-7.

[0225] Administration of the TfR-binding SGSH-Fc fusion protein resulted in approximately a 6-fold increase in cerebral uptake at 2 hours post-administration and approximately a 4-fold increase in cerebral concentration at 8 hours post-administration compared to the control SGSH-Fc fusion protein (Figure 7). Hepatic accumulation of the fusion protein was similar for both ETV:SGSH and SGSH:Fc at 2 hours, but decreased significantly (approximately 30-fold) at 8 hours post-administration, with ETV:SGSH showing lower levels compared to SGSH:Fc (Figure 6). Serum concentrations of the fusion protein were measured at 0.5, 1, 2, 4, and 8 hours post-administration using the sandwich ELISA-based assay described above. Serum PK was similar for both ETV:SGSH and SGSH:Fc at 2 hours, but ETV:SGSH showed lower levels compared to SGSH:Fc between 2 and 8 hours post-administration (Figure 5). Brain levels of TfR-binding SGSH-Fc fusion protein remained elevated for 8 hours compared to the control SGSH:Fc fusion protein, although faster peripheral clearance may be responsible for the decrease in brain and liver concentrations from 2 to 8 hours post-administration. In summary, these data indicate that the interaction between TfR-binding SGSH-Fc fusion protein and TfR significantly improves brain exposure while maintaining overall peripheral distribution.

[0226] ETV: Intravenous administration of SGSH reduces GAGs in the brain. To investigate whether the improvement in brain exposure observed for the TfR-binding SGSH-Fc fusion protein (hereinafter referred to as ETV:SGSH) prepared according to the above and Example 1 resulted in a corresponding decrease in accumulated substrates in the brain, a mouse model was created containing a sulfamidase mutation having a human TfR apical domain knocked into mouse TfR (hereinafter referred to as Sgsh). mps3a ×TfR mu / hu KI mouse, or alternatively SGSH D31N ;TfR mu / hu (Known as KI mice). Sgsh mice containing the novel sulfamidase mutation D31N. mps3aThe mouse was obtained from Jackson Laboratories (JAX stock #003780). In short, TfR mu / hu KI male mice, female Sgsh mps3a By crossing with heterozygous mice, TfR mu / hu Sgsh with KI homozygous background mps3a Homozygous mutant mice were generated. The mice used in this study were mixed and raised under a 12-hour light-dark cycle with free access to food (LabDiet JL irradiation 6F) and water.

[0227] Sgsh mps3a ×TfR mu / hu KI mice were administered a single dose of 40 mg / kg body weight of ETV:SGSH (Bizyme structure 1) or SGSH-Fc by intravenous injection, and the pharmacodynamic response was evaluated (see Example 1 for fusion proteins). In particular, Sgsh mps3a ×TfR mu / hu The effects of peripheral administration of ETV:SGSH on liver, brain, and CSF HS levels in KI mice were investigated in 3-month-old Sgsh mice that received intravenous (iv) injection of saline, SGSH-Fc (40 mg / kg body weight), or ETV:SGSH (40 mg / kg body weight). mps3a ×TfR mu / hu The determination was made using KI mice (n=8 / group). TfR in 3-month-old littermates. mu / hu KI mice were intravenously injected with physiological saline and used as controls. Sgsh mice injected with ETV:SGSH were euthanized 3 days after a single dose. mps3a ×TfR mu / hu With the exception of a subset of KI mice (n=4), all animals were euthanized 7 days after a single dose. Serum, CSF, liver, and brain were collected and rapidly frozen with dry ice.

[0228] Disaccharides derived from heparan sulfate were measured in vivo using the LC-MS / MS-based method described below. Briefly, after collecting all tissues and body fluids, they were immediately frozen and stored at -80°C. Tissue aliquots (50 mg) were homogenized in water (750 μL) using a Qiagen TissueLyzer II at 30 Hz for 3 minutes. The homogenate was transferred to a 96-well deep plate and sonicated with 10 × 1 second pulses using a 96-tip sonicator (Q Sonica). The sonicated homogenate was centrifuged at 2,500 × g for 30 minutes at 4°C to pellet cell debris. The resulting lysate was transferred to a clean 96-well deep plate and total protein was quantified by BCA. Heparan sulfate (HS) in the samples was digested into the corresponding disaccharides before LC-MS / MS analysis. 10 μg of total protein lysate or 3 μl of CSF was incubated in a PCR plate with heparinase I, II, and III in digestion buffer [111 mM NH4OAc, 0.11 mM CaOAc, 2 mM DTT, pH 7.0] for 3 hours with shaking at 30°C. After 3 hours, EDTA and 20 ng of the internal standard D4UA-2S-GlcNCOEt-6S (HD009, Iduron Ltd, Manchester, UK) were added to each sample, and the mixture was boiled at 95°C for 10 minutes to inactivate the enzymes. The digested samples were centrifuged at 3,364 × g for 5 minutes, and the supernatant was transferred to a cellulose acetate filter plate (Millipore, MSUN03010) and centrifuged at 3,364 × g for 5 minutes. The resulting eluate was mixed with an equal volume of acetonitrile in a glass vial and analyzed by mass spectrometry as follows.

