Enzyme-containing fusion protein for enzyme replacement therapy

Abstract

Provision of fusion proteins containing enzymes for enzyme replacement therapy. [Solution] The present invention provides an enzyme for enzyme replacement therapy and a fusion protein comprising an Fc region, as well as a method for treating lysosomal storage disorders using such a protein. The present invention also provides a method for transporting a drug across the blood-brain barrier. In some embodiments, the present invention provides a protein comprising (a) an enzyme for ERT, a first Fc polypeptide linked to an enzyme variant for ERT or a catalytically active fragment thereof, and (b) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Patent Application No. 62 / 566,898 filed on October 2, 2017, U.S. Provisional Patent Application No. 62 / 583,276 filed on November 8, 2017, U.S. Provisional Patent Application No. 62 / 626,365 filed on February 5, 2018, U.S. Provisional Patent Application No. 62 / 678,183 filed on May 30, 2018, and U.S. Provisional Patent Application No. 62 / 721,396 filed on August 22, 2018, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This application is filed electronically in ASCII format and includes a sequence listing which is incorporated herein by reference in its entirety. The ASCII copy created on September 28, 2018 is named 102342 - 000350PC - 1103949_SL.txt and has a size of 580,464 bytes.

Background Art

[0003] Lysosomal storage disorders (LSDs) are relatively rare genetic metabolic disorders caused by abnormal lysosomal function. LSDs typically result from the deficiency of one enzyme involved in the breakdown of metabolic products in lysosomes. The deficiency of enzyme activity leads to the accumulation of metabolic products, which can affect various organ systems, cause severe symptoms, and may lead to early death. In most LSDs, significant neurological elements ranging from progressive neurodegeneration and severe cognitive impairment to epileptic disorders, behavioral disorders, and psychiatric disorders are also seen. Although LSDs can be treated using recombinant forms of the enzymes that are deficient in LSDs, delivering recombinant enzymes across the blood - brain barrier (BBB) is difficult, and thus such therapies may have little effect on the brain.

Summary of the Invention

Means for Solving the Problems

[0004] The present invention provides a fusion protein comprising an enzyme for enzyme replacement therapy (ERT), and a method of using the protein for the purpose of treating lysosomal storage disorders (LSDs).

[0005] In some embodiments, the present invention provides (a) a first Fc polypeptide linked to an enzyme for ERT, an ERT enzyme variant or a catalytically active fragment thereof, and (b) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide comprising a protein.

[0006] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide do not contain immunoglobulin heavy and / or light chain variable region sequences, or antigen-binding portions thereof.

[0007] In some embodiments, the enzyme for ERT is iduronate-2-sulfatase (IDS), an IDS variant or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT comprises an amino acid sequence having at least 80%, 85%, 90% or 95% identity with any one of the amino acid sequences of SEQ ID NOs: 91, 92, 114, 230 and 234. In some embodiments, the enzyme for ERT comprises any one of the amino acid sequences of SEQ ID NOs: 91, 92, 114, 230 and 234.

[0008] In some embodiments, the enzyme for ERT is N-sulfo-glucosamine sulfohydrolase (SGSH), an SGSH variant or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT comprises an amino acid sequence having at least 80%, 85%, 90% or 95% identity with any one of the amino acid sequences of SEQ ID NOs: 119 and 120. In some embodiments, the enzyme for ERT comprises any one of the amino acid sequences of SEQ ID NOs: 119 and 120.

[0009] In some embodiments, the enzyme for ERT is acid sphingomyelinase (ASM), an ASM variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with any one of the amino acid sequences of SEQ ID NOs: 121, 122, and 123. In some embodiments, the enzyme for ERT includes any one of the amino acid sequences of SEQ ID NOs: 121, 122, and 123.

[0010] In some embodiments, the enzyme for ERT is β-glucocerebrosidase (GBA), a GBA variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with one of the amino acid sequences of SEQ ID NOs. 93 and 94. In some embodiments, the enzyme for ERT includes one of the amino acid sequences of SEQ ID NOs. 93 and 94.

[0011] In some embodiments, the first Fc polypeptide is a fusion polypeptide linked to its ERT enzyme, ERT enzyme variant, or catalytically active fragment by a peptide bond or polypeptide linker. In some embodiments, the polypeptide linker is a flexible polypeptide linker. In some embodiments, the flexible polypeptide linker is a glycine-rich linker. In some embodiments, the glycine-rich linker is G4S (SEQ ID NO: 239) or (G4S)2 (SEQ ID NO: 240). In some embodiments, the first Fc polypeptide is not linked to its ERT enzyme, ERT enzyme variant, or catalytically active fragment by a chemical crosslinking agent; for example, the fusion polypeptide does not contain a non-peptide bond or a non-polypeptide linker.

[0012] In a particular embodiment, the fusion polypeptide comprises, in order from the N-terminus to the C-terminus, its ERT enzyme, an ERT enzyme variant or its catalytically active fragment, its polypeptide linker, and a first Fc polypeptide.

[0013] In some embodiments, the second Fc polypeptide is linked to the ERT enzyme, ERT enzyme variant, or catalytically active fragment thereof. In some embodiments, the second Fc polypeptide is a fusion polypeptide linked to the ERT enzyme, ERT enzyme variant, or catalytically active fragment thereof by a peptide bond or polypeptide linker. In some embodiments, the polypeptide linker is a flexible polypeptide linker. In some embodiments, the flexible polypeptide linker is a glycine-rich linker. In some embodiments, the glycine-rich linker is G4S (SEQ ID NO: 239) or (G4S)2 (SEQ ID NO: 240). In some embodiments, the second Fc polypeptide is not linked to the ERT enzyme, ERT enzyme variant, or catalytically active fragment thereof by a chemical crosslinking agent; for example, the fusion polypeptide does not contain a non-peptide bond or a non-polypeptide linker.

[0014] In some embodiments, the N-terminus of the first Fc polypeptide and / or the N-terminus of the second Fc polypeptide are ligated to the ERT enzyme. In some embodiments, the N-terminus of the first Fc polypeptide is ligated to one ERT enzyme, and the N-terminus of the second Fc polypeptide is ligated to the other ERT enzyme.

[0015] In some embodiments, the C-terminus of the first Fc polypeptide and / or the C-terminus of the second Fc polypeptide are ligated to the ERT enzyme. In some embodiments, the C-terminus of the first Fc polypeptide is ligated to one ERT enzyme, and the C-terminus of the second Fc polypeptide is ligated to the other ERT enzyme.

[0016] In some embodiments, the N-terminus of the first Fc polypeptide is ligated to one ERT enzyme, and the C-terminus of the second Fc polypeptide is ligated to the other ERT enzyme.

[0017] In some embodiments, the protein of the present invention comprises one ERT enzyme, wherein the N-terminus or C-terminus of a first Fc polypeptide is ligated to the ERT enzyme. In some embodiments, the protein of the present invention comprises two ERT enzymes (e.g., exactly two ERT enzymes). In some embodiments, the protein of the present invention comprises exactly one or exactly two ERT enzymes, enzyme variants, or catalytically active fragments thereof.

[0018] In some embodiments, the first Fc polypeptide is a modified Fc polypeptide, and / or the second Fc polypeptide is a modified Fc polypeptide.

[0019] In some embodiments, the first Fc polypeptide and the second Fc polypeptide each include modifications that promote heterodimerization. In some embodiments, the Fc dimer is an Fc heterodimer. In some embodiments, according to EU numbering, one of the Fc polypeptides has the substitution T366W, and the other Fc polypeptide has the substitutions T366S, L368A, and Y407V. In some embodiments, the first Fc polypeptide includes the substitutions T366S, L368A, and Y407V, and the second Fc polypeptide includes the substitution T366W. In some embodiments, the first Fc polypeptide is linked to the enzyme IDS for ERT and includes one of the amino acid sequences of SEQ ID NOs: 117, 232, and 236. In some embodiments, the first Fc polypeptide includes the substitution T366W, and the second Fc polypeptide includes the substitutions T366S, L368A, and Y407V. In some embodiments, the first Fc polypeptide is linked to the enzyme IDS for ERT and contains one of the amino acid sequences of SEQ ID NOs: 118, 233, and 237.

[0020] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide contains a native FcRn binding site. In some embodiments, the first Fc polypeptide and the second Fc polypeptide do not have effector function. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide include modifications that reduce effector function. In some embodiments, the modification that reduces effector function is, according to EU numbering, a substitution of Ala at position 234 and Ala at position 235. In some embodiments, the modification that reduces effector function further includes a substitution of Gly at position 329, according to EU numbering. In some embodiments, the first Fc polypeptide is linked to the enzyme IDS for ERT and contains one of the amino acid sequences of SEQ ID NOs. 115, 231, and 235. In some embodiments, the first Fc polypeptide is linked to the enzyme SGSH for ERT and contains one of the amino acid sequences of SEQ ID NOs. 149, 150, 152, and 153.

[0021] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide are amino acid changes from the native Fc sequence that extend the serum half-life. In some embodiments, the amino acid changes include substitutions to Tyr at position 252, Thr at position 254, and Glu at position 256, according to EU numbering. Alternatively, in another embodiment, the amino acid changes include substitutions to Leu at position 428 and Ser at position 434, according to EU numbering. Or, in a further embodiment, the amino acid changes include substitutions to Ser or Ala at position 434, according to EU numbering.

[0022] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide specifically bind to the transferrin receptor (TfR).

[0023] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide include at least two substitutions at positions selected from the group consisting of positions 384, 386, 387, 388, 389, 390, 413, 416, and 421, according to EU numbering. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide include at least three, four, five, six, seven, eight, or nine substitutions at those positions.

[0024] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further include one, two, three, or four substitutions at positions including 380, 391, 392, and 415 according to EU numbering. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further include one, two, or three substitutions at positions including 414, 424, and 426 according to EU numbering.

[0025] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide contains Trp at position 388. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide contains an aromatic amino acid at position 421. In some embodiments, the aromatic amino acid at position 421 is Trp or Phe.

[0026] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes at least one position selected from position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu; and position 421 which is Phe.

[0027] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from position 380 being Trp, Leu, or Glu; position 384 being Tyr or Phe; position 386 being Thr; position 387 being Glu; position 388 being Trp; position 389 being Ser, Ala, Val, or Asn; position 390 being Ser or Asn; position 413 being Thr or Ser; position 415 being Glu or Ser; position 416 being Glu; and position 421 being Phe.

[0028] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes 11 positions: position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu; and position 421 which is Phe.

[0029] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide has a CH3 domain that has at least 85% identity, at least 90% identity, or at least 95% identity with any one of the amino acids at positions 111-217 of SEQ ID NOs. 34-38, 58, 60-90, 151, and 156-229. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 156-229. In some embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues at positions corresponding to EU index positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 of any one of sequence numbers 34-38, 58, 60-90, 151, and 156-229 are not deleted or substituted.

[0030] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes the amino acid sequence of SEQ ID NO: 157. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes the amino acid sequence of SEQ ID NO: 169. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes the amino acid sequence of SEQ ID NO: 181. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes the amino acid sequence of SEQ ID NO: 193. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes the amino acid sequence of SEQ ID NO: 205. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes the amino acid sequence of SEQ ID NO: 217.

[0031] In some embodiments, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 115, and the second Fc polypeptide comprises the amino acid sequence of either SEQ ID NO: 205 or 228 (e.g., SEQ ID NO: 228). In another embodiment, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 115, and the second Fc polypeptide comprises the amino acid sequence of either SEQ ID NO: 169 or 229 (e.g., SEQ ID NO: 229).

[0032] In some embodiments, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 231, and the second Fc polypeptide comprises the amino acid sequence of either SEQ ID NO: 205 or 228 (e.g., SEQ ID NO: 228). In another embodiment, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 231, and the second Fc polypeptide comprises the amino acid sequence of either SEQ ID NO: 169 or 229 (e.g., SEQ ID NO: 229).

[0033] In some embodiments, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 235, and the second Fc polypeptide comprises the amino acid sequence of either SEQ ID NO: 205 or 228 (e.g., SEQ ID NO: 228). In another embodiment, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 235, and the second Fc polypeptide comprises the amino acid sequence of either SEQ ID NO: 169 or 229 (e.g., SEQ ID NO: 229).

[0034] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide bind to the apical domain of TfR. In some embodiments, the binding of the protein to TfR does not substantially inhibit the binding of transferrin to TfR.

[0035] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide have at least 75% amino acid sequence identity to the corresponding wild-type Fc polypeptide, or at least 80%, 85%, 90%, 92%, or 95%. In some embodiments, the corresponding wild-type Fc polypeptide is the Fc polypeptide of human IgG1, IgG2, IgG3, or IgG4.

[0036] In some embodiments, the uptake of the ERT enzyme into the brain (e.g., using a suitable animal model as described herein) is greater than the uptake of the ERT enzyme in the absence of the first Fc polypeptide and / or the second Fc polypeptide, or in the case of modification of the first Fc polypeptide and / or the second Fc polypeptide that does not result in binding to TfR. In some embodiments, the uptake of the ERT enzyme into the brain is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times greater than the uptake of the ERT enzyme in the absence of the first Fc polypeptide and / or the second Fc polypeptide, or compared to the uptake of the ERT enzyme when the first Fc polypeptide and / or the second Fc polypeptide are modified but not to result in binding to TfR.

[0037] In some embodiments, the first Fc polypeptide is not modified to bind to blood-brain barrier (BBB) ​​receptors, and the second Fc polypeptide is modified to specifically bind to TfRs.

[0038] In some embodiments, the proteins of the present invention do not contain immunoglobulin heavy chain and / or light chain variable region sequences, or their antigen-binding portions.

[0039] In some embodiments, the present invention provides a polypeptide comprising an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof, wherein the Fc polypeptide comprises one or more modifications that promote heterodimerization with another Fc polypeptide.

[0040] In some embodiments, the enzyme for ERT is an IDS, an IDS variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with any one of the amino acid sequences of SEQ ID NOs: 91, 92, 114, 230, and 234. In some embodiments, the enzyme for ERT includes any one of the amino acid sequences of SEQ ID NOs: 91, 92, 114, 230, and 234.

[0041] In some embodiments, the enzyme for ERT is SGSH, an SGSH variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with one of the amino acid sequences of SEQ ID NOs: 119 and 120. In some embodiments, the enzyme for ERT includes one of the amino acid sequences of SEQ ID NOs: 119 and 120.

[0042] In some embodiments, the enzyme for ERT is ASM, an ASM variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with any one of the amino acid sequences of SEQ ID NOs: 121, 122, and 123. In some embodiments, the enzyme for ERT includes any one of the amino acid sequences of SEQ ID NOs: 121, 122, and 123.

[0043] In some embodiments, the enzyme for ERT is GBA, a GBA variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with one of the amino acid sequences of SEQ ID NOs: 93 and 94. In some embodiments, the enzyme for ERT includes one of the amino acid sequences of SEQ ID NOs: 93 and 94.

[0044] In some embodiments, the Fc polypeptide is a fusion polypeptide linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof by a peptide bond or a polypeptide linker. In some embodiments, the polypeptide linker is a flexible polypeptide linker. In some embodiments, the flexible polypeptide linker is a glycine-rich linker. In some embodiments, the glycine-rich linker is G4S (SEQ ID NO: 239) or (G4S)2 (SEQ ID NO: 240). In some embodiments, the Fc polypeptide is not linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof by a chemical crosslinking agent; for example, the fusion polypeptide does not contain a non-peptide bond or a non-polypeptide linker.

[0045] In a particular embodiment, the fusion polypeptide comprises, in order from the N-terminus to the C-terminus, an enzyme for ERT, an enzyme variant for ERT or its catalytically active fragment, a polypeptide linker, and a first Fc polypeptide.

[0046] In some embodiments, the Fc polypeptide contains the substitutions T366S, L368A, and Y407V according to EU numbering. In some embodiments, the polypeptide contains one amino acid sequence from SEQ ID NOs: 115, 117, 231, 232, 235, and 236. In some embodiments, the polypeptide contains one amino acid sequence from SEQ ID NOs: 149 and 150. In some embodiments, the Fc polypeptide contains the substitution T366W. In some embodiments, the polypeptide contains one amino acid sequence from SEQ ID NOs: 118, 233, and 237. In some embodiments, the polypeptide contains one amino acid sequence from SEQ ID NOs: 152 to 155. In some embodiments, the polypeptide further comprises another Fc polypeptide. In some embodiments, the other Fc polypeptide contains the substitution T366W or the substitutions T366S, L368A, and Y407V, and forms an Fc dimer with the enzyme-Fc fusion polypeptide for ERT.

[0047] In some embodiments, the Fc polypeptide includes a native FcRn binding site. In some embodiments, the Fc polypeptide does not have effector function. In some embodiments, the Fc polypeptide includes modifications that reduce effector function. In some embodiments, the modification that reduces effector function is, according to EU numbering, substitution of Ala at position 234 and Ala at position 235. In some embodiments, the modification that reduces effector function further includes substitution of Gly at position 329, according to EU numbering.

[0048] In some embodiments, the Fc polypeptide includes amino acid changes from the native Fc sequence that extend the serum half-life. In some 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.

[0049] In some embodiments, the Fc polypeptide specifically binds to TfR.

[0050] In some embodiments, the Fc polypeptide includes at least two substitutions at positions selected from the group consisting of positions 384, 386, 387, 388, 389, 390, 413, 416, and 421, according to EU numbering. In some embodiments, the Fc polypeptide includes at least three, four, five, six, seven, eight, or nine substitutions at those positions.

[0051] In some embodiments, the Fc polypeptide further includes one, two, three, or four substitutions at positions including positions 380, 391, 392, and 415 according to EU numbering. In some embodiments, the Fc polypeptide further includes one, two, or three substitutions at positions including positions 414, 424, and 426 according to EU numbering.

[0052] In some embodiments, the Fc polypeptide contains Trp at position 388. In some embodiments, the Fc polypeptide contains an aromatic amino acid at position 421. In some embodiments, the aromatic amino acid at position 421 is Trp or Phe.

[0053] In some embodiments, the Fc polypeptide includes at least one position selected from position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu; and position 421 which is Phe.

[0054] In some embodiments, the Fc polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from position 380 being Trp, Leu, or Glu; position 384 being Tyr or Phe; position 386 being Thr; position 387 being Glu; position 388 being Trp; position 389 being Ser, Ala, Val, or Asn; position 390 being Ser or Asn; position 413 being Thr or Ser; position 415 being Glu or Ser; position 416 being Glu; and position 421 being Phe.

[0055] In some embodiments, the Fc polypeptide includes 11 positions: position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu; and position 421 which is Phe.

[0056] In some embodiments, the Fc polypeptide has a CH3 domain that has at least 85% identity, at least 90% identity, or at least 95% identity with an amino acid at positions 111-217 of any one of SEQ ID NOs.34-38, 58, and 60-90. In some embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues at positions corresponding to EU index positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 of any one of SEQ ID NOs.34-38, 58, and 60-90 are not deleted or substituted.

[0057] In some embodiments, the Fc polypeptide binds to the apical domain of TfR. In some embodiments, the binding of the protein to TfR does not substantially inhibit the binding of transferrin to TfR.

[0058] In some embodiments, the Fc polypeptide has at least 75% amino acid sequence identity to the corresponding wild-type Fc polypeptide, or at least 80%, 85%, 90%, 92%, or 95%. In some embodiments, the corresponding wild-type Fc polypeptide is the Fc polypeptide of human IgG1, IgG2, IgG3, or IgG4.

[0059] In some embodiments, the Fc polypeptide does not contain immunoglobulin heavy chain and / or light chain variable region sequences, or their antigen-binding moieties.

[0060] In some embodiments, the present invention provides a polynucleotide comprising a nucleic acid sequence encoding a polypeptide containing an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof, wherein the Fc polypeptide comprises one or more modifications that promote heterodimerization with another Fc polypeptide. In some embodiments, the present invention provides a vector comprising the polynucleotide. In some embodiments, the present invention provides a host cell comprising the polynucleotide or vector. In some embodiments, the host cell further comprises a polynucleotide comprising a nucleic acid sequence encoding another Fc polypeptide. In some embodiments, the present invention provides a method for producing the polypeptide described herein, comprising culturing a host cell under conditions for expression of the polypeptide encoded by the polynucleotide.

[0061] In some embodiments, the present invention provides, (a) A first polypeptide chain comprising a modified Fc polypeptide that specifically binds to TfR, (b) A second polypeptide chain containing an Fc polypeptide (the first and second polypeptide chains form an Fc dimer), and (c) An enzyme for ERT, an enzyme variant for ERT, or a catalytic fragment linked to the modified Fc polypeptide of (a) or the Fc polypeptide of (b). It is a protein that contains [something].

[0062] In some embodiments, the enzyme for ERT is an IDS, an IDS variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with any one of the amino acid sequences of SEQ ID NOs: 91, 92, 114, 230, and 234. In some embodiments, the enzyme for ERT includes any one of the amino acid sequences of SEQ ID NOs: 91, 92, 114, 230, and 234.

[0063] In some embodiments, the enzyme for ERT is SGSH, an SGSH variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with one of the amino acid sequences of SEQ ID NOs: 119 and 120. In some embodiments, the enzyme for ERT includes one of the amino acid sequences of SEQ ID NOs: 119 and 120.

[0064] In some embodiments, the enzyme for ERT is ASM, an ASM variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with any one of the amino acid sequences of SEQ ID NOs: 121, 122, and 123. In some embodiments, the enzyme for ERT includes any one of the amino acid sequences of SEQ ID NOs: 121, 122, and 123.

[0065] In some embodiments, the enzyme for ERT is GBA, a GBA variant, or a catalytically active fragment thereof. In some embodiments, the enzyme for ERT includes an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with one of the amino acid sequences of SEQ ID NOs: 93 and 94. In some embodiments, the enzyme for ERT includes one of the amino acid sequences of SEQ ID NOs: 93 and 94.

[0066] In some embodiments, the ERT enzyme is linked to the modified Fc polypeptide of (a). In some embodiments, the ERT enzyme is linked to the Fc polypeptide of (b). In some embodiments, the Fc polypeptide of (b) is not modified to bind to the BBB receptor. In some embodiments, the Fc polypeptide of (b) is a modified Fc polypeptide that specifically binds to TfR.

[0067] In some embodiments, the enzyme for ERT is linked (e.g., fused) to the modified Fc polypeptide of (a) or the Fc polypeptide of (b) by a peptide bond or polypeptide linker to form a fusion polypeptide. In some embodiments, the polypeptide linker is a flexible polypeptide linker. In some embodiments, the flexible polypeptide linker is a glycine-rich linker. In some embodiments, the glycine-rich linker is G4S (SEQ ID NO: 239) or (G4S)2 (SEQ ID NO: 240). In some embodiments, the enzyme for ERT is not linked to the modified Fc polypeptide of (a) or the Fc polypeptide of (b) by a chemical crosslinking agent, for example, the fusion polypeptide does not contain a non-peptide bond or a non-polypeptide linker.

[0068] In some embodiments, the ERT enzyme is ligated to the N-terminus of the modified Fc polypeptide of (a) or the N-terminus of the Fc polypeptide of (b). In some embodiments, the ERT enzyme is ligated to the C-terminus of the modified Fc polypeptide of (a) or the C-terminus of the Fc polypeptide of (b).