[0229] Quantification of HS-derived disaccharides in body fluids and tissues was performed using electrospray mass spectrometry (Sciex 6500+QTRAP, Sciex, Framingham, MA, USA) and combined liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA). Each analysis used ACQUITY UPLC BEH Amide. The sample was injected into a 1.7 mm, 2.1 × 150 mm column (Waters Corporation, Milford, MA, USA) at a flow rate of 0.4 mL / min and a column temperature of 50°C. Mobile phase A consisted of water containing 10 mM ammonium formate and 0.1% formic acid, and mobile phase B consisted of acetonitrile containing 0.1% formic acid. The gradient was programmed as follows: 0.0–1.0 min at 85%B, 1.0–5.0 min from 85%B to 50%B, 5.0–6.0 min from 50%B to 85%B, and 6–8.0 min hold at 85%B. Electrospray ionization was performed in negative ionization mode with the following settings: curtain gas 30; collision gas moderate; ion spray voltage -4500; temperature 450°C; ion source gas 1 50; and ion source gas 2 60. Data acquisition was performed using Analyst 1.6.3 (Sciex) in multiple reaction monitoring mode (MRM) with the following settings: residence period 30 msec; collision energy -30; declustering potential -80; inlet potential -10; collision cell outlet potential -10. Individual disaccharides were identified based on retention time and MRM transitions using a commercially available reference standard (Iduron Ltd). The following disaccharide transitions were monitored: D0A0 (HS), m / z 378.1 > 87.0; D0S0 (HS), m / z 416.1 > 138.0; D4UA-2S-GlcNCOEt-6S (internal standard) m / z 472.0 > 97.0. The amount of disaccharides was normalized to the total protein level measured by the BCA assay or the amount of body fluid used per sample.

[0230] ETV: To determine whether SGSH reduces substrate levels in the brain, a single dose of the enzyme followed by SGSH mps3a ×TfR mu / huHS levels were evaluated in KI mice. SGSH-Fc had no effect on reducing brain HS levels after a single dose (Figure 9). However, ETV:SGSH reduced brain HS levels by approximately 50% and 57% on days 3 and 7, respectively, after a single dose (Figure 9). This resulted in a reduction of approximately 70% and 80% in CSF HS levels on days 3 and 7, respectively, after a single dose (Figure 10). Both molecules effectively reduced liver HS levels after one week (Figure 8), demonstrating that TfR binding does not adversely affect the pharmacodynamic response in these tissues. Data in Figures 8-10 are expressed as mean + / - standard error of the mean (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant). In summary, these data demonstrate that ETV:SGSH significantly increases enzyme exposure to the brain and reliably reduces substrate accumulation in both the periphery and the central nervous system (CNS).

[0231] Example 3: Product quality attributes of ETV:SGSH bizyme structure. Different bizyme structures of the ETV:SGSH fusion protein were evaluated in terms of product quality. For this study, ETV:SGSH bizyme structure 1 (Example 1) was compared with a structure having a different TfR-binding Fc region (ETV:SGSH bizyme structure 6, described below). Both structures were prepared as described in Example 1 using the additional purification steps described below.

[0232] result The human TfR affinity measured for Bizyme structure 1 and Bizyme structure 6 was equivalent (K D (These are approximately 290 nM versus approximately 245 nM, respectively.)

[0233] The expression titer of Bizyme structure 1 was determined to be approximately 30-40 mg / L, while the expression titer of Bizyme structure 6 was measured to be slightly lower (approximately 12-23 mg / L).