[0069] In some embodiments, the protein of the present invention comprises two ERT enzymes. In some embodiments, one ERT enzyme is ligated to the modified Fc polypeptide of (a), and the other ERT enzyme is ligated to the Fc polypeptide of (b). In some embodiments, both of these ERT enzymes are ligated to the N-terminus or the C-terminus of their respective Fc polypeptides. In some embodiments, one ERT enzyme is ligated to the N-terminus of the modified Fc polypeptide of (a), and the other ERT enzyme is ligated to the C-terminus of the Fc polypeptide of (b). In some embodiments, one ERT enzyme is ligated to the C-terminus of the modified Fc polypeptide of (a), and the other ERT enzyme is ligated to the N-terminus of the Fc polypeptide of (b).

[0070] In some embodiments, the Fc polypeptides of (a) and (b) each include modifications that promote heterodimerization. In some embodiments, according to EU numbering, one Fc polypeptide has the substitution T366W, and the other Fc polypeptide has the substitutions T366S, L368A, and Y407V. In some embodiments, the modified Fc polypeptide of (a) includes the substitution T366W, and the Fc polypeptide of (b) includes the substitutions T366S, L368A, and Y407V. In some embodiments, the Fc polypeptide of (b) is linked to the enzyme IDS for ERT and includes one of the amino acid sequences of SEQ ID NOs: 117, 232, and 236. In some embodiments, the modified Fc polypeptide of (a) includes the substitutions T366S, L368A, and Y407V, and the Fc polypeptide of (b) includes the substitution T366W. In some embodiments, the Fc polypeptide of (b) is linked to the enzyme IDS for ERT and contains one of the amino acid sequences of SEQ ID NOs: 118, 233, and 237.

[0071] In some embodiments, the modified Fc polypeptide of (a) and / or the Fc polypeptide of (b) contain a native FcRn binding site. In some embodiments, the modified Fc polypeptide of (a) and the Fc polypeptide of (b) do not have effector function. In some embodiments, the modified Fc polypeptide of (a) and / or the Fc polypeptide of (b) include modifications that reduce effector function. In some embodiments, the modifications that reduce effector function are substitutions of Ala at position 234 and Ala at position 235, according to EU numbering. In some embodiments, the modifications that reduce effector function further include a substitution of Gly at position 329, according to EU numbering. In some embodiments, the Fc polypeptide of (b) is linked to the enzyme IDS for ERT and contains one of the amino acid sequences of SEQ ID NOs: 115, 231, and 235. In some embodiments, the Fc polypeptide of (b) is linked to the enzyme SGSH for ERT and contains one of the amino acid sequences of SEQ ID NOs: 149, 150, 152, and 153.

[0072] In some embodiments, the modified Fc polypeptide of (a) and / or the Fc polypeptide of (b) are amino acid changes from the native Fc sequence that extend the serum half-life. In some 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.

[0073] In some embodiments, the modified Fc polypeptide includes at least two substitutions at positions selected from the group consisting of positions 384, 386, 387, 388, 389, 390, 413, 416, and 421, according to EU numbering. In some embodiments, the modified Fc polypeptide includes at least three, four, five, six, seven, eight, or nine substitutions at those positions.

[0074] In some embodiments, the modified Fc polypeptide further includes one, two, three, or four substitutions at positions including positions 380, 391, 392, and 415, according to EU numbering. In some embodiments, the modified Fc polypeptide further includes one, two, or three substitutions at positions including positions 414, 424, and 426, according to EU numbering.

[0075] In some embodiments, the modified Fc polypeptide contains Trp at position 388. In some embodiments, the modified Fc polypeptide contains an aromatic amino acid at position 421. In some embodiments, the aromatic amino acid at position 421 is Trp or Phe.

[0076] In some embodiments, the modified Fc polypeptide includes at least one position selected from position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu; and position 421 which is Phe.

[0077] In some embodiments, the modified Fc polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from position 380 being Trp, Leu, or Glu; position 384 being Tyr or Phe; position 386 being Thr; position 387 being Glu; position 388 being Trp; position 389 being Ser, Ala, Val, or Asn; position 390 being Ser or Asn; position 413 being Thr or Ser; position 415 being Glu or Ser; position 416 being Glu; and position 421 being Phe.

[0078] In some embodiments, the modified Fc polypeptide includes 11 positions: position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu; and position 421 which is Phe.

[0079] In some embodiments, the modified Fc polypeptide has a CH3 domain that has at least 85% identity, at least 90% identity, or at least 95% identity with any one of the amino acids at positions 111-217 of SEQ ID NOs.34-38, 58, 60-90, 151, and 156-229. In some embodiments, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs.156-229. In some embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues at positions corresponding to EU index positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 of any one of sequence numbers 34-38, 58, 60-90, 151, and 156-229 are not deleted or substituted.

[0080] In some embodiments, the modified Fc polypeptide includes the amino acid sequence of SEQ ID NO: 157. In some embodiments, the modified Fc polypeptide includes the amino acid sequence of SEQ ID NO: 169. In some embodiments, the modified Fc polypeptide includes the amino acid sequence of SEQ ID NO: 181. In some embodiments, the modified Fc polypeptide includes the amino acid sequence of SEQ ID NO: 193. In some embodiments, the modified Fc polypeptide includes the amino acid sequence of SEQ ID NO: 205. In some embodiments, the modified Fc polypeptide includes the amino acid sequence of SEQ ID NO: 217.

[0081] In some embodiments, the first polypeptide chain comprises the amino acid sequence of either SEQ ID NO: 205 or 228 (e.g., SEQ ID NO: 228), and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115. In another embodiment, the first polypeptide chain comprises the amino acid sequence of either SEQ ID NO: 169 or 229 (e.g., SEQ ID NO: 229), and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115.

[0082] In some embodiments, the first polypeptide chain comprises the amino acid sequence of either SEQ ID NO: 205 or 228 (e.g., SEQ ID NO: 228), and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 231. In another embodiment, the first polypeptide chain comprises the amino acid sequence of either SEQ ID NO: 169 or 229 (e.g., SEQ ID NO: 229), and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 231.

[0083] In some embodiments, the first polypeptide chain comprises the amino acid sequence of either SEQ ID NO: 205 or 228 (e.g., SEQ ID NO: 228), and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 235. In another embodiment, the first polypeptide chain comprises the amino acid sequence of either SEQ ID NO: 169 or 229 (e.g., SEQ ID NO: 229), and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 235.

[0084] In some embodiments, the modified Fc polypeptide binds to the apical domain of TfR. In some embodiments, the binding of the protein to TfR does not substantially inhibit the binding of transferrin to TfR.

[0085] In some embodiments, the modified Fc polypeptide has at least 75% amino acid sequence identity to the corresponding wild-type Fc polypeptide, or at least 80%, 85%, 90%, 92%, or 95%. In some embodiments, the corresponding wild-type Fc polypeptide is a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide.

[0086] In some embodiments, the uptake of the ERT enzyme into the brain (e.g., using a suitable animal model as described herein) is greater than the uptake of the ERT enzyme in the absence of the Fc polypeptide of the present invention, or when the Fc polypeptide has been modified in such a way that it does not result in binding to TfR. In some embodiments, the uptake of the ERT enzyme into the brain is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times greater than the uptake of the ERT enzyme in the absence of the Fc polypeptide of the present invention, or when the Fc polypeptide has been modified in such a way that it does not result in binding to TfR.

[0087] In some embodiments, the present invention provides a method for treating LSD, comprising administering the above-mentioned protein or polypeptide to a patient in need of treatment. In some embodiments, the method reduces the accumulation of toxic metabolites in the patient, for example, by reducing toxic metabolites in the patient's brain and / or cerebrospinal fluid (CSF).

[0088] In related embodiments, the present invention provides a method for reducing the accumulation of toxic metabolites in an LSD patient, comprising administering such protein or polypeptide to the patient. In some embodiments, the method reduces the accumulation of toxic metabolites in the patient's brain and / or CSF.

[0089] In some embodiments, the LSD is Hunter syndrome, and the enzyme for ERT is IDS. In some embodiments, the toxic metabolites include disaccharides derived from heparan sulfate and / or disaccharides derived from dermatan sulfate.

[0090] In some embodiments, the LSD is Sanfilippo syndrome A, and the enzyme for ERT is SGSH. In some embodiments, the toxic metabolites include oligosaccharides (e.g., hexasaccharides) derived from heparan sulfate.

[0091] In some embodiments, the LSD is Niemann-Pick disease, and the enzyme for ERT is ASM. In some embodiments, the toxic metabolites include sphingomyelin.

[0092] In some embodiments, the LSD is Gaucher disease or Parkinson's disease, and the enzyme for ERT is GBA. In some embodiments, the toxic metabolite includes glucosylceramide.

[0093] In some embodiments, the total amount of toxic metabolites is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to the total amount of toxic metabolites in the absence of the protein or polypeptide of the present invention. Exemplary assays for measuring the activity of the enzyme for ERT and the accumulation of substrates are described herein.

[0094] In some embodiments, the present invention provides a pharmaceutical composition comprising the above-described protein or polypeptide and a pharmaceutically acceptable carrier.

[0095] In some embodiments, the present invention provides a method for monitoring substrate accumulation and evaluating IDS activity, (a) Disrupting cells in cell or tissue samples or microvesicles in fluid samples derived from subjects to whom the above-mentioned proteins or polypeptides have been administered, and opening up the microvesicles to obtain a glycosaminoglycan (GAG) solution of the target for analysis, (b) Digesting the GAG ​​solution with at least one heparinase (e.g., heparinase I, heparinase II, and heparinase III) and chondroitinase B to obtain a disaccharide derived from the GAG, (c) Analyze the disaccharide derived from the GAG ​​by mass spectrometry (e.g., LC-MS / MS), (d) Determine the levels of disaccharides derived from heparan sulfate and / or dermatan sulfate, such that a lower level of disaccharides derived from heparan sulfate and / or dermatan sulfate compared to a control lacking IDS activity indicates that the IDS activity in the sample is elevated compared to the control. This method includes [something].

[0096] In some embodiments, the step of disrupting cells or microvesicles includes at least one freeze-thaw cycle and / or at least one sonication step. In some embodiments, the cells are derived from a tissue sample, and the method includes at least three, four, or five freeze-thaw cycles. In some embodiments, the subject is a mouse lacking IDS activity. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human patient with Hunter syndrome.

[0097] In some embodiments, the levels of disaccharides derived from heparan sulfate and / or dermatan sulfate are reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to the levels of disaccharides derived from heparan sulfate and / or dermatan sulfate in a control lacking IDS activity. In some embodiments, the control is a cell or tissue sample of the same type of tissue cells obtained from a subject before administration of the protein or polypeptide. In some embodiments, the control is a cell or tissue sample of the same type of tissue cells known to lack IDS activity. In some embodiments, the protein or polypeptide increases the IDS activity in the sample by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times compared to the IDS activity in the control.

[0098] In another aspect, the present invention provides a method for monitoring substrate accumulation and evaluating SGSH activity, (a) Disrupting cells in cell or tissue samples or microvesicles in fluid samples derived from subjects to whom the above-mentioned proteins or polypeptides have been administered, and opening up the microvesicles to obtain a glycosaminoglycan (GAG) solution of the target for analysis, (b) Digesting the GAG ​​solution with at least one heparinase to obtain a disaccharide derived from the GAG, (c) Analyze the disaccharide derived from the GAG ​​by mass spectrometry (e.g., LC-MS / MS), (d) Determine the level of disaccharides derived from heparan sulfate, and determine that the level of disaccharides derived from heparan sulfate is lower than that of a control lacking SGSH activity, thereby indicating that the SGSH activity in the sample is elevated compared to the control. This method includes [something].

[0099] In some embodiments, the step of disrupting cells or microvesicles includes at least one freeze-thaw cycle and / or at least one sonication step. In some embodiments, the cells are derived from a tissue sample, and the method includes at least three, four, or five freeze-thaw cycles. In some embodiments, the subject is a mouse lacking SGSH activity. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human patient with Sanfilippo syndrome A.

[0100] In some embodiments, the level of heparan sulfate-derived disaccharides is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to the level of heparan sulfate-derived disaccharides in a control lacking SGSH activity. In some embodiments, the control is a cell or tissue sample of the same type of tissue cells obtained from a subject before administration of the protein or polypeptide. In some embodiments, the control is a cell or tissue sample of the same type of tissue cells known to be lacking SGSH activity. In some embodiments, the protein or polypeptide increases the SGSH activity in the sample by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times compared to the SGSH activity in the control.

[0101] In another embodiment, the present invention provides a method for transporting a drug across the blood-brain barrier (BBB) ​​of a mammal, comprising exposing the BBB to a protein that binds to TfR with affinity of about 50 nM to about 250 nM, wherein the protein is ligated to the drug, and the ligated drug is transported across the BBB. In some embodiments, the highest concentration (C) of the drug in the brain of the mammal is provided. max ) increases. In some embodiments, the drug is useful in treating LSD.

[0102] In yet another embodiment, the present invention provides a method for treating LSD, comprising administering to a mammal a protein that binds to TfR with an affinity of about 50 nM to about 250 nM, wherein the protein is linked to a drug for treating LSD, thereby exposing the mammal's brain to the drug. In some embodiments, the protein has a higher affinity for the drug in the brain compared to a drug linked to a reference protein that binds to TfR with a weaker affinity than the protein. max This increases the affinity. In some embodiments, the reference protein binds to TfR with an affinity weaker than about 600 nM.

[0103] In some embodiments, the TfR is a primate TfR. In some embodiments, the primate TfR is a human TfR. In some embodiments, the protein binds to the TfR apical domain.

[0104] In some embodiments, the protein binds to TfR with an affinity of about 100 nM to about 200 nM. In some embodiments, the protein binds to TfR with an affinity of about 110 nM to about 150 nM.

[0105] In some embodiments, the therapeutically effective concentration of the drug is a concentration that treats one or more symptoms of LSD in the mammal. In some embodiments, the drug is a protein replacement therapy agent. In some embodiments, the drug or protein replacement therapy agent is an enzyme.

[0106] In some embodiments, when the enzyme is linked to the protein of the present invention, it reduces the accumulation of toxic metabolites in the mammalian brain of LSD to a greater extent than when the enzyme is linked to a reference protein. In some embodiments, the enzyme is IDS and the LSD is Hunter syndrome. In some embodiments, the toxic metabolites include disaccharides derived from heparan sulfate and / or disaccharides derived from dermatan sulfate. In some embodiments, the enzyme is SGSH and the LSD is Sanfilippo syndrome A. In some embodiments, the enzyme is ASM and the LSD is Niemann-Pick disease. In some embodiments, the enzyme is GBA and the LSD is Gaucher disease.

[0107] In some embodiments, the agent comprises an antibody variable region. In some embodiments, the agent comprises an antibody fragment. In some embodiments, the agent comprises Fab or scFv.

[0108] In some embodiments, the protein is a modified Fc polypeptide containing a non-native binding site capable of binding to TfR. In some embodiments, the protein contains an antibody variable region that specifically binds to TfR. In some embodiments, the protein contains an antibody fragment. In some embodiments, the protein contains Fab or scFv.

[0109] In some embodiments, the protein linked to the drug is administered as part of a pharmaceutically acceptable carrier. The present invention provides, for example, the following items. (Item 1) (a) an enzyme for enzyme replacement therapy (ERT), an enzyme variant for ERT, or a first Fc polypeptide linked to its catalytically active fragment, (b) A second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, A protein containing, The protein wherein the first Fc polypeptide and / or the second Fc polypeptide does not contain immunoglobulin heavy chain and / or light chain variable region sequences, or their antigen-binding portions. (Item 2) The protein described in item 1, wherein the enzyme for ERT is iduronate-2-sulfatase (IDS), an IDS variant, or a catalytically active fragment thereof. (Item 3) The protein according to item 2, wherein the enzyme for ERT comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with any one of the amino acid sequences of sequence numbers 91, 92, 114, 230, and 234. (Item 4) The protein described in item 3, wherein the ERT enzyme comprises one of the amino acid sequences of sequence numbers 91, 92, 114, 230, and 234. (Item 5) The protein described in item 1, wherein the enzyme for ERT is N-sulfoglucosamine sulfohydrolase (SGSH), an SGSH variant, or a catalytically active fragment thereof. (Item 6) The ERT enzyme contains an amino acid sequence that has at least 80%, 85%, 90%, or 95% identity with one of the amino acid sequences of SEQ ID NOs. 119 and 120. Hmm, the protein listed in item 5. (Item 7) The ERT enzyme is the protein described in item 6, wherein the ERT enzyme contains one of the amino acid sequences of sequence numbers 119 and 120. (Item 8) The protein described in item 1, wherein the enzyme for ERT is acid sphingomyelinase (ASM), an ASM variant, or a catalytically active fragment thereof. (Item 9) The protein according to item 8, wherein the enzyme for ERT comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with any one of the amino acid sequences of sequence numbers 121, 122, and 123. (Item 10) The protein described in item 9, wherein the ERT enzyme comprises one of the amino acid sequences of sequence numbers 121, 122, and 123. (Item 11) The protein described in item 1, wherein the enzyme for ERT is β-glucocerebrosidase (GBA), a GBA variant, or a catalytically active fragment thereof. (Item 12) The protein according to item 11, wherein the enzyme for ERT comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with any one of the amino acid sequences of sequence numbers 93 and 94. (Item 13) The protein described in item 12, wherein the enzyme for ERT contains one of the amino acid sequences of sequence numbers 93 and 94. (Item 14) The protein according to any one of items 1 to 13, wherein the first Fc polypeptide is a fusion polypeptide linked to the ERT enzyme, the ERT enzyme variant, or the catalytically active fragment thereof by a peptide bond or a polypeptide linker. (Item 15) The protein described in item 14, wherein the polypeptide linker is a flexible polypeptide linker. (Item 16) The protein described in item 15, wherein the flexible polypeptide linker is a glycine-rich linker. (Item 17) The protein described in item 16, wherein the glycine-rich linker is G4S (SEQ ID NO: 239) or (G4S)2 (SEQ ID NO: 240). (Item 18) The protein according to any one of items 1 to 17, wherein the second Fc polypeptide is linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof. (Item 19) The protein according to item 18, wherein the second Fc polypeptide is a fusion polypeptide linked to the ERT enzyme, the ERT enzyme variant, or the catalytically active fragment thereof by a peptide bond or a polypeptide linker. (Item 20) The protein according to item 18 or 19, wherein the N-terminus of the first Fc polypeptide and / or the N-terminus of the second Fc polypeptide are linked to the enzyme for ERT. (Item 21) The N-terminus of the first Fc polypeptide is linked to one ERT enzyme, and the N-terminus of the second Fc polypeptide is linked to the other ERT enzyme, item 2 Protein as described in 0. (Item 22) The protein according to item 18 or 19, wherein the C-terminus of the first Fc polypeptide and / or the C-terminus of the second Fc polypeptide are linked to the enzyme for ERT. (Item 23) The protein described in item 22, wherein the C-terminus of the first Fc polypeptide is linked to one ERT enzyme, and the C-terminus of the second Fc polypeptide is linked to the other ERT enzyme. (Item 24) The protein described in item 18 or 19, wherein the N-terminus of the first Fc polypeptide is linked to one ERT enzyme, and the C-terminus of the second Fc polypeptide is linked to the other ERT enzyme. (Item 25) The protein described in item 18 or 19, wherein the C-terminus of the first Fc polypeptide is linked to one ERT enzyme, and the N-terminus of the second Fc polypeptide is linked to the other ERT enzyme. (Item 26) The protein according to any one of items 1 to 17, wherein the protein comprises one ERT enzyme, and the N-terminus or C-terminus of the first Fc polypeptide is ligated to the ERT enzyme. (Item 27) A protein containing two ERT enzymes, as described in any one of items 18-25. (Item 28) The protein according to any one of items 14 to 17, wherein the fusion polypeptide comprises, in order from the N-terminus to the C-terminus, the ERT enzyme, the ERT enzyme variant or the catalytically active fragment thereof, the polypeptide linker, and the first Fc polypeptide. (Item 29) The protein according to any one of items 1 to 28, wherein the first Fc polypeptide is a modified Fc polypeptide, and / or the second Fc polypeptide is a modified Fc polypeptide. (Item 30) The protein according to item 29, wherein the first Fc polypeptide and the second Fc polypeptide each include modifications that promote heterodimerization. (Item 31) The protein described in item 30, wherein the Fc dimer is an Fc heterodimer. (Item 32) According to EU numbering, the protein described in item 30 or 31, wherein one of the Fc polypeptides has the substitution T366W, and the other Fc polypeptide has the substitutions T366S, L368A, and Y407V. (Item 33) The protein according to item 32, wherein the first Fc polypeptide has the substitutions T366S, L368A and Y407V, and the second Fc polypeptide contains the substitution T366W. (Item 34) The protein according to item 33, wherein the first Fc polypeptide is linked to the ERT enzyme and contains one of the amino acid sequences of sequence numbers 117, 232, and 236. (Item 35) The first Fc polypeptide contains the substitution T366W, and the second Fc polypeptide contains the substitutions T366S, L368A, and Y407V, item 32 The protein described above. (Item 36) The protein according to item 35, wherein the first Fc polypeptide is linked to the ERT enzyme and contains one of the amino acid sequences of sequence numbers 118, 233, and 237. (Item 37) The protein according to any one of items 29 to 36, wherein the first Fc polypeptide and / or the second Fc polypeptide contains a native FcRn binding site. (Item 38) The first Fc polypeptide and the second Fc polypeptide are proteins according to any one of items 29 to 37, wherein the first Fc polypeptide and the second Fc polypeptide do not have effector functions. (Item 39) The protein according to any one of items 29 to 37, wherein the first Fc polypeptide and / or the second Fc polypeptide includes modifications that reduce effector function. (Item 40) According to EU numbering, the protein described in item 39, in which the modification that reduces the effector function is the substitution of Ala at position 234 and Ala at position 235. (Item 41) The protein according to item 40, wherein the first Fc polypeptide is linked to the ERT enzyme and contains one of the amino acid sequences of SEQ ID NOs: 115, 231, and 235. (Item 42) The protein according to item 40, wherein the first Fc polypeptide is linked to the ERT enzyme and contains one of the amino acid sequences of sequence numbers 149, 150, 152, and 153. (Item 43) The protein according to any one of items 29 to 42, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises an amino acid change from the native Fc sequence that prolongs the serum half-life. (Item 44) According to EU numbering, the protein described in item 43, wherein the amino acid changes include substitutions of Tyr at position 252, Thr at position 254, and Glu at position 256. (Item 45) According to EU numbering, the protein described in item 43, wherein the amino acid changes include substitutions of Leu at position 428 and Ser at position 434. (Item 46) According to EU numbering, the protein described in item 43, wherein the amino acid change includes a substitution of Ser or Ala at position 434. (Item 47) The protein according to any one of items 29 to 46, wherein the first Fc polypeptide and / or the second Fc polypeptide specifically bind to a transferrin receptor (TfR). (Item 48) According to EU numbering, the protein described in item 47, wherein the first Fc polypeptide and / or the second Fc polypeptide contains at least two substitutions at positions selected from the group consisting of positions 384, 386, 387, 388, 389, 390, 413, 416, and 421. (Item 49) The first Fc polypeptide and / or the second Fc polypeptide have at least 3, 4, 5, 6, 7, 8 or 9 substitutions in the position of item 48. The protein described. (Item 50) According to EU numbering, the protein described in item 48 or 49, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprises one, two, three or four substitutions at positions including 380, 391, 392 and 415. (Item 51) According to EU numbering, the protein described in any one of items 48-50, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprises one, two, or three substitutions at positions including 414, 424, and 426. (Item 52) The protein according to any one of items 48 to 51, wherein the first Fc polypeptide and / or the second Fc polypeptide contains Trp at position 388. (Item 53) The protein according to any one of items 48 to 52, wherein the first Fc polypeptide and / or the second Fc polypeptide contains an aromatic amino acid at position 421. (Item 54) The protein described in item 53, wherein the aromatic amino acid at position 421 is Trp or Phe. (Item 55) The protein according to any one of items 48 to 54, wherein the first Fc polypeptide and / or the second Fc polypeptide includes at least one position selected from position 380 which is Trp, Leu, or Glu, position 384 which is Tyr or Phe, position 386 which is Thr, position 387 which is Glu, position 388 which is Trp, position 389 which is Ser, Ala, Val, or Asn, position 390 which is Ser or Asn, position 413 which is Thr or Ser, position 415 which is Glu or Ser, position 416 which is Glu, and position 421 which is Phe. (Item 56) The protein according to item 55, wherein the first Fc polypeptide and / or the second Fc polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from the following: position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu; and position 421 which is Phe. (Item 57) The protein according to item 56, wherein the first Fc polypeptide and / or the second Fc polypeptide include 11 positions: position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu; and position 421 which is Phe. (Item 58) The protein according to item 56 or 57, wherein the first Fc polypeptide and / or the second Fc polypeptide has a CH3 domain having at least 85% identity, at least 90% identity, or at least 95% identity with an amino acid at positions 111-217 of any one of SEQ ID NOs. 34-38, 58, 60-90, 151, and 156-229. (Item 59) One of sequence numbers 34-38, 58, 60-90, 151, and 156-229, ranked 380th, 384th, 386th, 387th, 388th, and 38th in the EU index. The protein described in item 58, wherein at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues at positions corresponding to 9, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 are not deleted or substituted. (Item 60) The protein according to item 58 or 59, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises one amino acid sequence from SEQ ID NOs. 156 to 229. (Item 61) The protein according to item 60, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises one of the amino acid sequences of SEQ ID NOs: 157, 169, 181, 193, 205, and 217. (Item 62) The protein according to item 60, wherein the first Fc polypeptide comprises one of the amino acid sequences of SEQ ID NOs: 115, 231, and 235, and the second Fc polypeptide comprises one of the amino acid sequences of SEQ ID NOs: 205 and 228. (Item 63) The protein according to item 60, wherein the first Fc polypeptide comprises one of the amino acid sequences of SEQ ID NOs: 115, 231, and 235, and the second Fc polypeptide comprises one of the amino acid sequences of SEQ ID NOs: 169 and 229. (Item 64) The protein according to any one of items 47 to 63, wherein the first Fc polypeptide and / or the second Fc polypeptide are bound to the apical domain of the TfR. (Item 65) The protein according to any one of items 47 to 64, wherein the binding of the protein to the TfR does not substantially inhibit the binding of transferrin to the TfR. (Item 66) The protein according to any one of items 47 to 65, wherein the amino acid sequence identity of the first Fc polypeptide and / or the second Fc polypeptide to the corresponding wild-type Fc polypeptide is at least 75%, or at least 80%, 85%, 90%, 92%, or 95%. (Item 67) The protein according to item 66, wherein the corresponding wild-type Fc polypeptide is a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide. (Item 68) The protein according to any one of items 47 to 67, wherein the uptake of the ERT enzyme into the brain is at least 10 times greater than the uptake of the ERT enzyme in the absence of the first Fc polypeptide and / or the second Fc polypeptide, or compared to the uptake of the ERT enzyme when the modification of the first Fc polypeptide and / or the second Fc polypeptide that results in binding to TfR is not made. (Item 69) The protein described in any one of items 1 to 68, 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 70) The protein according to any one of items 1 to 68, wherein the first Fc polypeptide is modified to specifically bind to TfR, and the second Fc polypeptide is not modified to bind to the BBB receptor. (Item 71) Proteins described in any one of items 1 to 70, which do not contain the heavy chain and / or light chain variable region sequences of immunoglobulins, or their antigen-binding moieties. (Item 72) A polypeptide comprising an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof, wherein the polypeptide comprises one or more modifications that promote heterodimerization of the Fc polypeptide with another Fc polypeptide. (Item 73) The polypeptide according to item 72, wherein the enzyme for ERT is iduronate-2-sulfatase (IDS), an IDS variant, or a catalytically active fragment thereof. (Item 74) The polypeptide according to item 72, wherein the enzyme for ERT is N-sulfoglucosamine sulfohydrolase (SGSH), an SGSH variant, or a catalytically active fragment thereof. (Item 75) The polypeptide according to item 72, wherein the enzyme for ERT is acid sphingomyelinase (ASM), an ASM variant, or a catalytically active fragment thereof. (Item 76) The polypeptide according to item 72, wherein the enzyme for ERT is β-glucocerebrosidase (GBA), a GBA variant, or a catalytically active fragment thereof. (Item 77) The polypeptide according to any one of items 72 to 76, wherein the Fc polypeptide is a fusion polypeptide linked to the ERT enzyme, the ERT enzyme variant, or the catalytically active fragment thereof by a peptide bond or a polypeptide linker. (Item 78) The polypeptide according to item 77, wherein the fusion polypeptide comprises, in order from the N-terminus to the C-terminus, the ERT enzyme, the ERT enzyme variant or the catalytically active fragment thereof, the polypeptide linker, and the first Fc polypeptide. (Item 79) According to EU numbering, the Fc polypeptide is a polypeptide according to any one of items 72 to 78, wherein the Fc polypeptide includes the substitutions T366S, L368A, and Y407V. (Item 80) A polypeptide as described in item 79, comprising any one of the amino acid sequences of sequence numbers 115, 117, 231, 232, 235, and 236. (Item 81) A polypeptide as described in item 79, comprising one of the amino acid sequences of sequence numbers 149 and 150. (Item 82) The polypeptide according to any one of items 72 to 78, wherein the Fc polypeptide includes the substitution T366W. (Item 83) A polypeptide as described in item 82, comprising any one of the amino acid sequences of sequence numbers 118, 233, and 237. (Item 84) A polypeptide as described in item 82, comprising one of the amino acid sequences from sequence numbers 152 to 155. (Item 85) The polypeptide according to any one of items 72 to 84, further comprising the other Fc polypeptide mentioned above. (Item 86) A polynucleotide comprising a nucleic acid sequence encoding a polypeptide described in any one of items 72-84. (Item 87) A vector containing the polynucleotides described in item 86. (Item 88) A host cell containing the polynucleotide described in item 86 or the vector described in item 87. (Item 89) The host cell according to item 88, further comprising a polynucleotide containing a nucleic acid sequence encoding the other Fc polypeptide. (Item 90) A method for producing a polypeptide comprising an ERT enzyme, an ERT enzyme variant, or an Fc polypeptide linked to a catalytically active fragment thereof, the method comprising culturing a host cell under conditions for expressing the polypeptide encoded by the polynucleotide described in item 86. (Item 91) A method for treating lysosomal storage disorder (LSD), comprising administering to a patient in need of treatment a protein described in any one of items 1 to 71, or a polypeptide described in any one of items 72 to 85. (Item 92) A method for reducing the accumulation of toxic metabolites in an LSD patient, comprising administering to the patient a protein described in any one of items 1 to 71, or a polypeptide described in any one of items 72 to 85. (Item 93) The method according to item 91 or 92, wherein the LSD is Hunter syndrome and the enzyme for ERT is IDS. (Item 94) The method according to item 93, wherein the toxic metabolite comprises a disaccharide derived from heparan sulfate and / or a disaccharide derived from dermatan sulfate. (Item 95) The method according to item 91 or 92, wherein the LSD is Sanfilippo syndrome A and the enzyme for ERT is SGSH. (Item 96) The method according to item 95, wherein the toxic metabolite includes an oligosaccharide derived from heparan sulfate. (Item 97) The method according to item 91 or 92, wherein the LSD is Niemann-Pick disease and the enzyme for ERT is ASM. (Item 98) The method according to item 97, wherein the toxic metabolite includes sphingomyelin. (Item 99) The method according to item 91 or 92, wherein the LSD is Gaucher disease or Parkinson's disease, and the enzyme for ERT is GBA. (Item 100) The method according to item 99, wherein the toxic metabolite includes glucosylceramide. (Item 101) A pharmaceutical composition comprising a protein as described in any one of items 1 to 71, or a polypeptide as described in any one of items 72 to 85, and a pharmaceutically acceptable carrier. (Item 102) A method for monitoring substrate accumulation and evaluating IDS activity, (a) Disrupt cells in a cell or tissue sample derived from a subject administered with any one of items 2-4, 14-41, and 43-71, or any one of items 72-73, 77-80, 82-83, and 85, thereby disrupting and opening the microvesicles to obtain a glycosaminoglycan (GAG) solution for analysis. , (b) Digesting the GAG ​​solution with at least one heparinase and chondroitinase B to obtain a disaccharide derived from GAG, (c) Analyze the disaccharide derived from the GAG ​​by mass spectrometry, (d) Determining the level of disaccharides derived from heparan sulfate and / or dermatan sulfate, wherein the level of disaccharides derived from heparan sulfate and / or dermatan sulfate is lower than that of a control lacking IDS activity, thereby indicating that the IDS activity in the sample is increased compared to the control. The method comprising the above. (Item 103) The method according to item 102, wherein the step of destroying the cells comprises at least one freeze-thaw cycle and at least one sonication step. (Item 104) The method according to item 103, wherein the cells are derived from a tissue sample, and the method comprises at least three, four, or five freeze-thaw cycles. (Item 105) The method according to any one of items 102 to 104, wherein the subject is a mouse lacking IDS activity. (Item 106) The method described in any one of items 102 to 104, wherein the subject is a primate other than a human. (Item 107) The method described in any one of items 102 to 104, wherein the subject is a human patient with Hunter syndrome. (Item 108) A method for monitoring substrate accumulation and evaluating SGSH activity, (a) Disrupt cells in a cell or tissue sample derived from a subject administered with any one of items 5-7, 14-33, 35, 37-40 and 42-71 or any one of items 72, 74, 77-79, 81-82 and 84-85, disrupt and open the microvesicles to obtain a glycosaminoglycan (GAG) solution of the target for analysis. (b) Digesting the GAG ​​solution with at least one heparinase to obtain a disaccharide derived from GAG, (c) Analyze the disaccharide derived from the GAG ​​by mass spectrometry, (d) Determining the level of disaccharides derived from heparan sulfate, wherein the level of disaccharides derived from heparan sulfate is lower than that of a control lacking SGSH activity, thereby indicating that the SGSH activity in the sample is higher than that of the control. The method comprising the above. (Item 109) The method according to item 108, wherein the step of destroying the cells comprises at least one freeze-thaw cycle and at least one sonication step. (Item 110) The method according to item 109, wherein the cells are derived from a tissue sample, and the method comprises at least three, four, or five freeze-thaw cycles. (Item 111) The method according to any one of items 108 to 110, wherein the subject is a mouse lacking SGSH activity. (Item 112) The subject is a primate other than a human, as described in one of items 108-110. The method. (Item 113) The method according to any one of items 108 to 110, wherein the subject is a human patient with Sanfilippo syndrome A. (Item 114) A method for transporting a drug across the blood-brain barrier (BBB) ​​of a mammal, comprising exposing the BBB to a protein that binds to TfR with an affinity of about 50 nM to about 250 nM, wherein the protein is ligated to the drug, and the method transports the ligated drug across the BBB. (Item 115) The highest concentration of the drug in the brain of the mammal (C max The method described in item 114 to improve ). (Item 116) The method described in item 114 or 115, wherein the aforementioned drug is useful for treating LSD. (Item 117) A method for treating LSD, comprising administering to a mammal a protein that binds to TfR with an affinity of about 50 nM to about 250 nM, wherein the protein is linked to a drug for treating LSD, and thereby exposing the brain of the mammal to the drug. (Item 118) Compared to the drug in which the protein is linked to a reference protein that binds to TfR with a weaker affinity than the aforementioned, the C of the drug in the brain max The method described in any one of items 114-117 to improve the following: (Item 119) Compared to a drug in which the aforementioned protein is linked to a reference protein that binds to TfR with weaker affinity than the aforementioned, in the mammal, the C of the aforementioned drug is present at a therapeutically effective concentration. max The method described in any one of items 114-118 to improve the following: (Item 120) The method according to any one of items 118 to 119, wherein the reference protein binds to the TfR with an affinity of about 600 nM or less. (Item 121) The method according to any one of items 114-120, wherein the TfR is a TfR of a primate. (Item 122) The method according to item 121, wherein the primate TfR is a human TfR. (Item 123) The method according to any one of items 114 to 122, wherein the protein binds to the TfR apical domain. (Item 124) The method according to any one of items 114 to 123, wherein the protein binds to the TfR with an affinity of approximately 100 nM to approximately 200 nM. (Item 125) The method according to any one of items 114 to 124, wherein the protein binds to the TfR with an affinity of approximately 110 nM to approximately 150 nM. (Item 126) The method according to any one of items 119 to 125, wherein the therapeutically effective concentration of the drug is a concentration that treats one or more symptoms of LSD in the mammal. (Item 127) The aforementioned drug is a protein replacement therapy agent, as described in any one of items 114-126. The method. (Item 128) The method according to any one of items 114 to 127, wherein the drug or protein replacement therapy agent is an enzyme. (Item 129) The method according to item 128, wherein when the enzyme is linked to the protein, the degree to which it reduces the accumulation of toxic metabolites in the brain of the mammal that is the LSD is greater than when the enzyme is linked to the reference protein. (Item 130) The method according to item 128 or 129, wherein the enzyme is IDS and the LSD is Hunter syndrome. (Item 131) The method according to item 130, wherein the toxic metabolite includes a disaccharide derived from heparan sulfate and / or a disaccharide derived from dermatan sulfate. (Item 132) The method according to item 128 or 129, wherein the enzyme is SGSH and the LSD is Sanfilippo syndrome A. (Item 133) The method according to item 128 or 129, wherein the enzyme is ASM and the LSD is Niemann-Pick disease. (Item 134) The method according to item 128 or 129, wherein the enzyme is GBA and the LSD is Gaucher disease. (Item 135) The method according to any one of items 114 to 126, wherein the drug includes an antibody variable region. (Item 136) The method according to item 135, wherein the drug comprises an antibody fragment. (Item 137) The method according to item 136, wherein the drug comprises Fab or scFv. (Item 138) The method according to any one of items 114 to 137, wherein the protein is a modified Fc polypeptide containing a non-natural binding site that can bind to TfR. (Item 139) The method according to any one of items 114 to 137, wherein the protein includes an antibody variable region that specifically binds to TfR. (Item 140) The method according to item 139, wherein the protein comprises an antibody fragment. (Item 141) The method according to item 140, wherein the protein comprises Fab or scFv. (Item 142) The method according to any one of items 114 to 141, wherein the protein linked to the drug is administered as part of a pharmaceutically acceptable carrier. [Brief explanation of the drawing]

[0110] [Figure 1] This paper shows the purification and analysis results of IDS-Fc fusion proteins, including Fc polypeptides linked to the iduronate-2-sulfatase (IDS) enzyme and modified Fc polypeptides that bind to the transferrin receptor (TfR). [Figure 2] Figure 1 shows the results of the binding affinity assay for the IDS-Fc fusion protein, demonstrating that the fusion protein binds to TfR. [Figure 3] Figure 1 shows data illustrating the in vitro IDS activity of the IDS-Fc fusion protein analyzed. [Figure 4] Evaluation using LC-MS / MS assays revealed elevated levels of heparan sulfate-derived disaccharides in IDS-deficient knockout cells, and the data suggests that IDS expression in IDS knockout (KO) cells rescues the knockout phenotype. [Figure 5-1]A shows that the IDS-Fc fusion protein, either as an N-terminal monozyme or a C-terminal monozyme containing the same TfR-binding Fc polypeptide (i.e., CH3C.35.21.17), reverses the accumulation of heparan sulfate and dermatan sulfate in IDS KO cells. B shows that the N-terminal monozyme ("ETV:IDS35.21.17") has cellular efficacy equivalent to IDS. C shows that the accumulation of S35-sulfate-labeled protein was dose-dependently reduced in fibroblasts (n=8) from MPS II patients treated with IDS-Fc fusion protein ("ETV:IDS") or IDS. D shows an evaluation of M6PR-dependent clearance of S35-labeled protein in fibroblasts (n=3) from MPS II patients treated with escalating concentrations of IDS-Fc fusion protein ("ETV:IDS") or IDS, in the presence or absence of 5 mM M6P. In Figures 5C-5D, "ETV:IDS" is ETV:IDS35.23.2, and the graph shows the mean ± standard error across repeated experiments. [Figure 5-2] A shows that the IDS-Fc fusion protein, either as an N-terminal monozyme or a C-terminal monozyme containing the same TfR-binding Fc polypeptide (i.e., CH3C.35.21.17), reverses the accumulation of heparan sulfate and dermatan sulfate in IDS KO cells. B shows that the N-terminal monozyme ("ETV:IDS35.21.17") has cellular efficacy equivalent to IDS. C shows that the accumulation of S35-sulfate-labeled protein was dose-dependently reduced in fibroblasts (n=8) from MPS II patients treated with IDS-Fc fusion protein ("ETV:IDS") or IDS. D shows an evaluation of M6PR-dependent clearance of S35-labeled protein in fibroblasts (n=3) from MPS II patients treated with escalating concentrations of IDS-Fc fusion protein ("ETV:IDS") or IDS, in the presence or absence of 5 mM M6P. In Figures 5C-5D, "ETV:IDS" is ETV:IDS35.23.2, and the graph shows the mean ± standard error across repeated experiments. [Figure 6]The data shows the levels of heparan sulfate and dermatan sulfate in serum from wild-type (WT) mice administered with the vehicle, or from IDS KO mice administered with IDS or IDS-Fc fusion protein ("ETV:IDS"). "ETV:IDS" is ETV:IDS35.21. [Figure 7] The data presented here show the total levels of disaccharides D0SO, DOA0, and D0a4 ("total sGAG levels") in peripheral tissues derived from IDS KO mice, evaluated 7 days after a single intravenous injection of 40 mg / kg of IDS-Fc fusion protein ("ETV:IDS") or 5.3 mg / kg of IDS, compared to vehicle-treated IDS KO mice and wild-type mice (n=8 in the IDS KO group, n=3 in the wild-type group). Data are presented as mean ± standard error, and p-values ​​are calculated using one-way ANOVA with Dunnett's multiple comparison test, where ** indicates p<0.01 and **** indicates p<0.0001. "ETV:IDS" is 35.21. [Figure 8] This data shows the concentration of IDS-Fc fusion protein in the brain of human TfR knock-in (TfRms / hu KI) mice after peripheral administration of either the IDS-Fc fusion protein ETV:IDS35.21 or a control IDS-Fc fusion protein ("IDS:Fc") that does not contain mutations that lead to TfR binding. [Figure 9] Graph A shows the concentration of IDS-Fc fusion protein in the brain of TfRms / hu KI mice after peripheral administration of either the IDS-Fc fusion protein ETV:IDS35.21.17.2 or ETV:IDS35.23.2, or a control IDS-Fc fusion protein ("IDS:Fc") that does not have mutations that lead to binding to TfR.Graph B shows the liver concentration of IDS-Fc fusion protein ETV:IDS35.21 or IDS:Fc in TfRms / hu KI mice after a single intravenous injection at a dose of 50 mg / kg (n=4-5). The graphs show the mean ± standard error. [Figure 10-1]Figures A-C show that ETV:IDS reduces GAGs in the brain and peripheral tissues of IDS KO×TfRms / hu KI mice. As described in Example 2, IDS KO×TfRms / hu KI mice were administered either 40 mg / kg of ETV:IDS or 14.2 mg / kg of IDS by a single intravenous injection, or administered once a week for four weeks. Serum (A) and tissue (B) concentrations of IDS were measured in IDS KO×TfRms / hu KI mice after a single dose. Tissue PK 2 hours after administration is shown (n=4). The graphs show the mean ± standard error, and the p-values ​​are from independent t-test analysis. C represents the levels of disaccharides D0SO, DOA0, and D0a4 ("total sGAG levels") in the brain, CSF, and peripheral tissues of IDS KO×TfRms / hu KI mice, measured after a single or multiple dose of ETV:IDS or IDS, and compared to vehicle-treated and wild-type mice (n=8 for the IDS KO×TfRms / hu KI group and n=5 for the wild-type group). The graph shows the mean ± standard error, and the p-values ​​are from one-way ANOVA with Dunnett's multiple comparison test, where ** is p<0.01, *** is p≦0.001, and **** is p≦0.0001. [Figure 10-2]Figures A-C show that ETV:IDS reduces GAGs in the brain and peripheral tissues of IDS KO×TfRms / hu KI mice. As described in Example 2, IDS KO×TfRms / hu KI mice were administered either 40 mg / kg of ETV:IDS or 14.2 mg / kg of IDS by a single intravenous injection, or administered once a week for four weeks. Serum (A) and tissue (B) concentrations of IDS were measured in IDS KO×TfRms / hu KI mice after a single dose. Tissue PK 2 hours after administration is shown (n=4). The graphs show the mean ± standard error, and the p-values ​​are from independent t-test analysis. C represents the levels of disaccharides D0SO, DOA0, and D0a4 ("total sGAG levels") in the brain, CSF, and peripheral tissues of IDS KO×TfRms / hu KI mice, measured after a single or multiple dose of ETV:IDS or IDS, and compared to vehicle-treated and wild-type mice (n=8 for the IDS KO×TfRms / hu KI group and n=5 for the wild-type group). The graph shows the mean ± standard error, and the p-values ​​are from one-way ANOVA with Dunnett's multiple comparison test, where ** is p<0.01, *** is p≦0.001, and **** is p≦0.0001. [Figure 11] This shows the purification and analysis results of an ASM-Fc fusion protein containing an Fc region linked to two acid sphingomyelinase (ASM) enzymes. [Figure 12] Figure 11 shows data illustrating the in vitro ASM activity of the ASM-Fc fusion protein analyzed. [Figure 13] Using imaging-based assays, we have data showing that the ASM-Fc fusion protein, as analyzed in Figure 11, reduces sphingomyelin accumulation in ASM KO cells. [Figure 14] Using an LC-MS / MS-based assay, data from Figure 11 shows that the ASM-Fc fusion protein, as analyzed, reduces sphingomyelin accumulation in ASM KO cells. [Figure 15]This shows data demonstrating the in vitro N-sulfoglucosamine sulfohydrolase (SGSH) activity of the SGSH-Fc fusion protein described in Example 6. [Figure 16] Data obtained using LC-MS / MS assays show elevated levels of heparan sulfate-derived disaccharides in SGSH-deficient knockout (KO) cells. The study involved 3-4 independent cell lines, and data are presented as mean ± standard error. [Figure 17] Figure 15 shows data indicating that the SGSH-Fc fusion protein analyzed reverses the accumulation of heparan sulfate in SGSH KO cells. [Figure 18] This graph shows the relationship between the hTfR affinity of modified TfR-binding polypeptides and brain exposure over time in TfRms / hu KI mice. The points represent the cumulative brain exposure (AUC) over time for various affinity variants of modified TfR-binding polypeptides after a single 50 mg / kg dose in TfRms / hu KI mice. Brain concentrations of the polypeptides (measured by huIgG1) were calculated at various post-administration intervals (ranging from 1 to 10 days). The data are a compilation of three independent studies, with 4-5 mice in each group in each study. [Figure 19] This graph shows the relationship between the hTfR affinity of modified TfR-binding polypeptides and their peak brain concentration in TfRms / hu KI mice. The dots represent the peak brain concentrations of various polypeptide affinity variants, measured on day 1 after a single dose of 50 mg / kg. The data is a compilation of three independent studies, with 4-5 mice per group in each study. [Figure 20]This graph shows the relationship between the hTfR affinity of modified TfR-binding polypeptides and the ratio of the polypeptide's brain concentration to its plasma concentration in TfRms / hu KI mice. The dots represent the ratio of the peak brain concentration to the plasma concentration of various polypeptide affinity variants, measured on day 1 after a single dose of 50 mg / kg. The data is a compilation of three independent studies, with 4-5 mice per group in each study. [Figure 21A] The plasma huIgG1 concentrations in TfRms / hu knock-in (KI) mice after a single systemic injection of 50 mg / kg of an anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusion are shown (mean ± standard error, n=5 per group). [Figure 21B] The following shows the huIgG1 concentration in the brain lysate of TfRms / hu knock-in (KI) mice after a single systemic injection of 50 mg / kg of an anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusion (mean ± standard error, n=5 per group). [Figure 21C] The endogenous mouse Aβ concentration in the brain lysate of TfRms / hu KI mice after a single systemic injection of 50 mg / kg of an anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusion is shown (mean ± standard error, n=5 per group). [Figure 21D] The results shown are the quantitative analysis of TfR protein in the brain lysate of TfRms / hu KI mice after a single systemic injection of 50 mg / kg of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusion, normalized to actin (mean ± standard error, n=5 per group). [Modes for carrying out the invention]

[0111] I. Introduction We have developed fusion proteins containing enzyme replacement therapy (ERT) enzymes linked to Fc polypeptides. These proteins can be used to treat lysosomal storage disorders (LSDs). In some cases, the protein contains a dimeric Fc polypeptide, one of which is linked to an ERT enzyme. These Fc polypeptides can extend the half-life of the enzyme and, in some cases, can be modified to confer additional functional properties to the protein. This specification also describes fusion proteins that facilitate the delivery of ERT enzymes across the blood-brain barrier (BBB). These proteins include dimeric Fc polypeptides and modified Fc polypeptides, as well as an ERT enzyme linked to their Fc region and / or modified Fc region. The modified Fc region can specifically bind to BBB receptors such as transferrin receptors (TfRs). In some embodiments, the ERT enzyme is iduronate-2-sulfatase (IDS), or a catalytically active variant or fragment of wild-type IDS, e.g., wild-type human IDS. In another embodiment, the enzyme for ERT is N-sulfoglucosamine sulfohydrolase (SGSH), acid sphingomyelinase (ASM), β-glucocerebrosidase (GBA), or wild-type SGSH, ASM, or GBA, for example, a catalytically active variant or fragment of wild-type human SGSH, ASM, or GBA.