[0234] The recovery rates of Protein A after chromatographic purification were evaluated for both Bizyme structure 1 and Bizyme structure 6. Analysis of the post-protein A pools of both Bizyme structure 1 and Bizyme structure 6 showed a purity of approximately 50–60% with at least approximately 80% intact ETV structure (maintenance of modified Fc dimers including knob-hole pairs) (measured by HPLC-SEC). The post-protein A pools of both bizyme structures were subjected to hydrophobic interaction chromatography (HIC) for further purification (described below). The HIC post-pool of Bizyme structure 1 achieved a purity level of >95% (measured by HPLC-SEC) with >90% intact ETV structure, while the HIC post-pool of Bizyme structure 6 achieved a purity level of approximately 85% (measured by HPLC-SEC) with >90% intact ETV structure. Achieving higher purity levels (>90%) with Bizyme structure 6 requires additional purification steps, which may reduce the yield and recovery rate of the purified protein.

[0235] Therefore, Bizyme structure 1 and its P329S variant (Bizyme structure 4) were identified as preferred structures for transitioning to larger-scale production.

[0236] Experimental method A sixth “N-terminal bizyme” SGSH-Fc fusion protein (“ETV:SGSH Bizyme Structure 6”) was generated, which contained a first SGSH-Fc fusion polypeptide having the sequence of either SEQ ID NOs. 61 or 63, and a second TfR-binding SGSH-Fc fusion polypeptide having the sequence of either SEQ ID NOs. 122 or 124. The SGSH-Fc fusion protein may also be further processed during cell culture generation so that the first SGSH-Fc fusion polypeptide has the sequence of SEQ ID NOs. 62 or 64, and / or the second TfR-binding SGSH-Fc fusion polypeptide has the sequence of SEQ ID NOs. 123 or 125. Therefore, as used herein, the term ETV:SGSH Bizyme Structure 6 can be used to refer to a protein molecule having a non-processing sequence (i.e., SEQ ID NOs: 61, 63, 122, and 124); a protein molecule containing one or more processing sequences (i.e., selected from SEQ ID NOs: 62, 64, 123, and 125); or a mixture containing processing and non-processing protein molecules.

[0237] ETV:SGSH Bizyme Structure 1 and ETV:SGSH Bizyme Structure 6 were expressed and purified with the following modifications as described in Example 1: The pooled protein fraction eluted from the protein A affinity column was neutralized to a target pH 6.0 using 1 M Tris pH 8.0. The neutralized protein A pool was then prepared with 1 M sodium citrate to a final concentration of 0.6 M sodium citrate. The pooled fraction was loaded onto a butyl HP hydrophobic interaction chromatography (HIC) column, washed with 0.6 M sodium citrate (pH 6.0), and eluted (i) via a 50% step gradient from 0.6 M sodium citrate (pH 6.0) to WFI over 10 CV, followed by (ii) via a 100% step gradient from 0.6 M sodium citrate (pH 6.0) to WFI over 5 CV.

[0238] The homogeneity of the ETV:SGSH fusion protein in the eluted fraction was evaluated using multiple techniques, including reduced SDS-PAGE and HPLC-SEC, as well as unreduced SDS-PAGE and HPLC-SEC. Affinity for human TfR was measured as described in Example 2.

[0239] Example 4: ETV:SGSH was administered intravenously with different bizyme structures to achieve equivalent reductions in GAGs in the brain. We evaluated the effects of different bizyme structures of the ETV:SGSH fusion protein on brain GAG levels in a mouse model of MPS III. In this study, ETV:SGSH Bizyme Structure 1 was compared to the corresponding structure (ETV:SGSH Bizyme Structure 4) containing the P329S mutation in the Fc region.

[0240] result The bizyme structures were analyzed for formylglycine (fGly) content, mannose-6-phosphate (M6P) content, and human TfR affinity using the method described in Example 2. Table 2 shows the analysis results for each bizyme structure. The post-HIC pooled fractions of Bizyme Structure 1 and Bizyme Structure 4 achieved a purity level of >95% (measured by HPLC-SEC) with >90% intact ETV structures. [Table 2]

[0241] To determine whether the ETV:SGSH structure reduces substrate levels in the brain, a single dose of the ETV:SGSH protein was administered followed by Sgsh mps3a ×TfR mu / huHS levels were evaluated in KI mice. Both ETV:SGSH Bizyme Structure 1 and ETV:SGSH Bizyme Structure 4 reduced brain HS levels by approximately 63% and 59%, respectively, at 7 days after a single dose (Figure 11). The data in Figure 11 are expressed as mean + / - mean standard error. This data demonstrates that both ETV:SGSH bizyme structures reliably reduced substrate accumulation in the brain. Brain uptake of both bizyme structures was detectable at 7 days post-administration and was quantified as greater than 0.5 nM in brain tissue sampled from each cohort.