[0112] We have also developed methods for transporting therapeutic agents linked to TfR-binding polypeptides and proteins across the blood-brain barrier (BBB) ​​for the treatment of diseases. We found that the desired TfR-binding affinity for transporting therapeutic agents across the BBB depends on the target of the therapeutic agent and the mechanism of action that gives rise to its efficacy in treating the disease. In particular, we found that the stronger the TfR affinity of the polypeptides and proteins used, the higher the C max Although it rises, we discovered that the clearance is fast.

[0113] In some therapies, such as protein replacement therapy that can be used for the treatment of, for example, LSD (including the use of ERT enzymes such as for the treatment of Hunter syndrome), it is desirable to increase the brain C of the therapeutic agent throughout the dosing range. max This is because the higher the extracellular concentration, the more the increase in intracellular protein concentration will be promoted. Once delivered into the cell, the intracellular half-life of the delivered protein is sustained for a longer period than the plasma residence time. In addition, max a high C can be beneficial for enzyme replacement. This is because a high enzyme concentration can promote an increase in the substrate turnover rate by the enzyme. To increase the brain C max , it is particularly useful to use polypeptides and proteins with a TfR affinity in the range of 50 - 250 nM.

[0114] II. Definitions As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" can mean that two or more such molecules are included.

[0115] As used herein, the terms "about" and "approximately" are used when modifying an amount defined by a numerical value or range, and indicate that the numerical value, and reasonable deviations from that value known to those skilled in the art (e.g., ±20%, ±10% or ±5%), are within the contemplated meaning of the recited value.

[0116] "Enzyme for enzyme replacement therapy" or "Enzyme for ERT" refers to the enzyme that is deficient in lysosomal storage disorders. "Enzyme variant for ERT" refers to a functional variant (including allelic variants and splice variants) or fragment of the wild-type enzyme for ERT, which, when assayed under identical conditions, 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%, or at least 95% of the activity of the corresponding wild-type enzyme for ERT or fragment. A "catalytically active fragment" of an ERT enzyme refers to a portion of the full-length ERT enzyme or its variant, and the catalytically active fragment, when assayed under identical conditions, for example, 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 ERT enzyme or its variant.

[0117] When used herein, “iduronate sulfatase,” “iduronate-2-sulfatase,” or “IDS” refers to iduronate-2-sulfatase (EC 3.1.6.13), an enzyme involved in the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. Deficiency of IDS is associated with mucopolysaccharidosis type II, also known as Hunter syndrome. When used herein as a component of a protein containing an Fc polypeptide, the term “IDS” encompasses catalytic activity and includes functional variants (including allele variants and splice variants) or fragments of wild-type IDS. The sequence of human IDS isoform I (a human sequence designated as the canonical sequence) is available under UniProt registry number P22304 and is encoded by the human IDS gene located at Xq28. Its full-length sequence is shown as Sequence ID No. 91. As used herein, a “mature” IDS sequence refers to a polypeptide chain form lacking the signal and propeptide sequences of the natural full-length polypeptide chain. The amino acid sequence of a mature human IDS polypeptide is shown as SEQ ID NO: 92, corresponding to amino acids 34–550 of its full-length human sequence. As used herein, a “truncated” IDS sequence refers to a catalytically active fragment of its natural full-length polypeptide chain. The amino acid sequence of an exemplary truncated human IDS polypeptide is shown as SEQ ID NO: 114, corresponding to amino acids 26–550 of its full-length human sequence. The structures of human IDS are well-characterized. Exemplary structures are available under PDB accession code 5FQL. Their structures are also described in Nat.Comm.8:15786doi:10.1038 / ncomms15786,2017. IDS sequences for non-human primates, including chimpanzees (UniProt registry number K7BKV4) and rhesus macaques (UniProt registry number H9FTX2), are also described. Mouse IDS sequences are available under UniProt registry number Q08890.The IDS variant 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 wild-type IDS or its fragment when assayed under identical conditions. The catalytically active IDS fragment 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 IDS or its variant when assayed under identical conditions.

[0118] "Sulfoglucosamine sulfohydrolase," "N-sulfoglucosamine sulfohydrolase," or "SGSH" as used herein refers to N-sulfoglucosamine sulfohydrolase (EC3.10.1.1), an enzyme involved in the lysosomal degradation of heparan sulfate. Mutations in this gene have been associated with Sanfilippo syndrome A (a type of mucopolysaccharidosis type III, a lysosomal storage disorder) resulting from impaired heparan sulfate degradation. When used herein as a component of a protein containing an Fc polypeptide, the term "SGSH" encompasses catalytic activity and includes functional variants (including allele variants and splice variants) or fragments of wild-type SGSH. The sequence of human SGSH is available under UniProt registry number P51688 and is encoded by the human SGSH gene located at 17q25.3. Its full-length sequence is shown as Sequence ID No. 119. When used herein, the "mature" SGSH sequence refers to a polypeptide chain form lacking the signal sequence of its native full-length polypeptide chain. The amino acid sequence of the mature human SGSH polypeptide is shown as SEQ ID NO: 120, corresponding to amino acids 21–502 of its full-length human sequence. The “truncate” SGSH sequence, as used herein, refers to the catalytically active fragment of its native full-length polypeptide chain. The structure of human SGSH is well-characterized. Exemplary structures are available under PDB accession code 4MHX. SGSH sequences from non-human primates, including chimpanzee (UniProt registry number K7C218), are also described. The mouse SGSH sequence is available under UniProt registry number Q9EQ08. SGSH variants exhibit 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, for example.The catalytically active SGSH fragment, when assayed under identical conditions, for example, 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.

[0119] "Acidic sphingomyelinase," "sphingomyelin phosphodiesterase," or "ASM" as used herein refers to sphingomyelin phosphodiesterase 1 (EC 3.1.4.12), a lysosomal enzyme that converts sphingomyelin to ceramide. Diseases associated with ASM deficiency include Niemann-Pick disease (e.g., type A or B). When used herein as a component of a protein containing an Fc polypeptide, the term "ASM" encompasses catalytic activity and includes functional variants (including allele variants and splice variants) or fragments of wild-type ASM. The sequence of human ASM isoform 1 (a human sequence designated as the canonical sequence) is available under UniProt registry number P17405 and is encoded by the human SMPD1 gene located at 11p15.4. Its full-length sequence is shown as Sequence ID No. 121. "Mature" ASM sequence, as used herein, refers to a polypeptide chain form lacking the signal sequence of its native full-length polypeptide chain. The amino acid sequence of a mature human ASM polypeptide is shown as SEQ ID NO: 122, corresponding to amino acids 47–629 of its full-length human sequence. "Truncate" ASM sequences, as used herein, refer to catalytically active fragments of their native full-length polypeptide chains. An exemplary truncated human ASM polypeptide amino acid sequence is shown as SEQ ID NO: 123, corresponding to amino acids 47–620 of its full-length human sequence. The structure of human ASMs is well-characterized. Exemplary structures are available under PDB accession code 5I81. ASM sequences of non-human primates, including chimpanzee (UniProt registry number H2Q319), are also described. Mouse ASM sequences are available under UniProt registry number Q04519. The ASM variant, when assayed under identical conditions, for example, 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 wild-type ASM or its fragment.The catalytically active ASM fragment, when assayed under identical conditions, for example, 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 ASM or its variant.

[0120] "β-glucocerebrosidase" or "GBA" is also known as glucosylceramidase (EC 3.2.1.45). As used herein, the term refers to a lysosomal enzyme that possesses glucosylceramidase activity and catalyzes the breakdown of glucosylceramide into ceramide and glucose. GBA deficiency is associated with Gaucher disease and Parkinson's disease. As used herein, the term "GBA" refers to a component of a protein containing an Fc polypeptide, encompassing catalytic activity and including functional variants (including allele variants and splice variants) or fragments of wild-type GBA. The sequence of the human GBA long isoform (defined as the canonical sequence) is available under UniProt registry number P04062-1 and is encoded by the human GBA gene located at 1q22. Its full-length sequence is shown as Sequence ID No. 93. As used herein, the "mature" GBA sequence refers to a polypeptide chain form lacking the signal and propeptide sequences of its native full-length polypeptide chain. The amino acid sequence of the mature human GBA polypeptide is shown as SEQ ID NO: 94, corresponding to amino acids 40–536 of its full-length human sequence. The “truncate” GBA sequence, as used herein, refers to the catalytically active fragment of its native full-length polypeptide chain. The structure of human GBA is well-characterized. Nearly 20 crystal structures of GBA are available. GBA sequences from non-human primates, including chimpanzee (UniProt registry number Q9BDT0) and orangutan (UniProt registry number Q5R8E3), have also been described. The mouse GBA sequence is available under UniProt registry number P17439. GBA variants exhibit 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 GBA or fragment when assayed, for example, under identical conditions.The catalytically active GBA fragment, when assayed under identical conditions, for example, 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 GBA or its variant.

[0121] As used herein, “transferrin receptor” or “TfR” refers to transferrin receptor protein 1. The polypeptide sequence of human transferrin receptor 1 is shown in SEQ ID NO: 96. Sequences of transferrin receptor protein 1 in 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, chicken: NP_990587.1). The term “transferrin receptor” also includes exemplary reference sequences encoded by genes located at the transferrin receptor protein 1 locus, such as allele variants of the human sequence. The full-length transferrin receptor protein contains a short N-terminal intracellular domain, a transmembrane domain, and a large extracellular domain. Its 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 shown in SEQ ID NO: 238.

[0122] "Fusion protein" or "[ERT enzyme]-Fc fusion protein" as used herein refers to a dimerized protein comprising a first Fc polypeptide (i.e., "[ERT]-Fc fusion polypeptide") linked (e.g., fused) to the ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof, 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 the ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof. The first Fc polypeptide and / or the second Fc polypeptide may be linked to the ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof by a peptide bond or a polypeptide linker. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that facilitate heterodimerization with the other Fc polypeptide. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that result in 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. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that extend serum half-life.

[0123] "Fusion polypeptide" or "[ERT enzyme]-Fc fusion polypeptide" as used herein refers to an Fc polypeptide linked (e.g., fused) to an ERT enzyme, an ERT enzyme variant, or its catalytically active fragment. The Fc polypeptide may be linked to the ERT enzyme, an ERT enzyme variant, or its catalytically active fragment by a peptide bond or a polypeptide linker. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that promote heterodimerization with another Fc polypeptide. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that result in binding to a transferrin receptor. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that reduce effector function. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that extend its serum half-life.

[0124] As used herein, the term “Fc polypeptide” refers to the C-terminal region of a native immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. An Fc polypeptide comprises a constant region sequence containing at least a CH2 domain and / or a CH3 domain, and may also include at least a portion of a hinge region. Generally, Fc polypeptides do not contain a variable region.

[0125] 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 entire Ig fold or structure of the native Fc polypeptide.

[0126] The term "FcRn" refers to the fetal Fc receptor. When an Fc polypeptide binds to FcRn, its clearance is reduced and its serum half-life is prolonged. 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.

[0127] As used herein, "FcRn binding site" refers to the region of an Fc polypeptide that binds to FcRn. In human IgG, FcRn binding sites, when 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 of Sequence ID No. 1.

[0128] As used herein, the term "natural FcRn binding site" refers to a region of the Fc polypeptide that binds to FcRn and has the same amino acid sequence as the region of the natural Fc polypeptide that binds to FcRn.

[0129] As used herein, the terms "CH3 domain" and "CH2 domain" refer to polypeptides of the constant region domain of immunoglobulins. For the purposes of this application, a CH3 domain polypeptide refers to the amino acid segment from approximately 341 to 447, as numbered according to the EU, and a CH2 domain polypeptide refers to the amino acid segment from approximately 231 to 340, as numbered according to the EU numbering scheme, and does not include the hinge region sequence. CH2 domain 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 numbers for CH2 domains are 1 to 110 and the numbers for CH3 domains are 1 to 107. The CH2 domain and CH3 domain are part of the Fc region of immunoglobulins. The Fc region refers to the amino acid segment from approximately position 231 to approximately position 447, when numbered according to the EU numbering scheme, but as used herein, it may include at least a portion of the hinge region of an antibody. An example hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 95).

[0130] With respect to the CH3 and CH2 domains, the terms “wild type,” “natural type,” and “natural” are used herein to refer to domains having naturally occurring sequences.

[0131] As used herein, the term “variant” is used synonymously with “variant” with respect to a mutant polypeptide or mutant polynucleotide. With respect to a given wild-type CH3 or CH2 domain reference sequence, a variant may include a native allele variant. A “non-naturally occurring” CH3 or CH2 domain refers to a variant or mutant domain that does not exist in cells in nature and is produced by genetic modification (e.g., genetic modification using genetic engineering techniques or mutagenesis) of a native CH3 or CH2 domain polynucleotide or polypeptide. A “variant” includes any domain containing at least one amino acid mutation from the wild type. Mutations may include substitutions, insertions, and deletions.

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

[0133] Natural amino acids are those encoded by the genetic code, as well as those that are later modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). "Amino acid analogs" refer to compounds that have the same basic chemical structure as natural 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 natural 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 similar way to natural amino acids.

[0134] Natural 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. Stereoisomers of natural α-amino acids include 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), and D-leucine ( Examples include, but are not limited to, D-Leu, D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0135] In this specification, amino acids may be represented by either the commonly known three-letter abbreviation or the one-letter abbreviation recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0136] The terms “polypeptide” and “peptide” are used herein synonymously to refer to single-chain amino acid residue polymers. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as to natural amino acid polymers and non-natural amino acid polymers. Amino acid polymers may consist of L-amino acids only, D-amino acids only, or mixtures of L-amino acids and D-amino acids.

[0137] As used herein, the term "protein" refers to a polypeptide, or a dimer (i.e., two) or polymer (i.e., three or more) of a single-chain polypeptide. The single-chain polypeptides of a protein may be linked by covalent bonds, such as disulfide bonds, or by non-covalent interactions.

[0138] The terms "conservative substitution," "conservative mutation," or "conservative variant" refer to changes in which one amino acid is substituted for another amino acid that can be classified as having similar characteristics. Examples of classifications of such conservative amino acid groups include the "charged / polar group" which includes Glu (glutamic acid, i.e. E), Asp (aspartic acid, i.e. D), Asn (asparagine, i.e. N), Gln (glutamine, i.e. Q), Lys (lysine, i.e. K), Arg (arginine, i. R), and His (histidine, i.e. H), as well as Phe (phenylalanine, i.e. F), Tyr (tyrosine, i. Y), and Trp (tryptogen). Examples include the "aromatic group" containing phan (i.e., W) and histidine (i.e., H), and the "aliphatic group" containing Gly (glycine, i.e., G), Ala (alanine, i.e., A), Val (valine, i.e., V), Leu (leucine, i.e., L), Ile (isoleucine, i.e., I), Met (methionine, i.e., M), Ser (serine, i.e., S), Thr (threonine, i.e., T), and Cys (cysteine, i.e., C). Within each group, subgroups can also be identified. For example, the group of charged or polar amino acids can be subdivided into subgroups containing the "positively charged subgroup" containing Lys, Arg, and His, the "negatively charged subgroup" containing Glu and Asp, and the "polar subgroup" containing Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups containing the "nitrogen ring subgroup" containing Pro, His, and Trp, and the "phenyl subgroup" containing Phe and Tyr. In yet another example, the aliphatic group can be subdivided into subgroups such as the “aliphatic nonpolar subgroup,” which includes Val, Leu, Gly, and Ala, and the “aliphatic weakly polar subgroup,” which includes Met, Ser, Thr, and Cys.Examples of the classification of conservative mutations include amino acid substitutions within the above subgroups, such as substitution of Lys to Arg or Arg to Lys to maintain a positive charge, substitution of Glu to Asp or Asp to Glu to maintain a negative charge, substitution of Ser to Thr or Thr to Ser to maintain free -OH, and substitution of Gln to Asn or Asn to Gln to maintain free -NH2, but are not limited to these. In some embodiments, to maintain hydrophobicity, for example, in the active site, a native hydrophobic amino acid is substituted with a hydrophobic amino acid.

[0139] In relation to two or more polypeptide sequences, the term “identical” or “identity” % refers to two or more sequences or subsequences that are identical when compared and aligned to the greatest extent possible across a comparison window or a given region, as measured by a sequence comparison algorithm or by manual alignment and visual inspection, or two or more sequences or subsequences that have a predetermined proportion of identical amino acid residues across a given region (e.g., identity of 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%, or more).

[0140] In polypeptide sequence comparison, typically, a single amino acid sequence serves as a reference sequence for comparison with 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 obtain the best possible alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). Those skilled in the art can determine the parameters used during alignment to obtain the best possible alignment. For the purposes of this application, when performing polypeptide sequence comparison, the Standard Protein BLAST of the BLASTP algorithm for aligning two protein sequences is used with default parameters.

[0141] The terms "corresponds to," "determined based on," or "numbered based on" refer to the position of a residue in a given amino acid sequence when that given amino acid sequence is maximally aligned to a given reference sequence and compared to that reference sequence. Therefore, for example, when an amino acid residue in a modified Fc polypeptide is aligned to the amino acid in SEQ ID NO: 1 and optimally aligned to SEQ ID NO: 1, that amino acid residue "corresponds" to the amino acid in SEQ ID NO: 1. The polypeptide being aligned to a reference sequence does not need to be the same length as that reference sequence.

[0142] As used herein, "binding affinity" refers to the strength of a non-covalent interaction between two molecules, for example, a binding site on a polypeptide and a target to which that site binds, such as a transferrin receptor. Therefore, for example, unless otherwise indicated or evident from the context, this term may refer to a 1:1 interaction between a polypeptide and its target. Binding affinity is determined by the equilibrium dissociation constant (K). DThis can be quantified by measuring K D This refers to the dissociation rate constant (k d ,time -1 ) and the association rate constant (k a ,time -1 M -1 This refers to the result of dividing by ). D This can be determined, for example, by measuring the rates of complex formation and dissociation using surface plasmon resonance (SPR), binding equilibrium exclusion assays such as the Biacore® system and KinExA®, and biolayer interferometry (e.g., using the ForteBio® Octet® platform). As used herein, "binding affinity" includes not only formal binding affinity (such as binding affinity reflecting a 1:1 interaction between a polypeptide and its target) but also K, which may reflect strong binding. D This also includes the apparent affinity that is calculated.

[0143] 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 binds to a target with higher affinity, higher binding activity, and / or longer duration than when binding to structurally different targets, as described herein. In a typical embodiment, the manipulated TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody of the present invention has at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold or greater affinity to a specific target, e.g., TfR, compared to an unrelated target, when assayed under the same affinity assay conditions. The terms “specifically bind to” a particular target (e.g., TfR), “specifically bind to” a particular target (e.g., TfR), or “specific to” a particular target (e.g., TfR), as used herein, refer to, for example, the equilibrium dissociation constant K for the target to which it binds. D For example, 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 Applicable to molecules that are M. 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), such as an epitope that is conserved between non-human primates and humans (e.g., structurally conserved between non-human primates and humans). In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide or TfR-binding antibody can bind only to human TfR.

[0144] The terms "variable region" or "variable domain" refer to domains of the heavy or light chains of an antibody that are derived from germline variable (V), diversity (D), or joining (J) genes (not from the constant (Cμ and Cδ) gene segments) and that confer on the antibody its binding specificity for an antigen. Typically, an antibody variable region comprises four conserved "framework" regions that sandwich three hypervariable "complementary determining regions".

[0145] The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and refer to one or more antibody fragments that retain the ability to specifically bind an antigen through their variable regions. 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 containing two Fab fragments linked by a disulfide bond 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).

[0146] As used herein, terms such as "treatment", "treating", etc. are generally used for the purpose of meaning to obtain the desired pharmacological and / or physiological effects. "Treating" or "treatment" means any objective or subjective parameter, including alleviation, remission, improvement of patient survival, improvement of life span or survival rate, reduction of symptoms, improvement of the patient's tolerance to the disorder, reduction of the rate of deterioration or debilitation, or improvement of the patient's physical or mental health status, and may refer to any sign of efficacy or improvement in the treatment of lysosomal storage disorders, such as Hunter syndrome, Sanfilippo syndrome A, Niemann-Pick disease, Gaucher disease or Parkinson's disease. Treatment or improvement of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared with that of an untreated individual or population, or the same patient at different times before or during treatment.

[0147] As used herein, the terms "subject", "individual" and "patient" are used synonymously for the purpose of referring to mammals (including, but not limited to, humans, non-human primates, rodents (such as rats, mice and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species). In one embodiment, the patient is human.

[0148] The term "pharmaceutically acceptable excipient" refers to an inactive pharmaceutical ingredient (such as, but not limited to, buffers, carriers or preservatives) that is biologically or pharmacologically compatible for use in humans or animals.

[0149] As used herein, a "therapeutic amount", "therapeutically effective amount" or "therapeutically effective concentration" of a drug is the amount or concentration of the drug that treats the signs or symptoms of a disease (such as LSD) in a subject (such as a mammal).

[0150] The term "administer" refers to a method of delivering a drug, compound, or composition to a desired biological site of action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the polypeptide described herein is administered intravenously.

[0151] III. Enzymes for Enzyme Replacement Therapy (ERT) Lysosomal storage disorders (LSDs) are hereditary metabolic disorders characterized by the accumulation of undigested or partially digested macromolecules, which ultimately lead to cellular dysfunction and clinical abnormalities. Classically, LSDs have been defined as lysosomal dysfunctions generally classified by the substances they accumulate, including sphingolipidosis, oligosaccharidosis, mucolipidosis, mucopolysaccharidosis, lipoprotein storage disorders, and neuronal ceroid lipofuscinosis. The classification of these disorders has recently been expanded to include other deficiencies or abnormalities in proteins that accumulate macromolecules (such as proteins essential for the normal post-translational modification of lysosomal enzymes or proteins important for proper lysosomal transport).

[0152] In some embodiments, the fusion protein described herein comprises (i) an Fc polypeptide which may include a modification (e.g., one or more modifications that promote heterodimerization) or which may be a wild-type Fc polypeptide, and an enzyme for ERT; and (ii) an Fc polypeptide which may include a modification (e.g., one or more modifications that promote heterodimerization) or which may be a wild-type Fc polypeptide, and optionally an enzyme for ERT. In some embodiments, one or both Fc polypeptides may include a modification that results in binding to a blood-brain barrier (BBB) ​​receptor, such as a transferrin receptor (TfR). The enzyme for ERT may be any enzyme that is deficient in LSD. The enzyme for ERT incorporated into the fusion protein has catalytic activity, i.e., retains the enzyme activity that is deficient in LSD. In some embodiments, the enzyme for ERT is iduronate-2-sulfatase (IDS) which is deficient in Hunter syndrome. In some embodiments, the enzyme for ERT is N-sulfoglucosamine sulfohydrolase (SGSH) which is deficient in Sanfilippo syndrome. In some embodiments, the enzyme for ERT is acid sphingomyelinase (ASM), which is deficient in Niemann-Pick disease. In some embodiments, the enzyme for ERT is β-glucocerebrosidase (GBA), which is deficient in Gaucher disease and Parkinson's disease.

[0153] In some embodiments, the fusion protein comprising the ERT enzyme and optionally a modified Fc polypeptide that binds to the BBB receptor, such as a TfR-binding Fc polypeptide, comprises a catalytically active fragment or variant of the wild-type IDS. In some embodiments, the IDS enzyme is a variant or catalytically active fragment of the IDS protein comprising any one of the amino acid sequences of SEQ ID NOs: 91, 92, 114, 230, and 234. In some embodiments, the catalytically active variant or fragment of the IDS enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more activity than that of the wild-type IDS enzyme.

[0154] In some embodiments, the fusion protein comprising an ERT enzyme and optionally a modified Fc polypeptide that binds to the BBB receptor, such as a TfR-binding Fc polypeptide, comprises a catalytically active fragment or variant of wild-type SGSH. In some embodiments, the SGSH enzyme is a variant or catalytically active fragment of the SGSH protein comprising either one of the amino acid sequences of SEQ ID NOs. 119 and 120. 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.

[0155] In some embodiments, the fusion protein comprising the enzyme for ERT and optionally a modified Fc polypeptide that binds to the BBB receptor, such as a TfR-binding Fc polypeptide, comprises a catalytically active fragment or variant of wild-type ASM. In some embodiments, the ASM enzyme is a variant or catalytically active fragment of the ASM protein comprising any one of the amino acid sequences of SEQ ID NOs: 121, 122, and 123. In some embodiments, the catalytically active variant or fragment of the ASM 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 activity than that of the wild-type ASM enzyme.

[0156] In some embodiments, the fusion protein comprising the enzyme for ERT and optionally a modified Fc polypeptide that binds to the BBB receptor, such as a TfR-binding Fc polypeptide, comprises a catalytically active fragment or variant of wild-type GBA. In some embodiments, the GBA enzyme is a variant or catalytically active fragment of the GBA protein comprising either one of the amino acid sequences of SEQ ID NOs. 93 and 94. In some embodiments, the catalytically active variant or fragment of the GBA 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 GBA enzyme.

[0157] In some embodiments, the ERT enzyme present in the fusion protein described herein, such as IDS, SGSH, ASM, or GBA, or its catalytic variant or fragment, retains at least 25% of its activity compared to its activity when not bound to an Fc polypeptide or TfR-linked Fc polypeptide. In some embodiments, the ERT 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 bound to an Fc polypeptide or TfR-linked Fc polypeptide. In some embodiments, the ERT enzyme, or its catalytic variant or fragment, retains at least 80%, 85%, 90%, or 95% of its activity compared to its activity when not bound to an Fc polypeptide or TfR-linked Fc polypeptide. In some embodiments, fusion to an Fc polypeptide does not reduce the activity of its ERT enzyme, such as IDS, SGSH, ASM, or GBA, or its catalytic variant or fragment. In some embodiments, fusion to a TfR-bound Fc polypeptide does not reduce the activity of its ERT enzyme.

[0158] IV. Modification of Fc polypeptides for binding to blood-brain barrier (BBB) ​​receptors In some embodiments, the present invention provides 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 types of cells and tissues. In some embodiments, the BBB receptor is a transferrin receptor (TfR).

[0159] The amino acid residues shown in the various Fc modifications (including amino acid residues introduced into modified Fc polypeptides that bind to BBB receptors, e.g., TfR) are numbered herein using EU numbering. Any Fc polypeptide, e.g., IgG1, IgG2, IgG3, or IgG4, may have modifications, e.g., amino acid substitutions, at one or more positions as described herein.

[0160] Modified Fc polypeptides present in the fusion proteins described herein (e.g., Fc polypeptides modified to promote heterodimerization and / or binding to the BBB receptor) may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with the native Fc region sequence or fragment thereof, for example, fragments of at least 50 amino acids, 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, or at least 95% identity with the amino acids at positions 1 to 110 of SEQ ID NO: 1, or the amino acids at positions 111 to 217 of SEQ ID NO: 1, or fragments thereof, for example, fragments of at least 50 amino acids, at least 100 amino acids or longer.

[0161] In some embodiments, the modified Fc polypeptide (e.g., modified to promote heterodimerization and / or binding to the BBB receptor) contains at least 50 amino acids, at least 60, 65, 70, 75, 80, 85, 90, 95 or more amino acids, 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, at least 30, 35, 40 or 45 consecutive amino acids, 50 consecutive amino acids, 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 (e.g., SEQ ID NO: 1).

[0162] In some embodiments, the domain modified for BBB receptor binding activity is the human Ig CH3 domain (such as the IgG1 CH3 domain). This CH3 domain can be derived from any of the IgG subtypes, namely IgG1, IgG2, IgG3, or IgG4. In the case of IgG1 antibodies, the CH3 domain refers to the amino acid segment from approximately positions 341 to 447, when numbered according to the EU numbering scheme.

[0163] In some embodiments, the domain modified for BBB receptor binding activity is the human Ig CH2 domain (such as the IgG CH2 domain). This CH2 domain can be derived from any of the IgG subtypes, namely IgG1, IgG2, IgG3, or IgG4. In the case of IgG1 antibodies, the CH2 domain refers to the amino acid segment from approximately positions 231 to 340, when numbered according to the EU numbering scheme.

[0164] In some embodiments, the modified (e.g., BBB receptor-binding) Fc polypeptide present in the fusion protein described herein comprises at least one, two, or three substitutions, and in some embodiments, comprises at least four, five, six, seven, eight, nine, or ten substitutions at amino acid positions including positions 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 according to the EU numbering scheme.

[0165] In some embodiments, the modified (e.g., BBB receptor-binding) Fc polypeptide present in the fusion protein described herein comprises at least one, two, or three substitutions, and in some embodiments, comprises at least four, five, six, seven, eight, or nine substitutions at amino acid positions including positions 274, 276, 283, 285, 286, 287, 288, 289, and 290 according to the EU numbering scheme.

[0166] In some embodiments, the modified (e.g., BBB receptor-binding) Fc polypeptide present in the fusion protein described herein comprises at least one, two, or three substitutions, and in some embodiments, comprises at least four, five, six, seven, eight, nine, or ten substitutions at amino acid positions including positions 268, 269, 270, 271, 272, 292, 293, 294, 296, and 300 according to the EU numbering scheme.

[0167] In some embodiments, the modified (e.g., BBB receptor-binding) Fc polypeptide present in the fusion protein described herein comprises at least one, two, or three substitutions, and in some embodiments, comprises at least four, five, six, seven, eight, or nine substitutions at amino acid positions including positions 272, 274, 276, 322, 324, 326, 329, 330, and 331 according to the EU numbering scheme.

[0168] In some embodiments, the modified (e.g., binds to a BBB receptor) Fc polypeptide present in the fusion proteins described herein comprises at least 1, 2, or 3 substitutions, and in some embodiments, according to the EU numbering scheme, at least 4, 5, 6, or 7 substitutions at amino acid positions including positions 345, 346, 347, 349, 437, 438, 439, and 440.

[0169] In some embodiments, the modified (e.g., binds to a BBB receptor) Fc polypeptide present in the fusion proteins described herein comprises at least 1, 2, or 3 substitutions, and in some embodiments, according to the EU numbering scheme, at least 4, 5, 6, 7, 8, or 9 substitutions at amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and 421.

[0170] FcRn binding site In certain aspects, the modified (e.g., binds to a BBB receptor) Fc polypeptide present in the fusion proteins described herein or an Fc polypeptide that does not specifically bind to a BBB receptor can also include an FcRn binding site. In some embodiments, the FcRn binding site is within the Fc polypeptide or a fragment thereof.

[0171] In some embodiments, the FcRn binding site comprises a native FcRn binding site. In some embodiments, the FcRn binding site does not include amino acid changes relative to the amino acid sequence of the native FcRn binding site. In some embodiments, the native FcRn binding site is an IgG binding site, e.g., a human IgG binding site. In some embodiments, the FcRn binding site includes modifications that alter binding to FcRn.

[0172] In some embodiments, the FcRn binding site has mutations, for example, one or more substituted amino acid residues, and these mutations (or multiple mutations) either prolong the serum half-life or do not substantially shorten it (i.e., when assayed under the same conditions, the serum half-life is shortened by 25% or less compared to the modified Fc polypeptide of the counterpart 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.

[0173] 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 the positions 252, 254, and 256. In some embodiments, the mutations are M252Y, S254T, and T256E. In some embodiments, the modified Fc polypeptide further includes the mutations M252Y, S254T, and T256E. In some embodiments, the modified Fc polypeptide includes substitutions at one, two, or all three of the 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 the mutations T307A, E380A, and N434A. In some embodiments, the modified Fc polypeptide includes substitutions at the T250 and M428 positions according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide includes the mutation T250Q and / or M428L. In some embodiments, the modified Fc polypeptide includes substitutions at the M428 and N434 positions according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide includes the mutations M428L and N434S. In some embodiments, the modified Fc polypeptide includes the mutation N434S or N434A.

[0174] V. Transferrin receptor-binding FC polypeptide This section describes the preparation of modified Fc polypeptides described herein that can bind to transferrin receptors (TfRs) and be transported across the blood-brain barrier (BBB).

[0175] TfR-binding Fc polypeptide containing a mutation in the CH3 domain 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) that has been modified for TfR binding activity. The CH3 domain may be derived from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. With respect to IgG antibodies, the CH3 domain refers to the amino acid segment from approximately position 341 to approximately position 447, when numbered according to the EU numbering scheme.

[0176] In some embodiments, a modified Fc polypeptide that specifically binds to TfR can bind to the apical domain of TfR and bind to TfR without blocking or otherwise inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is substantially uninhibited. In some embodiments, the inhibition of transferrin binding to TfR is less than about 50% (e.g., less than about 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%). In some embodiments, the inhibition of transferrin binding to TfR occurs in less than approximately 20% of cases (e.g., less than approximately 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, 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%).

[0177] In some embodiments, the modified Fc polypeptide that specifically binds to TfR contains at least two, three, four, five, six, seven, eight, or nine substitutions at positions 384, 386, 387, 388, 389, 390, 413, 416, and 421, according to the EU numbering scheme. Exemplary substitutions that can be introduced at these positions are shown in Tables 4 and 5. In some embodiments, the amino acid at position 388 and / or 421 is an aromatic amino acid, e.g., Trp, Phe, or Tyr. In some embodiments, the amino acid at position 388 is Trp. In some embodiments, the aromatic amino acid at position 421 is Trp or Phe.

[0178] In some embodiments, at least one position is substituted, such as Leu, Tyr, Met, or Val at position 384; Leu, Thr, His, or Pro at position 386; Val, Pro, or an acidic amino acid at position 387; an aromatic amino acid, e.g., Trp, at position 388; Val, Ser, or Ala at position 389; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 413; Thr, or an acidic amino acid at position 416; or Trp, Tyr, His, or Phe at position 421. In some embodiments, the modified Fc polypeptide may contain, in one or more positions within the above set, a conservative substitution of a given amino acid, e.g., an amino acid within the same charge group, the same hydrophobic group, the same side-chain ring structure group (e.g., an aromatic amino acid), or the same size group and / or the same polar or nonpolar group. Thus, for example, Ile may be present at positions 384, 386, and / or 413. In some embodiments, one, two, or each of the acidic amino acids at positions 387, 413, and 416 are Glu. In other embodiments, one, two, or each of the acidic amino acids at positions 387, 413, and 416 are Asp. In some embodiments, two, three, four, five, six, seven, or eight of positions 384, 386, 387, 388, 389, 413, 416, and 421 have amino acid substitutions as defined in this paragraph.

[0179] In some embodiments, the modified Fc polypeptide, as described in the two paragraphs above, contains the native Asn at position 390. In some embodiments, the modified Fc polypeptide contains Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 390. In some embodiments, the modified Fc polypeptide further includes one, two, three, or four substitutions at positions including 380, 391, 392, and 415 according to the EU numbering scheme. In some embodiments, Trp, Tyr, Leu, or Gln may be present at position 380. In some embodiments, Ser, Thr, Gln, or Phe may be present at position 391. In some embodiments, Gln, Phe, or His may be present at position 392. In some embodiments, Glu may be present at position 415.

[0180] In certain embodiments, the modified Fc polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu; and / or position 421 which is Phe. In some embodiments, the modified Fc polypeptide includes all 11 elements: position 380 which is Trp, Leu, or Glu; position 384 which is Tyr or Phe; position 386 which is Thr; position 387 which is Glu; position 388 which is Trp; position 389 which is Ser, Ala, Val, or Asn; position 390 which is Ser or Asn; position 413 which is Thr or Ser; position 415 which is Glu or Ser; position 416 which is Glu and / or position 421 which is Phe.

[0181] In certain embodiments, the modified Fc polypeptide contains Leu or Met at position 384, Leu, His or Pro at position 386, Val at position 387, Trp at position 388, Val or Ala at position 389, Pro at position 413, Thr at position 416, and / or Trp at position 421. In some embodiments, the modified Fc polypeptide further contains Ser, Thr, Gln or Phe at position 391. In some embodiments, the modified Fc polypeptide further contains Trp, Tyr, Leu or Gln at position 380 and / or Gln, Phe or His at position 392. In some embodiments, Trp is present at position 380 and / or Gln is present at position 392. In some embodiments, the modified Fc polypeptide does not have Trp at position 380.

[0182] In another embodiment, the modified Fc polypeptide contains Tyr at position 384, Thr at position 386, Glu or Val at position 387, Trp at position 388, Ser at position 389, Ser or Thr at position 413, Glu at position 416, and / or Phe at position 421. In some embodiments, the modified Fc polypeptide contains native Asn at position 390. In certain embodiments, the modified Fc polypeptide further contains Trp, Tyr, Leu or Gln at position 380, and / or Glu at position 415. In some embodiments, the modified Fc polypeptide further contains Trp at position 380 and / or Glu at position 415.

[0183] In additional embodiments, the modified Fc polypeptide further includes one, two, or three substitutions at positions including 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.

[0184] In some embodiments, the modified Fc polypeptide contains one or more substitutions according to the EU numbering scheme, namely Trp at position 380, Thr at position 386, Trp at position 388, Val at position 389, Thr or Ser at position 413, Glu at position 415, and / or Phe at position 421.

[0185] 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 or at least 95% identity with any one of the amino acids at positions 111-217 of SEQ ID NOs. 4-90, 97-100 and 105-108 (e.g., SEQ ID NOs. 34-38, 58 and 60-90). In some embodiments, the modified Fc polypeptide contains an amino acid at EU index positions 384-390 and / or 413-421 of any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide contains an amino acid at EU index positions 380-390 and / or 413-421 of any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide contains an amino acid at EU index positions 380-392 and / or 413-426 of any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90).

[0186] In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90), and when numbered according to the EU Index, the 380th position is Trp, Tyr, Leu, Gln, or Glu; the 384th position is Leu, Tyr, Met, or Val; the 386th position is Leu, Thr, His, or Pro; the 387th position is Val, Pro, or an acidic amino acid; the 388th position is an aromatic amino acid, e.g., Trp; and the 388th position is Val, Ser... The molecule further comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 positions from the following locations: position 389, which is Ala; position 390, which is Ser or Asn; position 391, which is Ser, Thr, Gln, or Phe; position 392, which is Gln, Phe, or His; position 413, which is an acidic amino acid, Ala, Ser, Leu, Thr, or Pro; position 414, which is Lys, Arg, Gly, or Pro; position 415, which is Glu or Ser; position 416, which is Thr or an acidic amino acid; position 421, which is Trp, Tyr, His, or Phe; position 424, which is Ser, Thr, Glu, or Lys; and position 426, which is Ser, Trp, or Gly.

[0187] In some embodiments, the modified Fc polypeptide contains one of the amino acid sequences from SEQ ID NOs: 34-38, 58, and 60-90. In other embodiments, the modified Fc polypeptide contains one of the amino acid sequences from SEQ ID NOs: 34-38, 58, and 60-90, but with one, two, or three amino acids substituted.

[0188] In some embodiments, the modified Fc polypeptide includes additional mutations such as those described in Section VI below, including, but not limited to, knob mutations (e.g., T366W when numbered according to EU numbering), whole mutations (e.g., T366S, L368A, and Y407V when numbered according to EU numbering), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) when numbered according to EU numbering), and / or mutations that improve serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to the EU numbering scheme). As an example, Sequence IDs 156-229 show non-limiting examples of modified Fc polypeptides having mutations in the CH3 domain that include one or more of these additional mutations (e.g., clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, and CH3C.35.23).

[0189] In some embodiments, the modified Fc polypeptide includes a knob mutation (e.g., T366W when numbered according to EU numbering) and has at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 156, 168, 180, 192, 204, and 216. In some embodiments, the modified Fc polypeptide includes any one of sequence numbers 156, 168, 180, 192, 204, and 216.

[0190] In some embodiments, the modified Fc polypeptide includes a knob mutation (e.g., T366W when numbered according to EU numbering) and a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) when numbered according to EU numbering), and has at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 157, 158, 169, 170, 181, 182, 193, 194, 205, 206, 217, 218, 228, and 229. In some embodiments, the modified Fc polypeptide includes any one of sequence numbers 157, 158, 169, 170, 181, 182, 193, 194, 205, 206, 217, and 218.

[0191] In some embodiments, the modified Fc polypeptide includes a knob mutation (e.g., T366W when numbered according to EU numbering) and a mutation that improves serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to the EU numbering scheme), and has at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 159, 171, 183, 195, 207, and 219. In some embodiments, the modified Fc polypeptide includes any one of sequence numbers 159, 171, 183, 195, 207, and 219.

[0192] In some embodiments, the modified Fc polypeptide may have knob mutations (e.g., T366W when numbered based on EU numbering), mutations that modulate effector function (e.g., L234A, L235A and / or P329G (e.g., L234A and L235A) when numbered based on EU numbering), and mutations that improve serum stability or serum half-life (e.g., (i) based on EU numbering (ii) N434S, with or without M428L, when numbered according to the EU numbering scheme, comprising M252Y, S254T, and T256E, or (ii) N434S, with or without M428L, when numbered according to the EU numbering scheme, having at least 85% identity, at least 90% identity, or at least 95% identity with any one of the sequences of SEQ ID NOs. 160, 161, 172, 173, 184, 185, 196, 197, 208, 209, 220, and 221. In some embodiments, the modified Fc polypeptide comprises any one of the sequences of SEQ ID NOs. 160, 161, 172, 173, 184, 185, 196, 197, 208, 209, 220, and 221.

[0193] In some embodiments, the modified Fc polypeptide includes whole mutations (e.g., T366S, L368A, and Y407V when numbered according to EU numbering) and has at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 162, 174, 186, 198, 210, and 222. In some embodiments, the modified Fc polypeptide includes any one of sequence numbers 162, 174, 186, 198, 210, and 222.

[0194] In some embodiments, the modified Fc polypeptide includes whole mutations (e.g., T366S, L368A, and Y407V when numbered according to EU numbering) and mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) when numbered according to EU numbering), and has at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 163, 164, 175, 176, 187, 188, 199, 200, 211, 212, 223, and 224. In some embodiments, the modified Fc polypeptide includes one of the sequences 163, 164, 175, 176, 187, 188, 199, 200, 211, 212, 223, and 224.

[0195] In some embodiments, the modified Fc polypeptide includes whole mutations (e.g., T366S, L368A, and Y407V when numbered according to EU numbering), as well as mutations that improve serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to the EU numbering scheme), and has at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 165, 177, 189, 201, 213, and 225. In some embodiments, the modified Fc polypeptide includes any one of sequence numbers 165, 177, 189, 201, 213, and 225.

[0196] In some embodiments, the modified Fc polypeptide may have whole mutations (e.g., T366S, L368A, and Y407V when numbered based on EU numbering), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) when numbered based on EU numbering), and mutations that improve serum stability or serum half-life (e.g., (i) EU numbering (ii) N434S, with or without M428L, when numbered based on the G sequence, and having at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 166, 167, 178, 179, 190, 191, 202, 203, 214, 215, 226, and 227. In some embodiments, the modified Fc polypeptide includes any one of sequence numbers 166, 167, 178, 179, 190, 191, 202, 203, 214, 215, 226, and 227.