[0242] Experimental method ETV:SGSH Bizyme structure 1 was expressed and purified as described in Example 3.

[0243] ETV:SGSH Bizyme structure 4 was expressed from a stable CHO cell line transfected with the relevant DNA construct and selected by evaluation of expression titer, stability, and the activity of the expressed and purified protein. Briefly, the CHO-K1 GS knockout cell line (Horizon Discovery) was transfected with the relevant DNA construct (simultaneous transfection of the fusion protein and the plasmid encoding SUMF1), and then selected to generate a stable cell line expressing the target gene. The cell line was then placed in commercially available CHO cell culture medium for fed-batch generation (e.g., BalanCD CHO medium (Irvine Scientific) supplemented with BalanCD CHO Feed 4 (Irvine Scientific) in some cases). The culture was maintained at 37°C for 5 days, after which the temperature was changed to 32°C. On day 12, the cell culture was harvested, centrifuged, and the supernatant was sterile filtered through a commercially available (0.8 μm / 0.2 μm membrane filter) and stored at 4°C. The fusion protein was purified from the cell culture supernatant using protein A affinity and hydrophobic interaction chromatography. The supernatant was loaded onto a preparative scale MabSelect SuRe LX protein A affinity column (GE Healthcare Life Sciences, using the Akta Pure System). The column was then washed with 2 column volumes (CV) of PBS, followed by 4 CVs of 0.4 M potassium phosphate pH 7.0, and then 3 CVs of PBS. The bound protein was eluted using 50 mM citrate / NaOH buffer pH 3.7. Immediately after elution, the fraction was neutralized to a target pH of 6.0 using 1.5 M Tris pH 11. Prior to hydrophobic interaction chromatography, the neutralized protein A pool was adjusted with 1 M sodium citrate pH 6.0 in a 1:1.3 ratio. The prepared protein A pool was loaded onto a butyl HP hydrophobic interaction chromatography (HIC) column, washed with 0.6 M sodium citrate at pH 6.0, and then eluted by a 20–55% gradient from 0.6 M sodium citrate to WFI over 25 CV.The homogeneity of the fusion protein in the eluted fraction was evaluated using multiple techniques, including reduced SDS-PAGE and HPLC-SEC, as well as non-reduced SDS-PAGE and HPLC-SEC.

[0244] The fusion protein was analyzed for formylglycine (fGly) content, M6P content, and TfR affinity using the method described in Example 2.

[0245] Sgsh mps3a ×TfR mu / hu KI mice (Example 2) were administered a single dose of ETV:SGSH Bizyme structure 1 or ETV:SGSH Bizyme structure 4 by intravenous injection, and brain exposure and pharmacodynamic responses were evaluated. mps3a ×TfR mu / hu The effect of peripheral administration of ETV:SGSH bizyme structures on brain HS levels in KI mice was investigated in 9-month-old Sgsh mice that received intravenous (iv) injections of physiological saline, ETV:SGSH Bizyme Structure 1 (15 mg / kg body weight), or ETV:SGSH Bizyme Structure 4 (15 mg / kg body weight). mps3a ×TfR mu / hu The determination was made using KI mice (n=4-5 / group). TfR was determined in 9-month-old littermates who received intravenous injection of physiological saline. mu / hu KI mice (non-MPS III mice) were used as a control. All animals were euthanized 7 days after a single dose. Brain tissue was collected and rapidly frozen with dry ice. Brain uptake of ETV:SGSH and heparan sulfate-derived disaccharides was measured as described in Example 2. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21 Table 3-22 [Table 3-23] [Table 3-24] [Table 3-25] [Table 3-26] [Table 3-27] [Table 3-28]

[0246] All publications, patents, and patent documents are incorporated herein by reference as they are individually incorporated by reference. This disclosure has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many modifications and alterations are possible while remaining within the spirit and scope of the invention.

Claims

[Claim 1] The invention described in the specification.

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