[0197] In some embodiments, the modified Fc polypeptide that specifically binds to TfR contains at least two, three, four, five, six, seven, or eight substitutions at positions 345, 346, 347, 349, 437, 438, 439, and 440, according to the EU numbering scheme. Exemplary modified Fc polypeptides are shown in SEQ ID NOs: 124-128. In some embodiments, the modified Fc polypeptide contains Gly at position 437, Phe at position 438, and / or Asp at position 440. In some embodiments, Glu is present at position 440. In certain embodiments, the modified Fc polypeptide includes at least one substitution, such as at position 345 being Phe or Ile, at position 346 being Asp, Glu, Gly, Ala or Lys, at position 347 being Tyr, Met, Leu, Ile or Asp, at position 349 being Thr or Ala, at position 437 being Gly, at position 438 being Phe, at position 439 being His, Tyr, Ser or Phe, or at position 440 being Asp. In some embodiments, all two, three, four, five, six, seven or eight of positions 345, 346, 347, 349, 437, 438, 439 and 440 have substitutions as defined in this paragraph. In some embodiments, the modified Fc polypeptide may contain, in one or more positions within the above set, a conservative substitution of a predetermined amino acid, for example, an amino acid within the same charge group, the same hydrophobic group, the same side-chain ring structure group (e.g., aromatic amino acids), the same size group, and / or polar or nonpolar group.

[0198] 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 or at least 95% identity with any one of the amino acids from positions 111 to 217 of SEQ ID NOs. 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 or at least 95% identity with SEQ ID NOs. In some embodiments, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 124 to 128. In another embodiment, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 124 to 128, but with one, two, or three amino acids substituted.

[0199] TfR-binding Fc polypeptide containing a mutation in the CH2 domain In some embodiments, the modified Fc polypeptide that specifically binds to TfR includes a substitution in the CH2 domain. In some embodiments, the modified Fc polypeptide includes a human Ig CH2 domain (such as an IgG CH2 domain) that has been modified for TfR binding activity. The CH2 domain may be derived from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. With respect to IgG antibodies, the CH2 domain refers to the amino acid segment from approximately positions 231 to 340, when numbered according to the EU numbering scheme.

[0200] In some embodiments, a modified Fc polypeptide that specifically binds to TfR can bind to the apical domain of TfR and bind to TfR without blocking or otherwise inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is substantially uninhibited. In some embodiments, the inhibition of transferrin binding to TfR is less than about 50% (e.g., less than about 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%). In some embodiments, the inhibition of transferrin binding to TfR occurs in less than approximately 20% of cases (e.g., less than approximately 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, 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%).

[0201] In some embodiments, the modified Fc polypeptide that specifically binds to TfR includes at least two, three, four, five, six, seven, eight, or nine substitutions at positions 274, 276, 283, 285, 286, 287, 288, and 290, according to the EU numbering scheme. Exemplary modified Fc polypeptides are described in SEQ ID NOs: 129-133. In some embodiments, the modified Fc polypeptide includes Glu at position 287 and / or Trp at position 288. In some embodiments, the modified Fc polypeptide includes at least one substitution, such as at position 274 which is Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp; at position 276 which is Ile, Val, Asp, Glu, Thr, Ala, or Tyr; at position 283 which is Asp, Pro, Met, Leu, Ala, Asn, or Phe; at position 285 which is Arg, Ser, Ala, or Gly; at position 286 which is Tyr, Trp, Arg, or Val; at position 287 which is Glu; at position 288 which is Trp or Tyr; at position 289 which is Gln, Tyr, His, Ile, Phe, Val, or Asp; or at position 290 which is Leu, Trp, Arg, Asn, Tyr, or Val. In some embodiments, two, three, four, five, six, seven, eight, or nine of the positions 274, 276, 283, 285, 286, 287, 288, and 290 have substitutions as defined in this paragraph. In some embodiments, the modified Fc polypeptide may have, in one or more of the positions in the above set, a conservative substitution of a given amino acid, e.g., an amino acid within the same charge group, the same hydrophobic group, the same side-chain ring structure group (e.g., aromatic amino acids), the same size group, and / or an amino acid within the same polar or nonpolar group.

[0202] In some embodiments, the modified Fc polypeptide contains Glu, Gly, Gln, Ser, Ala, Asn, or Tyr at position 274, Ile, Val, Asp, Glu, Thr, Ala, or Tyr at position 276, Asp, Pro, Met, Leu, Ala, or Asn at position 283, Arg, Ser, or Ala at position 285, Tyr, Trp, Arg, or Val at position 286, Glu at position 287, Trp at position 288, Gln, Tyr, His, Ile, Phe, or Val at position 289, and / or Leu, Trp, Arg, Asn, or Tyr at position 290. In some embodiments, the modified Fc polypeptide contains Arg at position 285, Tyr or Trp at position 286, Glu at position 287, Trp at position 288, and / or Arg or Trp at position 290.

[0203] 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 or at least 95% identity with any one of the amino acids from positions 1 to 110 of SEQ ID NOs. 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 or at least 95% identity with SEQ ID NOs. In some embodiments, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 129 to 133. In another embodiment, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 129 to 133, but with one, two, or three amino acids substituted.

[0204] In some embodiments, the modified Fc polypeptide that specifically binds to TfR includes at least two, three, four, five, six, seven, eight, nine, or ten substitutions at positions 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299, according to the EU numbering scheme. Exemplary modified Fc polypeptides are shown in SEQ ID NOs: 134-138. In some embodiments, the modified Fc polypeptide includes Pro at position 270, Glu at position 295, and / or Tyr at position 297. In some embodiments, the modified Fc polypeptide includes at least one substitution, such as at position 266 being Pro, Phe, Ala, Met, or Asp; at position 267 being Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr; at position 268 being Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu; at position 269 being Pro, Val, Ala, Thr, or Asp; at position 270 being Pro, Val, or Phe; at position 271 being Trp, Gln, Thr, or Glu; at position 295 being Glu, Val, Thr, Leu, or Trp; at position 297 being Tyr, His, Val, or Asp; at position 298 being Thr, His, Gln, Arg, Asn, or Val; or at position 299 being Tyr, Asn, Asp, Ser, or Pro. In some embodiments, two, three, four, five, six, seven, eight, nine, or ten of the positions 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 have substitutions as defined in this paragraph. In some embodiments, the modified Fc polypeptide may have, in one or more of the positions in the above set, a conservative substitution of a given amino acid, e.g., an amino acid within the same charge group, the same hydrophobic group, the same side-chain ring structure group (e.g., aromatic amino acids), the same size group, and / or an amino acid within the same polar or nonpolar group.

[0205] In some embodiments, the modified Fc polypeptide includes Pro, Phe, or Ala at position 266, Gln, Pro, Arg, Lys, Ala, or Ile at position 267, Thr, Ser, Gly, Met, Val, Phe, or Trp at position 268, Pro, Val, or Ala at position 269, Pro at position 270, Trp, or Gln at position 271, Glu at position 295, Tyr at position 297, Thr, His, or Gln at position 298, and / or Tyr, Asn, Asp, or Ser at position 299.

[0206] In some embodiments, the modified Fc polypeptide includes Met at position 266, Leu or Glu at position 267, Trp at position 268, Pro at position 269, Val at position 270, Thr at position 271, Val or Thr at position 295, His at position 197, His, Arg or Asn at position 198, and / or Pro at position 299.

[0207] In some embodiments, the modified Fc polypeptide includes Asp at position 266, Asp at position 267, Leu at position 268, Thr at position 269, Phe at position 270, Gln at position 271, Val or Leu at position 295, Val at position 297, Thr at position 298, and / or Pro at position 299.

[0208] 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 or at least 95% identity with any one of the amino acids from positions 1 to 110 of SEQ ID NOs. 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 or at least 95% identity with SEQ ID NOs. In some embodiments, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 134 to 138. In another embodiment, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 134 to 138, but with one, two, or three amino acids substituted.

[0209] In some embodiments, modified Fc polypeptides that specifically bind to TfR contain at least two, three, four, five, six, seven, eight, nine, or ten substitutions at positions 268, 269, 270, 271, 272, 292, 293, 294, and 300, according to the EU numbering scheme. Exemplary modified Fc polypeptides are shown in SEQ ID NOs: 139-143. In some embodiments, the modified Fc polypeptide includes at least one substitution, such as at position 268 being Val or Asp, at position 269 being Pro, Met or Asp, at position 270 being Pro or Trp, at position 271 being Arg, Trp, Glu or Thr, at position 272 being Met, Tyr or Trp, at position 292 being Leu or Trp, at position 293 being Thr, Val, Ile or Lys, at position 294 being Ser, Lys, Ala or Leu, at position 296 being His, Leu or Pro, or at position 300 being Val or Trp. In some embodiments, all two, three, four, five, six, seven, eight, nine or ten of positions 268, 269, 270, 271, 272, 292, 293, 294 and 300 have substitutions as defined in this paragraph. In some embodiments, the modified Fc polypeptide may contain, in one or more positions within the above set, a conservative substitution of a predetermined amino acid, for example, an amino acid within the same charge group, the same hydrophobic group, the same side-chain ring structure group (e.g., an aromatic amino acid), the same size group, and / or the same polar or nonpolar group.

[0210] In some embodiments, the modified Fc polypeptide includes Val at position 268, Pro at position 269, Pro at position 270, Arg or Trp at position 271, Met at position 272, Leu at position 292, Thr at position 293, Ser at position 294, His at position 296, and / or Val at position 300.

[0211] In some embodiments, the modified Fc polypeptide includes Asp at position 268, Met or Asp at position 269, Trp at position 270, Glu or Thr at position 271, Tyr or Trp at position 272, Trp at position 292, Val, Ile or Lys at position 293, Lys, Ala or Leu at position 294, Leu or Pro at position 296, and / or Trp at position 300.

[0212] 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 or at least 95% identity with any one of the amino acids from positions 1 to 110 of SEQ ID NOs. 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 or at least 95% identity with SEQ ID NOs. In some embodiments, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 139 to 143. In another embodiment, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 139 to 143, but with one, two, or three amino acids substituted.

[0213] In some embodiments, modified Fc polypeptides that specifically bind to TfR have at least two, three, four, five, six, seven, eight, nine, or ten substitutions at positions 272, 274, 276, 322, 324, 326, 329, 330, and 331, according to the EU numbering scheme. Exemplary modified Fc polypeptides are shown in SEQ ID NOs: 144-148. In some embodiments, the modified Fc polypeptide contains Trp at position 330. In some embodiments, the modified Fc polypeptide includes at least one substitution, such as at position 272 being Trp, Val, Ile, or Ala; at position 274 being Trp or Gly; at position 276 being Tyr, Arg, or Glu; at position 322 being Ser, Arg, or Gln; at position 324 being Val, Ser, or Phe; at position 326 being Ile, Ser, or Trp; at position 329 being Trp, Thr, Ser, Arg, or Asp; at position 330 being Trp; or at position 331 being Ser, Lys, Arg, or Val. In some embodiments, all two, three, four, five, six, seven, eight, or nine of positions 272, 274, 276, 322, 324, 326, 329, 330, and 331 have substitutions as defined in this paragraph. In some embodiments, the modified Fc polypeptide may contain, in one or more positions within the above set, a conservative substitution of a predetermined amino acid, for example, an amino acid within the same charge group, the same hydrophobic group, the same side-chain ring structure group (e.g., an aromatic amino acid), the same size group, and / or the same polar or nonpolar group.

[0214] In some embodiments, the modified Fc polypeptide includes 2, 3, 4, 5, 6, 7, 8, or 9 positions selected from position 272 being Trp, Val, Ile, or Ala, position 274 being Trp or Gly, position 276 being Tyr, Arg, or Glu, position 322 being Ser, Arg, or Gln, position 324 being Val, Ser, or Phe, position 326 being Ile, Ser, or Trp, position 329 being Trp, Thr, Ser, Arg, or Asp, position 330 being Trp, and position 331 being Ser, Lys, Arg, or Val. In some embodiments, the modified Fc polypeptide includes Val or Ile at position 272, Gly at position 274, Arg at position 276, Arg at position 322, Ser at position 324, Ser at position 326, Thr, Ser or Arg at position 329, Trp at position 330, and / or Lys or Arg at position 331.

[0215] 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 or at least 95% identity with any one of the amino acids from positions 1 to 110 of SEQ ID NOs. 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 or at least 95% identity with SEQ ID NOs. In some embodiments, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 144 to 148. In another embodiment, the modified Fc polypeptide contains any one of the amino acid sequences from SEQ ID NOs. 144 to 148, but with one, two, or three amino acids substituted.

[0216] VI. Additional Fc polypeptide mutations In some embodiments, the fusion proteins described herein may each comprise independently selected modifications or two Fc polypeptides, each of which may be a wild-type Fc polypeptide, e.g., human IgG1 Fc polypeptide. In some embodiments, one or both Fc polypeptides comprise one or more modifications resulting in binding to blood-brain barrier (BBB) ​​receptors, e.g., transferrin receptors (TfRs). Non-limiting examples of other mutations that may be introduced into one or both Fc polypeptides include, for example, mutations to improve serum stability or serum half-life, mutations to modulate effector function, mutations to affect glycosylation, mutations to reduce immunogenicity in humans, and / or mutations to heterodimerize the knobs and holes of those Fc polypeptides.

[0217] In some embodiments, the Fc polypeptides present in the fusion protein of the present invention independently have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the corresponding wild-type Fc polypeptide (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide).

[0218] In some embodiments, the Fc polypeptide present in the fusion protein of the present invention includes knob and hole mutations to promote heterodimer formation and inhibit homodimer formation. Generally, the modification involves introducing a bump ("knob") at the interface of the first polypeptide and a corresponding depression ("hole") at the interface of the second polypeptide, thereby positioning the bump within the depression to promote heterodimer formation and consequently inhibit homodimer formation. The bump is constructed by replacing a small amino acid side chain (e.g., tyrosine or tryptophan) at the interface of the first polypeptide. A depression that counteracts the bump, and is the same size as or similar to the bump, is created at the interface of the second polypeptide by replacing a large amino acid side chain (e.g., alanine or threonine). In some embodiments, such additional mutations are located at positions on the Fc polypeptide that do not adversely affect the polypeptide's binding to BBB receptors, such as TfR.

[0219] In an exemplary embodiment of the knob and hole approach for dimerization, one of the Fc polypeptides present in the fusion protein of the present invention has tryptophan at position 366 (numbered according to the EU numbering scheme) instead of native threonine. The other Fc polypeptide in 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 native threonine at position 366 (numbered according to the EU numbering scheme) is replaced with serine, and native leucine at position 368 (numbered according to the EU numbering scheme) is replaced with alanine. Thus, one of the Fc polypeptides in the fusion protein described herein has a knob mutation T366W, and the other Fc polypeptide has a mutation Y407V, typically accompanied by whole mutations T366S and L368A.

[0220] In some embodiments, modifications can be introduced to improve 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, when numbered according to the EU numbering scheme. Thus, one or both Fc polypeptides may have the substitutions M252Y, S254T, and T256E. Alternatively, one or both Fc polypeptides may have the substitutions M428L and N434S, when numbered according to the EU numbering scheme. Alternatively, one or both Fc polypeptides may have the substitutions N434S or N434A.

[0221] 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 specific biological functions when bound to Fc receptors expressed on effector cells that mediate that 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 activation of B cells. Effector functions may vary 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.

[0222] In some embodiments, one or both Fc polypeptides present in the fusion protein described herein may be manipulated to include other modifications for heterodimerization (e.g., electrostatic manipulation of contact residues at the CH3-CH3 interface that are naturally charged, or modification of hydrophobic patches).

[0223] 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.

[0224] 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 within the CH2 domain, for example, 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 have the substitutions L234A and L235A (LALA).

[0225] Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, mutations at position 329 in which proline is substituted with a glycine, arginine, or 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. Additional exemplary substitutions, according to the EU numbering scheme, include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may exist (for example, according to the EU numbering scheme, L234A and L235A in the human IgG1 Fc region, L234A, L235A and P329G in the human IgG1 Fc region, S228P and L235E in the human IgG4 Fc region, L234A and G237A in the human IgG1 Fc region, L234A, L235A and G237A in the human IgG2 Fc region, V234A and G237A in the human IgG4 Fc region, L235A, G237A and E318A in the human IgG4 Fc region, and S228P and L236E in the human IgG4 Fc region). In some embodiments, one or both Fc polypeptides may have one or more amino acid substitutions that modulate ADCC, for example, substitutions at positions 298, 333 and / or 334 according to the EU numbering scheme.

[0226] Exemplary Fc polypeptides including additional mutations As a non-limiting example, one or both Fc polypeptides present in the fusion proteins described herein may include additional mutations, including knob mutations (e.g., T366W when numbered according to the EU numbering scheme), whole mutations (e.g., T366S, L368A, and Y407V when numbered according to the EU numbering scheme), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) when numbered according to the EU numbering scheme), and / or mutations that improve serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to the EU numbering scheme).

[0227] In some embodiments, the Fc polypeptide may have a knob mutation (e.g., T366W when numbered according to the EU numbering scheme) and may have at least 85% identity, at least 90% identity, or at least 95% identity with any one sequence of sequence numbers 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one sequence of sequence numbers 1, 4-90, and 124-148 may be modified to have a knob mutation.

[0228] In some embodiments, the Fc polypeptide may have a knob mutation (e.g., T366W when numbered according to the EU numbering scheme), a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) when numbered according to the EU numbering scheme), and may have at least 85% identity, at least 90% identity, or at least 95% identity with any one sequence of SEQ ID NOs: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one sequence of SEQ ID NOs: 1, 4-90, and 124-148 may be modified to have a knob mutation and a mutation that modulates effector function.

[0229] In some embodiments, the Fc polypeptide may have a knob mutation (e.g., T366W when numbered according to the EU numbering scheme), a mutation that improves serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to the EU numbering scheme), and may have at least 85% identity, at least 90% identity, or at least 95% identity with any one sequence of SEQ ID NOs: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one sequence of SEQ ID NOs: 1, 4-90, and 124-148 may be modified to have a knob mutation and a mutation that improves serum stability or serum half-life.

[0230] In some embodiments, the Fc polypeptide may have a knob mutation (e.g., T366W when numbered according to the EU numbering scheme), a mutation that modulates effector function (e.g., L234A, L235A and / or P329G (e.g., L234A and L235A) when numbered according to the EU numbering scheme), or a mutation that improves serum stability or serum half-life (e.g., (i) M252Y, S254T and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to the EU numbering scheme), and may have at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 1, 4-90 and 124-148. In some embodiments, an Fc polypeptide having any one sequence from sequence numbers 1, 4-90 and 124-148 may be modified to have a knob mutation, a mutation that modulates effector function, or a mutation that improves serum stability or serum half-life.

[0231] In some embodiments, the Fc polypeptide may have a whole mutation (e.g., T366S, L368A, and Y407V when numbered according to the EU numbering scheme) and may have at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one of sequence numbers 1, 4-90, and 124-148 may be modified to have a whole mutation.

[0232] In some embodiments, the Fc polypeptide may have hole mutations (e.g., T366S, L368A, and Y407V when numbered according to the EU numbering scheme), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) when numbered according to the EU numbering scheme), and may have at least 85% identity, at least 90% identity, or at least 95% identity with any one sequence of SEQ ID NOs: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one sequence of SEQ ID NOs: 1, 4-90, and 124-148 may be modified to have hole mutations and mutations that modulate effector function.

[0233] In some embodiments, the Fc polypeptide may have whole mutations (e.g., T366S, L368A, and Y407V when numbered according to the EU numbering scheme), mutations that improve serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to the EU numbering scheme), and may have at least 85% identity, at least 90% identity, or at least 95% identity with any one sequence of SEQ ID NOs: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one sequence of SEQ ID NOs: 1, 4-90, and 124-148 may be modified to have whole mutations and mutations that improve serum stability or serum half-life.

[0234] In some embodiments, the Fc polypeptide may have whole mutations (e.g., T366S, L368A, and Y407V when numbered according to the EU numbering scheme), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) when numbered according to the EU numbering scheme), mutations that improve serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to the EU numbering scheme), and may have at least 85% identity, at least 90% identity, or at least 95% identity with any one of sequence numbers 1, 4-90, and 124-148. In some embodiments, an Fc polypeptide having any one sequence from sequence numbers 1, 4-90 and 124-148 may be modified to have a whole mutation, a mutation that modulates effector function, or a mutation that improves serum stability or serum half-life.

[0235] Exemplary fusion protein containing enzyme for VII.ERT In some embodiments, the fusion protein described herein comprises an enzyme for enzyme replacement therapy (ERT), a first Fc polypeptide linked to an ERT enzyme variant or a catalytically active fragment thereof, and a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide. 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 ERT enzyme is IDS, SGSH, ASM, or GBA. In some embodiments, the first Fc polypeptide is a modified Fc polypeptide, and / or the second Fc polypeptide is a modified Fc polypeptide. In some embodiments, the second Fc polypeptide is a modified Fc polypeptide. In some embodiments, the modified Fc polypeptide includes one or more modifications that promote heterodimerization with the other Fc polypeptide. In some embodiments, the modified Fc polypeptide includes one or more modifications that reduce effector function. In some embodiments, the modified Fc polypeptide includes one or more modifications that extend its serum half-life. In some embodiments, the modified Fc polypeptide includes one or more modifications that result in binding to blood-brain barrier (BBB) ​​receptors, such as transferrin receptors (TfRs).

[0236] In another embodiment, the fusion protein described herein comprises a first polypeptide chain containing a modified Fc polypeptide that specifically binds to a BBB receptor, such as TfR, and a second polypeptide chain containing an Fc polypeptide that dimerizes with the modified Fc polypeptide to form an Fc dimer. The enzyme for ERT may be linked to either the first polypeptide chain or the second polypeptide chain. In some embodiments, the enzyme for ERT is IDS, SGSH, ASM, or GBA. In some embodiments, the enzyme for ERT is linked to the second polypeptide chain. In some embodiments, the protein of the present invention comprises two enzymes for ERT, each linked to one of their polypeptide chains. In some embodiments, the Fc polypeptide may be a BBB receptor-binding polypeptide that specifically binds to the same BBB receptor as the modified Fc polypeptide of the first polypeptide chain. In some embodiments, the Fc polypeptide does not specifically bind to the BBB receptor.

[0237] In some embodiments, the fusion protein described herein comprises a first polypeptide chain containing a modified Fc polypeptide that specifically binds to TfR, and a second polypeptide chain containing an Fc polypeptide, wherein the modified Fc polypeptide and the Fc polypeptide dimerize to form an Fc dimer. In some embodiments, the enzyme for ERT is IDS, SGSH, ASM, or GBA. In some embodiments, the enzyme for ERT is linked to the first polypeptide chain. In some embodiments, the enzyme for ERT is linked to the second polypeptide chain. In some embodiments, the Fc polypeptide does not specifically bind to a BBB receptor, such as TfR.

[0238] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a modified Fc polypeptide bound to TfR and including a T366W (knob) substitution, and a second polypeptide chain comprising an Fc polypeptide including T366S, L368A, and Y407V (hole) substitutions. In some embodiments, the modified Fc polypeptide and / or Fc polypeptide further comprises L234A and L235A (LALA) substitutions. In some embodiments, the modified Fc polypeptide and / or Fc polypeptide further comprises M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or Fc polypeptide further comprises L234A and L235A (LALA) substitutions, as well as M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or Fc polypeptide contains human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0239] In some embodiments, the modified Fc polypeptide includes knob mutations, LALA mutations and YTE mutations as defined in any one of sequence numbers 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209 and 216-221, and has at least 85% identity, at least 90% identity or at least 95% identity with each of these sequences, or includes any one of sequence numbers 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209 and 216-221. In some embodiments, the Fc polypeptide includes a hole mutation, an LALA mutation, and a YTE mutation as defined in any one of SEQ ID NOs: 101-104, and has at least 85% identity, at least 90% identity, or at least 95% identity with each of its sequences, or includes any one of the sequences from SEQ ID NOs: 101-104. In some embodiments, the modified Fc polypeptide includes any one of SEQ ID NOs: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, and the Fc polypeptide includes any one of SEQ ID NOs: 101-104. In some embodiments, the N-terminus of the modified Fc polypeptide and / or Fc polypeptide includes a portion of the IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 113). In some embodiments, the modified Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of sequence numbers 116, 228, and 229, or contains the sequence of any one of sequence numbers 116, 228, and 229.

[0240] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a modified Fc polypeptide that binds to TfR and includes T366S, L368A, and Y407V (hole) substitutions, and a second polypeptide chain comprising an Fc polypeptide including a T366W (knob) substitution. In some embodiments, the modified Fc polypeptide and / or Fc polypeptide further comprises L234A and L235A (LALA) substitutions. In some embodiments, the modified Fc polypeptide and / or Fc polypeptide further comprises M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or Fc polypeptide further comprises L234A and L235A (LALA) substitutions, as well as M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or Fc polypeptide contains human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0241] In some embodiments, the modified Fc polypeptide includes a hole mutation, an LALA mutation, and a YTE mutation as defined in any one of sequence numbers 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, having at least 85% identity, at least 90% identity, or at least 95% identity with each of the sequences, or includes any one of sequence numbers 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227. In some embodiments, the Fc polypeptide includes a knob mutation, an LALA mutation, and a YTE mutation as defined in any one of SEQ ID NOs: 109-112, having at least 85% identity, at least 90% identity, or at least 95% identity with each of its sequences, or includes any one of the sequences from SEQ ID NOs: 109-112. In some embodiments, the modified Fc polypeptide includes any one of SEQ ID NOs: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, and the Fc polypeptide includes any one of SEQ ID NOs: 109-112. In some embodiments, the N-terminus of the modified Fc polypeptide and / or Fc polypeptide includes a portion of the IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 113).

[0242] In some embodiments, the ERT enzyme present in the fusion protein described herein, e.g., IDS, SGSH, ASM, or GBA, contains an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NOs: 101-104, or is linked to a polypeptide chain containing the sequence of any one of SEQ ID NOs: 101-104 (e.g., as a fusion polypeptide). In some embodiments, the ERT enzyme, e.g., IDS, SGSH, ASM, or GBA, is linked to its Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or part thereof (e.g., DKTHTCPPCP, SEQ ID NO: 113). In some embodiments, the ERT enzyme contains an IDS sequence having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 114, 230, and 234, or contains the sequence of any one of SEQ ID NOs: 114, 230, and 234. In some embodiments, the IDS sequence linked to the Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with one of sequence numbers 115, 117, 231, 232, 235, and 236, or includes one of sequence numbers 115, 117, 231, 232, 235, and 236. In some embodiments, the enzyme for ERT includes an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with sequence number 120, or includes the sequence of sequence number 120. In some embodiments, the SGSH sequence linked to the Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with one of sequence numbers 149 and 150, or includes one of sequence numbers 149 and 150.In some embodiments, the fusion protein of the present invention comprises a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, or comprises any one of SEQ ID NOs: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221. In some embodiments, the N-terminus of the Fc polypeptide and / or modified Fc polypeptide comprises a portion of the IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 113). In some embodiments, the modified Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of sequence numbers 116, 228, and 229, or contains the sequence of any one of sequence numbers 116, 228, and 229.

[0243] In some embodiments, the fusion protein of the present invention comprises an IDS-Fc fusion polypeptide containing the sequence of SEQ ID NO: 115, and a modified Fc polypeptide containing either one of the sequences of SEQ ID NO: 205 and 228. In another embodiment, the fusion protein of the present invention comprises an IDS-Fc fusion polypeptide containing the sequence of SEQ ID NO: 115, and a modified Fc polypeptide containing either one of the sequences of SEQ ID NO: 169 and 229.

[0244] In some embodiments, the fusion protein of the present invention comprises an IDS-Fc fusion polypeptide containing the sequence of SEQ ID NO: 231, and a modified Fc polypeptide containing either one of the sequences of SEQ ID NOs: 205 and 228. In another embodiment, the fusion protein of the present invention comprises an IDS-Fc fusion polypeptide containing the sequence of SEQ ID NO: 231, and a modified Fc polypeptide containing either one of the sequences of SEQ ID NOs: 169 and 229.

[0245] In some embodiments, the fusion protein of the present invention comprises an IDS-Fc fusion polypeptide containing the sequence of SEQ ID NO: 235, and a modified Fc polypeptide containing either one of the sequences of SEQ ID NO: 205 and 228. In another embodiment, the fusion protein of the present invention comprises an IDS-Fc fusion polypeptide containing the sequence of SEQ ID NO: 235, and a modified Fc polypeptide containing either one of the sequences of SEQ ID NO: 169 and 229.

[0246] In some embodiments, the ERT enzyme present in the fusion protein described herein, e.g., IDS, SGSH, ASM, or GBA, contains an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 109-112, or is linked to a polypeptide chain containing the sequence of any one of SEQ ID NOs: 109-112 (e.g., as a fusion polypeptide). In some embodiments, the ERT enzyme, e.g., IDS, SGSH, ASM, or GBA, is linked to its Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or part thereof (e.g., DKTHTCPPCP, SEQ ID NO: 113). In some embodiments, the ERT enzyme contains an IDS sequence having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 114, 230, and 234, or contains the sequence of any one of SEQ ID NOs: 114, 230, and 234. In some embodiments, the IDS sequence linked to the Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 118, 233, and 237, or includes the sequence of any one of SEQ ID NOs: 118, 233, and 237. In some embodiments, the enzyme for ERT includes an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 120, or includes the sequence of SEQ ID NO: 120. In some embodiments, the SGSH sequence linked to the Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 152 and 153, or includes the sequence of any one of SEQ ID NOs: 152 and 153.In some embodiments, the fusion protein of the present invention comprises a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, or comprises any one of SEQ ID NOs: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227. In some embodiments, the N-terminus of the Fc polypeptide and / or modified Fc polypeptide comprises a portion of the IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 113).

[0247] In some embodiments, the enzymes for ERT present in the fusion proteins described herein, such as IDS, SGSH, ASM, or GBA, include a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, or (for example, as a fusion polypeptide) a polypeptide chain containing any one of SEQ ID NOs: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221. In some embodiments, the ERT enzyme, e.g., IDS, SGSH, ASM, or GBA, is linked to its modified Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or part thereof (e.g., DKTHTCPPCP, SEQ ID NO: 113). In some embodiments, the ERT enzyme includes an IDS sequence having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 114, 230, and 234, or includes a sequence of any one of SEQ ID NOs: 114, 230, and 234. In some embodiments, the ERT enzyme includes an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 120, or includes a sequence of SEQ ID NO: 120. In some embodiments, the SGSH sequence linked to the modified Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with one of SEQ ID NOs: 154 and 155, or contains the sequence of one of SEQ ID NOs: 154 and 155. In some embodiments, the fusion protein of the present invention contains an Fc polypeptide that has at least 85%, at least 90%, or at least 95% identity with one of SEQ ID NOs: 101-104, 149, and 150, or contains the sequence of one of SEQ ID NOs: 101-104, 149, and 150.In some embodiments, the N-terminus of the modified Fc polypeptide and / or Fc polypeptide includes a portion of the IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 113).

[0248] In some embodiments, the enzymes for ERT present in the fusion proteins described herein, such as IDS, SGSH, ASM, or GBA, include a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, or (for example, as a fusion polypeptide) a polypeptide chain containing any one of SEQ ID NOs: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227. In some embodiments, the ERT enzyme, e.g., IDS, SGSH, ASM, or GBA, is linked to its modified Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or part thereof (e.g., DKTHTCPPCP, SEQ ID NO: 113). In some embodiments, the ERT enzyme includes an IDS sequence having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 114, 230, and 234, or includes a sequence of any one of SEQ ID NOs: 114, 230, and 234. In some embodiments, the ERT enzyme includes an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 120, or includes a sequence of SEQ ID NO: 120. In some embodiments, the fusion protein of the present invention comprises an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 109-112, or comprises a sequence of any one of SEQ ID NOs: 109-112. In some embodiments, the fusion protein of the present invention comprises an SGSH sequence linked to an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NOs: 152 and 153, or comprising a sequence of any one of SEQ ID NOs: 152 and 153.In some embodiments, the N-terminus of the modified Fc polypeptide and / or Fc polypeptide includes a portion of the IgG1 hinge region (e.g., DKTHTCPPCP, SEQ ID NO: 113).

[0249] VIII. Measurement of binding reaction rate, affinity, brain concentration, and brain exposure 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 blood-brain barrier (BBB) ​​receptors, such as transferrin receptors (TfRs), in the range of 1 pM to 10 μM. In some embodiments, the affinity for TfRs is in the range of 1 nM to 5 μM or 10 nM to 1 μM. In some embodiments, the affinity for TfRs is in the range of about 50 nM to about 250 nM.

[0250] In some embodiments, the affinity of the TfR-binding polypeptide can be measured in a monovalent form. In other embodiments, the affinity can be measured in a bivalent form, for example, as a dimer containing a polypeptide-Fab fusion protein.

[0251] Methods for analyzing binding affinity, binding reaction rate, and cross-reactivity to BBB receptors, such as TfR, are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore® (GE Healthcare, Piscataway, NJ)), binding equilibrium exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), biolayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding reaction rate, and / or cross-reactivity. In some embodiments, binding equilibrium exclusion assays are used to determine binding affinity, binding reaction rate, and / or cross-reactivity. In some embodiments, a biolayer interference assay is used to determine binding affinity, binding reaction rate, and / or cross-reactivity.

[0252] A non-limiting example of a method for determining binding affinity (e.g., binding affinity to TfR) is described in Example 13 below, in which affinity was determined by surface plasmon resonance (SPR) using a Biacore® instrument. In this method, the target manipulated TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is captured on a sensor chip, and serial dilutions of TfR are injected onto the sensor chip at a predetermined flow rate (e.g., 30 μL / min) and temperature (e.g., room temperature). The sample is analyzed using predetermined binding and dissociation times (e.g., 45 seconds binding time and 180 seconds dissociation time), and then the sensor chip is regenerated. The binding response can be corrected by subtracting the binding response measurement from a control (e.g., using unrelated IgG at a similar density), and then the steady-state affinity can be determined by using software to fit the equilibrium response to concentration.

[0253] The concentrations of engineered TfR-binding polypeptides, TfR-binding peptides, TfR-binding antibodies, or drugs (e.g., drugs linked to the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody) in the brain and / or plasma can be measured, for example, using a human transferrin receptor (hTfR) knock-in mouse model. Using such a model, for example, the peak brain concentration (C) can be measured. max ) and / or measure and / or compare brain exposure, for example, C max It can be determined whether the level is elevated and / or whether brain exposure is prolonged. Human apical TfR (TfR ms / huThe preparation of the knock-in mouse model is described in Example 12 below. To prepare a suitable model, mice can be created using the CRISPR / Cas9 system that express the human Tfrc apical domain within the mouse Tfrc gene (e.g., in vivo expression under the control of an endogenous promoter). Specifically, Cas9, a single guide RNA, and donor DNA (e.g., a human apical domain coding sequence codon-optimized for expression in mice) can be introduced into mouse embryos (e.g., by pronuclear injection). Subsequently, these embryos can be transferred to pseudopregnant female mice. Male founders derived from the offspring of the female to whom the embryos were transplanted can be mated with wild-type females to obtain F1 heterozygous mice. Subsequently, homozygous mice can be obtained by mating the F1 generation heterozygous mice.

[0254] To evaluate the brain and / or plasma concentrations or exposure of an engineered TfR-binding polypeptide, TfR-binding peptide, TfR-binding antibody, or drug (e.g., a drug linked to the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody), the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody (e.g., linked to a drug) is used in a mouse model (e.g., TfR ms / hu) can be administered to the mouse. After a suitable period, a plasma sample can be taken from the mouse and then perfused with a suitable fluid for its vascular system. After perfusion, the brain (or a portion thereof) can be removed, homogenized, and lysed. Subsequently, the concentration of the drug in the plasma and / or brain lysate can be determined using standard methods understood by those skilled in the art. As a non-limiting example, the concentration can be measured using an ELISA-based assay, such as that described in Example 3 below. Briefly, a sandwich ELISA can be used to quantify the concentration of a drug, engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody (e.g., in plasma or lysate). A capture antibody (e.g., anti-Fc capture antibody) can be coated onto a plate (e.g., a 384-well MaxiSorp® plate) at the desired concentration (e.g., about 3 μg / mL). The plate is blocked (e.g., with 5% BSA) and then incubated with diluted plasma (e.g., 1:1,000 or 1:10,000). Next, the detection antibody is added at the desired concentration (e.g., approximately 0.5 μg / mL), followed by the addition of a secondary antibody (such as an anti-goat HRP antibody). Subsequently, the plate is color-developed (e.g., using a TMB substrate), stopped (e.g., with sulfuric acid), and the absorbance at an appropriate wavelength (e.g., 450 nm) is measured using a plate reader (e.g., a BioTek plate reader). A calibration curve can be created using an appropriate (e.g., 4-fold) dilution series and fitted using an algorithm such as a four-parameter logistic regression.

[0255] Calibration curves can be created by administering various doses to the knock-in mouse model. By administering to the knock-in mouse model drugs linked to differently manipulated TfR-binding polypeptides, TfR-binding peptides, or TfR-binding antibodies (e.g., those with different TfR affinities), or drugs linked to reference polypeptides or reference proteins (e.g., those with a weaker affinity for TfR than the polypeptide or protein under test), the manipulated TfR-binding polypeptides, TfR-binding peptides, or TfR-binding antibodies of the present invention can be used to determine the brain exposure to the drug and / or the C15 of the drug in the brain.max It is possible to compare the effects on the values.

[0256] ERT enzyme linked to IX.Fc polypeptide In some embodiments, the fusion protein described herein comprises two Fc polypeptides as described herein, one or both of which may further comprise part or all of a hinge region. The hinge region may be derived from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is an IgG hinge region, such as the IgG1 hinge region, e.g., the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 95) or a portion thereof (e.g., DKTHTCPPCP, SEQ ID NO: 113). In some embodiments, the hinge region is located in the N-terminal region of the Fc polypeptide.

[0257] In some embodiments, the Fc polypeptide is linked to the ERT enzyme by a linker, such as a peptide linker. In some embodiments, the Fc polypeptide is linked to the ERT enzyme by a peptide bond or a peptide linker, such as a fusion polypeptide. The peptide linker may be configured to rotate the ERT enzyme with respect to the Fc polypeptide linked to the ERT enzyme and / or to be 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 containing amino acids such as Gly, Asn, Ser, Thr, Ala, etc. Such a linker is designed using known parameters and may be of any length and may contain any number of repeating units of any length (e.g., repeating units of Gly and Ser residues). For example, the linker may have a repeating structure such as two, three, four, five or more Gly4-Ser(SEQ ID NO: 239) repeats, or a single Gly4-Ser(SEQ ID NO: 239). In some embodiments, the peptide linker may include a protease cleavage site that can be cleaved by enzymes present in the central nervous system, for example.

[0258] In some embodiments, the ERT enzyme is linked to the N-terminus of its Fc polypeptide by, for example, a Gly4-Ser linker (SEQ ID NO: 239) or a (Gly4-Ser)2 linker (SEQ ID NO: 240). In some embodiments, the Fc polypeptide may contain a hinge sequence or a portion of a hinge sequence at the N-terminus linked to the linker or directly linked to the ERT enzyme.

[0259] In some embodiments, the ERT enzyme is linked to the C-terminus of its Fc polypeptide by, for example, a Gly4-Ser linker (SEQ ID NO: 239) or a (Gly4-Ser)2 linker (SEQ ID NO: 240). In some embodiments, the C-terminus of its Fc polypeptide is directly linked to the ERT enzyme.

[0260] In some embodiments, the enzyme for ERT is linked to its Fc polypeptide by a chemical crosslinking agent. Such conjugates can be prepared using well-known chemical crosslinking reagents and protocols. For example, there are many chemical crosslinking agents known to those skilled in the art that are useful for crosslinking polypeptides with target drugs. For example, these 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 protein conjugation, thereby reducing the generation of unwanted by-reactants such as homoprotein polymers. Examples include 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 succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). A wide variety of heterobifunctional crosslinking agents are known in the art, including 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4-succinimidyloxycarbonyl-α-methyl-α-(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 improved water solubility. In addition, these 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 many other crosslinking agents, including homobifunctional crosslinking agents and photoreactive crosslinking agents.Disuccinimidyl suberate (DSS), bismaleimide hexane (BMH), and dimethylpimerimidate 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.

[0261] X. Evaluation of protein activity The activity of fusion proteins described herein, for example, fusion proteins containing ERT enzymes such as IDS, SGSH, ASM, or GBA, can be evaluated using a variety of assays, including assays that measure activity in vitro using artificial substrates (such as those described in the Examples section). An exemplary protocol for measuring IDS activity in vitro is shown in Example 2. An exemplary protocol for measuring ASM activity in vitro is shown in Example 5. Exemplary protocols for measuring SGSH activity in vitro are shown in Examples 7 and 8.

[0262] In some embodiments, IDS activity is assessed by assaying samples such as cell samples, tissue samples, or fluid samples (e.g., CSF or urine) for the amount of heparan sulfate and dermatan sulfate, which are glycosaminoglycans (GAGs) that accumulate due to IDS deficiency. The amounts of heparan sulfate and dermatan sulfate are determined by digesting the GAGs present in the sample with heparinase and chondroitinase. Subsequently, the resulting disaccharides can be assayed by mass spectrometry (e.g., LC-MS / MS). Samples with high levels of heparan sulfate and dermatan sulfate accumulation will have increased amounts of disaccharides derived from heparan sulfate and dermatan sulfate. Therefore, the level of disaccharides is inversely proportional to IDS enzyme activity.

[0263] Mass spectrometry (e.g., LC-MS / MS) assays can be performed on any sample in which GAG accumulates, including cell samples, tissue samples, and fluid samples. Such samples can be evaluated to monitor the activity of IDS-containing proteins described herein, for example, administered in vitro to cells, or, in some embodiments, in vivo to a subject. The subject may be an animal such as a rodent, e.g., a mouse, or a non-human primate. In some embodiments, the subject is a human patient, such as a Hunter syndrome patient, receiving treatment with IDS therapy, in which case the assay is used to monitor IDS activity in the patient. In some embodiments, the human patient is receiving treatment with a fusion protein described herein.

[0264] For cellular or tissue samples, such as cells, the assay involves disrupting the cells and disrupting and opening the microvesicles. Disrupting the cells or disrupting and opening the microvesicles may be done by freeze-thaw and / or sonication to obtain an extract containing GAG (e.g., a cell extract). Subsequently, the GAG ​​is treated with heparinase (e.g., any of those described herein) and chondroitinase to degrade the heparan sulfate and dermatan sulfate GAG. After digestion, a supernatant containing GAG disaccharides is obtained, and the disaccharide products are analyzed by mass spectrometry (e.g., LC-MS / MS). An exemplary protocol is shown in Example 2.

[0265] In some embodiments, the cell sample to be assayed for IDS activity is washed and frozen. The cell pellet is sonicated in a disaccharide digestion buffer. Subsequently, the desired amount of total protein obtained from the sonicated sample is added to a digestion buffer containing heparinase I, heparinase II, heparinase III, and chondroitinase B (chondroitinase B is specific to dermatan sulfate). After digestion at 30°C for approximately 3 hours, for example, the enzymes are inactivated by EDTA and boiling. Subsequently, the sample is centrifuged, for example at 16,000 × G, the supernatant is transferred to a centrifuge filter, and centrifuged again at approximately 14,000 × G. Subsequently, the disaccharides are resuspended in a 1:1 (v / v ratio) mixture of assay buffer:acetonitrile and further analyzed by liquid chromatography combined with electrospray mass spectrometry, for example, as described in Example 2. Based on the retention time compared to the retention time of a commercially available reference standard, the disaccharide products derived from GAGs can be identified. Examples of heparan sulfate-derived disaccharides include D0S0 and D2S0 (named according to Lawrence et al., Nat. Methods, 5:291-292 (2008)).

[0266] In another embodiment, SGSH activity is assessed by assaying a sample, such as a cell or tissue sample, for the amount of heparan sulfate glycosaminoglycans (GAGs) that accumulate due to SGSH deficiency. The amount of heparan sulfate is determined by digesting the GAGs present in the sample with heparinase (e.g., any of those described herein). The resulting disaccharide can then be assayed by mass spectrometry (e.g., LC-MS / MS). Samples with high levels of heparan sulfate accumulation will have an increased amount of disaccharides derived from heparan sulfate. Therefore, the level of disaccharides is inversely proportional to SGSH enzyme activity.

[0267] Mass spectrometry (e.g., LC-MS / MS) assays can be performed on any sample in which GAG accumulates, including cell samples, tissue samples, and fluid samples. Such samples can be evaluated to monitor the activity of the SGSH-containing proteins described herein, for example, administered in vitro to cells, or, in some embodiments, in vivo to a subject. The subject may be an animal such as a rodent, e.g., a mouse, or a non-human primate. In some embodiments, the subject is a human patient, such as a patient with Sanfilippo syndrome A, being treated with SGSH therapy, in which case the assay is used to monitor SGSH activity in the patient. In some embodiments, the human patient is being treated with the fusion protein described herein.

[0268] For cellular or tissue samples, such as cells, the assay involves disrupting the cells and / or disrupting and opening the microvesicles. Disrupting the cells or disrupting and opening the microvesicles may be done by freeze-thaw and / or sonication to obtain an extract containing GAG (e.g., a cell extract). Subsequently, the GAG ​​is treated with heparinase (e.g., any of those described herein) to decompose the heparan sulfate GAG. After digestion, a supernatant containing the GAG ​​disaccharide is obtained, and the disaccharide product is analyzed by mass spectrometry (e.g., LC-MS / MS). An exemplary protocol is shown in Example 7.

[0269] In some embodiments, the cell sample to be assayed for SGSH activity is washed and frozen. The cell pellet is sonicated in a disaccharide digestion buffer. Subsequently, the desired amount of total protein obtained from the sonicated sample is added to a digestion buffer containing heparinase I, heparinase II, and / or heparinase III. After digestion at 30°C for approximately 3 hours, for example, the enzymes are inactivated by EDTA and boiling. Subsequently, the sample is centrifuged, for example at 16,000 × G, the supernatant is transferred to a centrifuge filter, and centrifuged again at approximately 14,000 × G. Subsequently, the disaccharides are resuspended in a 1:1 (v / v ratio) mixture of assay buffer:acetonitrile and further analyzed by liquid chromatography combined with electrospray mass spectrometry, for example, as described in Example 7. Based on the retention time compared to the retention time of a commercially available reference standard, the disaccharide products derived from GAG can be identified. Examples of heparan sulfate-derived disaccharides include D0S0 and D2S0 (named according to Lawrence et al., Nat. Methods, 5:291-292 (2008)).

[0270] In some embodiments, tissue samples are evaluated. Tissue samples can be evaluated using assays similar to those described above, except that they typically involve performing multiple freeze-thaw cycles, e.g., two, three, four, five, or more, prior to a sonication step that breaks down and opens the microvesicles.

[0271] Samples that can be evaluated by the assays described herein include the brain, liver, kidneys, lungs, spleen, plasma, serum, cerebrospinal fluid (CSF), and urine. In some embodiments, CSF samples taken from patients administered with the enzyme-Fc fusion proteins described herein (e.g., IDS-Fc or SGSH-Fc fusion proteins) can be evaluated.

[0272] XI. Nucleic acids, vectors, and host cells Polypeptide chains contained in fusion proteins as 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 Fc polypeptides as described herein, and host cells into which the nucleic acids have been introduced, to be used for replicating and / or expressing the polypeptide-coding nucleic acid. In some embodiments, the host cells are eukaryotic cells, such as human cells.

[0273] In another embodiment, a polynucleotide comprising a nucleotide sequence encoding a polypeptide chain described herein is provided. 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.

[0274] In some embodiments, the polynucleotide is contained within a nucleic acid construct. 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.

[0275] In some embodiments, the polynucleotide is functionally ligated to one or more regulatory nucleotide sequences within the expression construct. In a series of embodiments, the nucleic acid expression construct is adapted for use as a surface expression library. In some embodiments, the library is adapted for surface expression in yeast. In some embodiments, the library is adapted for surface expression in phages. In another series of embodiments, the nucleic acid expression construct is adapted to express polypeptides in a system that allows for the isolation of polypeptides 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.

[0276] Plasmids and other vectors can be used as expression vehicles for producing recombinant polypeptides. For example, suitable vectors include pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as E. coli. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for eukaryotic cell transfection. Alternatively, viral derivatives 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. Further expression systems include adenovirus expression systems, adeno-associated virus expression systems, and expression systems for other viruses.

[0277] The vector can be introduced into any suitable host cell. In some embodiments, the host cell, for example, a bacterial cell or a yeast cell, may be adapted to be used as a surface expression library. In some cells, the vector is expressed in the host cell to express a relatively large amount of polypeptide. Examples of such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cell is a mammalian cell 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.

[0278] Host cells transfected with expression vectors encoding one or more Fc polypeptide chains as described herein can be cultured under conditions suitable for expressing one or more polypeptides. The polypeptides can be secreted and isolated from a mixture of cells and culture medium containing the polypeptides. Alternatively, the polypeptides may be retained in the cytoplasm or membrane fraction, and the cells may be recovered and lysed, and the polypeptides isolated using a method of choice.

[0279] XII. Treatment method LSD may be treated using therapeutically modified TfR-binding polypeptides, TfR-binding peptides, or fusion proteins or agents (e.g., therapeutic agents) linked to TfR-binding antibodies as described herein. In some embodiments, patients with Hunter syndrome are treated with modified TfR-binding polypeptides, TfR-binding peptides, or fusion proteins or agents linked to TfR-binding antibodies, including IDS. In some embodiments, patients with Sanfilippo syndrome A are treated with modified TfR-binding polypeptides, TfR-binding peptides, or fusion proteins or agents linked to TfR-binding antibodies, including SGSH. In some embodiments, patients with Niemann-Pick disease are treated with modified TfR-binding polypeptides, TfR-binding peptides, or fusion proteins or agents linked to TfR-binding antibodies, including ASM. In some embodiments, patients with Gaucher disease or Parkinson's disease are treated with modified TfR-binding polypeptides, TfR-binding peptides, or fusion proteins or agents linked to TfR-binding antibodies, including GBA.

[0280] Fusion proteins described herein, which include an ERT enzyme such as IDS, SGSH, ASM, or GBA, are administered to the subject in a therapeutically effective amount or dose. Exemplary daily doses may range from approximately 0.01 mg / kg to approximately 500 mg / kg, approximately 0.1 mg / kg to approximately 200 mg / kg, approximately 1 mg / kg to approximately 100 mg / kg, or approximately 10 mg / kg to approximately 50 mg / kg. In some embodiments, the protein has an enzymatic activity of at least about 500 units (U) / mg, about 1,000 U / mg, or at least about 1,500 U / mg, 2,000 U / mg, 2,500 U / mg, 3,000 U / mg, 3,500 U / mg, 4,000 U / mg, 4,500 U / mg, 5,000 U / mg, 6,000 U / mg, 7,000 U / mg, 8,000 U / mg, 9,000 U / mg, or 10,000 U / mg. In some embodiments, the enzyme activity is at least about 11,000 U / mg, or at least about 12,000 U / mg, 13,000 U / mg, 14,000 U / mg, 15,000 U / mg, 16,000 U / mg, 17,000 U / mg, 18,000 U / mg, 19,000 U / mg, 20,000 U / mg, 25,000 U / mg, 30,000 U / mg, 35,000 U / mg, 40,000 U / mg, 45,000 U / mg, or 50,000 U / mg, or any value within the range of about 500 U / mg to about 50,000 U / mg. However, the dosage may vary depending on several factors, including the chosen route of administration, the composition of the formula, the patient's response, the severity of the condition, the patient's body weight, and the prescribing physician's judgment. The dosage can be increased or decreased over time as needed by the individual patient. In some embodiments, the patient is initially given a small dose, which is then increased to an effective dose that the patient can tolerate. Determining the effective dose is well within the capabilities of those skilled in the art.

[0281] In various embodiments, the fusion proteins described herein are administered parenterally. In some embodiments, the proteins are administered intravenously. Intravenous administration may be by infusion over a period of about 10 to about 30 minutes, or over a period of at least 1, 2, or 3 hours. In some embodiments, the proteins are administered as an intravenous bolus. A combination of infusion and bolus administration may also be used.

[0282] In some parenteral embodiments, the fusion protein or drug (e.g., therapeutic agent) linked to the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, the protein or drug linked to the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is administered intradermally or intramuscularly. In some embodiments, the protein or drug linked to the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is administered intrathecally (e.g., epidural or intraventricular).

[0283] In another embodiment, a fusion protein or drug (e.g., a therapeutic agent) linked to an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody may be administered orally, pulmonaryly, intranasally, intraocularly, or topically. Pulmonary administration may also be employed, for example, by using a formulation having an inhaler or nebulizer and an aerosolizing agent.

[0284] XIII. Protein Supplementation Methods In another embodiment, the present invention provides a method for transporting a drug (e.g., a drug useful for treating lysosomal storage disorders (LSDs)) across the mammalian blood-brain barrier (BBB). In some embodiments, the method involves exposing the BBB to a polypeptide or protein that binds (e.g., specifically binds) to the transferrin receptor (TfR) with an affinity of about 50 nM to about 250 nM. In some embodiments, the polypeptide or protein is ligated to the drug and transports the ligated drug across the BBB. In some embodiments, the highest concentration (C) of the drug in the mammalian brain is max ) improves (for example, increases).

[0285] In another embodiment, the present invention provides a method for treating LSD. In some embodiments, the method involves administering a polypeptide or protein that binds to TfR (e.g., specifically) with an affinity of about 50 nM to about 250 nM to a mammal. In some embodiments, the polypeptide or protein is linked to a drug for treating LSD, thereby exposing the brain of the mammal to the drug.

[0286] In some embodiments, the polypeptide or protein binds to TfR (e.g., specifically) with an affinity of about 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, or 250 nM. In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 100 nM to about 200 nM or about 110 nM to about 150 nM.

[0287] In some embodiments, the polypeptide or protein (e.g., the one linked to the drug) has a weaker affinity for TfR compared to a drug linked to a reference polypeptide or reference protein that specifically binds to TfR.max To improve (e.g., increase) something.

[0288] In some embodiments, the drug is detected in the brain. max Compared to drugs linked to a reference polypeptide or reference protein (e.g., one that binds to TfR with weaker affinity), the drug is improved (e.g., increased) by at least approximately 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 times or more.

[0289] In some embodiments, the mammalian brain is exposed to a therapeutically effective concentration of the drug (e.g., a concentration sufficient to treat one or more signs or symptoms of LSD) for a shorter period than the drug linked to a reference polypeptide or reference protein. In some embodiments, the brain exposure period is shortened by at least about 5%, 10%, 25%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, or 98%.

[0290] In some embodiments, brain exposure is quantified by plotting brain exposure (e.g., drug concentration in the brain) as a function of time and calculating the area under the curve (AUC). A decrease in AUC can represent a decrease in brain exposure or a reduction in brain exposure time. In some embodiments, the time of brain exposure to a drug (e.g., a therapeutically effective concentration of the drug) is reduced.

[0291] In some embodiments, the reference polypeptide or reference protein binds to TfR (e.g., specifically) with an affinity of about 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, or 600 nM, or weaker than that. In some embodiments, the reference polypeptide or reference protein binds to TfR with an affinity of about 600 nM, or weaker than that.

[0292] In some embodiments, the mammal is a primate (e.g., a human). In some embodiments, the human is a patient in need of treatment for LSD. In some embodiments, the patient has one or more signs or symptoms of LSD.

[0293] In some embodiments, the polypeptide or protein binds to a primate TfR (e.g., specifically). In some embodiments, the primate TfR is a human TfR. In some embodiments, the polypeptide or protein binds to the TfR apical domain.

[0294] In some embodiments, the agent (e.g., a therapeutic agent) is linked to an engineered TfR-binding polypeptide. In some embodiments, the engineered TfR-binding polypeptide includes a CH3 or CH2 domain having a modification that specifically links the polypeptide to TfR. Non-limiting examples of suitable engineered TfR-binding polypeptides are described herein. In some embodiments, the agent is linked to an engineered TfR-binding polypeptide listed in Table 4 or Table 5. In some embodiments, the agent is linked to an engineered TfR-binding polypeptide selected from the group consisting of CH3C.35.20.2, CH3C.35.23.2, CH3C.35.23.5, CH3C.35.21.17, and CH3C.35.21.17.2.

[0295] In some embodiments, the drug (e.g., a therapeutic agent) is linked to a TfR-binding peptide. In some embodiments, the TfR-binding peptide is a short peptide having a length of approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Methods for preparing, screening, and identifying suitable peptides (i.e., peptides that bind to TfR with a desired range of affinity) are known in the art. For example, suitable peptides can be identified using a phage display method that alternates between negative and positive selection for several rounds. This strategy is described, for example, in Lee et al., Eur. J. Biochem., 268:2004-2012 (2001), which is incorporated herein by reference in its entirety for all purposes.

[0296] In some embodiments, the drug (e.g., a therapeutic agent) is linked to a TfR-conjugating antibody. Non-limiting examples of suitable TfR-conjugating antibodies include the OX26 anti-TfR antibodies disclosed in Thom et al., Mol. Pharm., 15(4):1420-1431 (2018) (i.e., OX26 anti-TfR antibodies with affinity values ​​of approximately 76 nM, 108 nM, and 174 nM). In some embodiments, the drug is linked to a protein containing an antibody variable region that specifically binds to TfR. In some cases, the protein includes Fab or scFv.

[0297] In some embodiments, the agent (e.g., therapeutic agent) is a protein (e.g., enzyme). In some embodiments, the agent is a protein replacement therapy agent. In some embodiments, the agent is a protein or enzyme that is deficient (e.g., expressed or absent) in mammalian cells or tissues (e.g., nerve cells or tissues). In some embodiments, the agent is a protein or enzyme that is endogenous or expressed in normal, healthy mammalian cells or tissues (e.g., nerve cells or tissues) but is deficient in mammals receiving LSD treatment (e.g., in the corresponding cells or tissues).

[0298] In some embodiments, the protein replacement therapy agent is an enzyme. For the treatment of various LSDs, any number of agents (e.g., protein replacement therapy agents such as enzymes) can be linked to a polypeptide or protein (e.g., one that binds to TfR). In some embodiments, the agent is an enzyme that, when linked to the polypeptide or protein, significantly reduces the accumulation of toxic metabolites in the brain of mammals with LSD compared to when the enzyme is linked to a reference polypeptide or reference protein. In some embodiments, the enzyme is iduronate-2-sulfatase (IDS), and the LSD is Hunter syndrome. In some cases, the toxic metabolites include disaccharides derived from heparan sulfate and / or disaccharides derived from dermatan sulfate. In some embodiments, the enzyme is N-sulfoglucosamine sulfohydrolase (SGSH), and the LSD is Sanfilippo syndrome. In some embodiments, the enzyme is acid sphingomyelinase (ASM), and the LSD is Niemann-Pick disease. In some embodiments, the enzyme is β-glucocerebrosidase (GBA), and the LSD is Gaucher disease.

[0299] In some embodiments, the agent (e.g., a therapeutic agent) includes an antibody variable region. In some embodiments, the agent includes an antibody fragment. In some embodiments, the agent includes a Fab or scFv. In some embodiments, the agent does not include an antibody variable region. In some cases, the agent does not include anti-β-secretase 1 (BACE1)Fab.

[0300] Additional embodiments and linker Polypeptides (e.g., modified CH3 or CH2 domain polypeptides as further described herein) may be ligated to another domain in the Fc region. In some embodiments, a modified CH3 domain polypeptide is ligated to a CH2 domain (which may be a natural CH2 domain or a variant CH2 domain), typically at the C-terminus of that CH2 domain. In some embodiments, a modified CH2 domain polypeptide is ligated to a CH3 domain (which may be a natural CH3 domain or a CH3 variant domain), typically at the N-terminus of that CH3 domain. In some embodiments, a polypeptide comprising a modified CH2 domain ligated to a CH3 domain, or a polypeptide comprising a modified CH3 domain ligated to a CH2 domain, further comprises part or all of the hinge region of the antibody, resulting in a form in which the modified CH3 domain polypeptide or modified CH2 domain polypeptide is part of the Fc region having part or all of the hinge region. The hinge region may originate from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is the IgG hinge region (such as the IgG1 hinge region), for example, the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 95).

[0301] In some embodiments, the manipulated TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is fused to peptides or proteins useful for protein purification, such as polyhistidine, epitope tags (e.g., FLAG, c-Myc, hemagglutinin tags, etc.), glutathione S transferase (GST), thioredoxin, protein A, protein G, or maltose-binding protein (MBP). In some cases, the peptide or protein to which the manipulated TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is fused may contain protease cleavage sites, such as factor Xa or thrombin cleavage sites.

[0302] In the methods of this disclosure, a drug (e.g., a therapeutic agent) is linked to a polypeptide or protein (e.g., an engineered TfR-binding polypeptide, a TfR-binding peptide, or a TfR-binding antibody). The linker can be any linker suitable for linking the drug to the polypeptide or protein. In some embodiments, the linkage is enzymatically cleavable. In certain embodiments, the linkage is cleavable by an enzyme present in the central nervous system.

[0303] In some embodiments, the linker is a peptide linker. The peptide linker may be configured to rotate the drug (e.g., a therapeutic agent) and the polypeptide or protein relative to each other and / or to be resistant to digestion by proteases. In some embodiments, the linker may be a flexible linker containing amino acids such as Gly, Asn, Ser, Thr, Ala, etc. Such a linker is designed using known parameters. For example, the linker may have repeats such as Gly-Ser repeats.

[0304] In various embodiments, the linking of the drug (e.g., a therapeutic agent) to the polypeptide or protein (e.g., a manipulated TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody) can be carried out using well-known chemical crosslinking reagents and protocols. For example, there are many chemical crosslinkers known to those skilled in the art that are useful for crosslinking the polypeptide or protein with the drug of interest. For example, such crosslinkers are heterobifunctional crosslinkers that can be used to link molecules stepwise. Heterobifunctional crosslinkers allow for the design of more specific coupling methods for conjugating proteins, thereby reducing the generation of unwanted by-reactants such as homoprotein polymers.

[0305] The drug (e.g., a therapeutic agent) may be ligated to the N-terminal or C-terminal region of the polypeptide or protein, or to any region of the polypeptide or protein (e.g., a modified TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody), provided that the drug does not impair the binding of the polypeptide or protein to the transferrin receptor.

[0306] XIV. Pharmaceutical Compositions and Kits In another embodiment, pharmaceutical compositions and kits comprising the fusion proteins described herein are provided.

[0307] Pharmaceutical composition Instructions for preparing formulations for use in this disclosure can be found in any number of handbooks for pharmaceutical preparations and formulations known to those skilled in the art.

[0308] In some embodiments, the pharmaceutical composition comprises a fusion protein as 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 that is physiologically compatible and does not interfere with or otherwise inhibit the activity of the activator.

[0309] In some embodiments, the carrier is suitable for intravenous, intrathecal, ocular, intracerebroventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. A pharmaceutically acceptable carrier may include, for example, one or more physiologically acceptable compounds that act to stabilize the composition of the present invention or to increase or decrease the absorption of the polypeptide of the present invention. Examples of physiologically acceptable compounds include carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; compositions that reduce the removal or hydrolysis of activators; excipients; or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are also available in the art.

[0310] The pharmaceutical compositions described herein can be prepared, for example, by conventional mixing processes, dissolution processes, granulation processes, sugar coating processes, emulsification processes, encapsulation processes, inoculation processes, or freeze-drying processes. The methods and excipients described below are illustrative.

[0311] For oral administration, the fusion proteins described herein can be formulated by combining them with pharmaceutically acceptable carriers known in the art. With such carriers, the fusion proteins of the present invention can be formulated as tablets, pills, sugar-coated tablets, capsules, emulsions, lipophilic suspensions, hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc., for oral administration by patients under treatment. Pharmaceutical preparations for oral use can be obtained by mixing the fusion protein of the present invention with a solid excipient, optionally grinding the resulting mixture, and, if desired, adding a suitable adjuvant, and then processing the granular mixture to obtain a tablet or core tablet of a sugar-coated tablet. Suitable excipients include, for example, fillers such as sugars containing lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts (such as sodium alginate) may be added.

[0312] As disclosed above, fusion proteins as described herein can be formulated for parenteral administration by injection, e.g., bolus injection, or continuous infusion. For injection, the fusion proteins of the present invention can be formulated into preparations by dissolving, suspending, or emulsifying the protein in an aqueous or non-aqueous solvent such as vegetable oil or other similar oil, synthetic fatty acid glycerides, higher fatty acids, or propylene glycol esters, and optionally with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the fusion proteins of the present invention can be formulated in an aqueous solution such as a physiologically compatible buffer, non-limiting examples of such aqueous solutions include Hanks' solution, Ringer's solution, and saline buffer. Formulations for injection can be in unit dosage forms, e.g., in ampoules, or in multi-dose containers to which preservatives are added. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0313] In some embodiments, fusion proteins as described herein are prepared to be delivered in sustained-release formulations, controlled-release formulations, sustained-release formulations, time-release formulations, or delayed-release formulations, for example, in a semipermeable matrix of a solid hydrophobic polymer containing an activator. Various types of sustained-release substances are established and are well known to those skilled in the art. Examples of sustained-release formulations include film-coated tablets, multiply-particle or pellet systems, matrix technologies using hydrophilic or lipophilic substances, and wax-based tablets containing pore-forming excipients. Typically, sustained-release formulations can be prepared using natural or synthetic polymers, such as polymeric vinylpyrrolidones like polyvinylpyrrolidone, carboxyvinyl hydrophilic polymers, hydrophobic and / or hydrophilic hydrocolloids such as methylcellulose, ethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose, and carboxypolymethylene.

[0314] Typically, pharmaceutical compositions for in vivo administration are sterile. Sterilization can be carried out according to methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation sterilization.

[0315] The dosages and desired drug concentrations of the pharmaceutical compositions described herein may vary depending on the specific intended use. Preferred dosages are also described in Section XII above.

[0316] kit In some embodiments, kits are provided for use in the treatment of LSD, for example, Hunter syndrome, Sanfilippo syndrome A, Niemann-Pick disease, Gaucher disease, or Parkinson's disease, and each kit includes a fusion protein as described herein.

[0317] 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 used for the treatment of neurological symptoms of LSD, including a fusion protein as described herein. In some embodiments, the kit further comprises instructional materials including procedures (i.e., protocols) for carrying out the methods described herein (e.g., instructions for using the kit to administer a fusion protein containing an ERT enzyme across the blood-brain barrier). Such instructional materials typically include, but are not limited to, written or printed materials. The disclosure envisions any medium in which such instructions can be stored and communicated to end users. Such mediums include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips) and optical media (e.g., CD-ROMs). Such media may include addresses of internet sites providing such instructional materials. [Examples]

[0318] XV. Example The present disclosure will be described in more detail by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present disclosure in any way. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to produce essentially the same results. With regard to the numerical values ​​used (e.g., quantities, temperatures, etc.), efforts have been made to ensure accuracy, but some degree of experimental error and deviation may exist. In carrying out the present disclosure, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology will be used within the scope of the art of the present art. Such techniques are well described in the literature. In addition, it will be obvious to those skilled in the art that procedures applied to a particular library can also be applied to other libraries described herein.

[0319] Example 1. Construction of a fusion protein containing iduronate-2-sulfatase (IDS) Design and cloning We designed IDS-Fc fusion proteins comprising (i) a fusion polypeptide in which a mature human IDS enzyme is fused to a human IgG1 fragment containing an Fc region ("IDS-Fc fusion polypeptide"), and (ii) a modified human IgG1 fragment containing a mutation in the Fc region that leads to binding to the transferrin receptor (TfR) ("modified Fc polypeptide"). Specifically, we created IDS-Fc fusion polypeptides by fusioning the IDS fragment to either the N-terminus or C-terminus of the human IgG1 Fc region. In some cases, a linker was placed between the IDS and the IgG1 fragment to reduce any steric hindrance between the two fragments. In all constructs, to promote secretion, a signal peptide derived from amino acids at positions 1-20 of the κ chain V-III (UniProtKB ID-P01661) was inserted upstream of the fusion site, truncating the IDS to consist of amino acids S26-P550 (UniProtKB ID-P22304). The human IgG1 Fc region fragment used corresponds to amino acids D104–K330 of the UniProtKB ID P01857 sequence (positions 221–447 in EU numbering, including 10 hinge amino acids (positions 221–230)). In some embodiments, a second Fc polypeptide derived from human IgG1 residues D104–K330 but without fusion to IDS was cotransfected with the IDS-Fc fusion polypeptide to create a heterodimer fusion protein ("monozyme") containing one IDS enzyme. In some constructs, additional mutations were added to the IgG1 fragment to promote heterodimerization of the two Fc regions. Control IDS-Fc fusion proteins without mutations resulting in TfR binding were designed and constructed in the same manner as these proteins, except that they did not contain mutations resulting in TfR binding. As an additional control, we prepared an IDS (amino acid S26-P550) containing a C-terminal hexahistidine tag (SEQ ID NO: 241) to facilitate detection and purification.

[0320] The TfR-binding IDS-Fc fusion protein used in the examples is a dimer formed from an IDS-Fc fusion polypeptide and a modified Fc polypeptide that binds to TfR. In the dimer, where the IDS enzyme is linked to the N-terminus of the Fc region, the IDS-Fc fusion polypeptide may have one of the sequences of SEQ ID NOs. 115, 231, and 235. In these sequences, the IDS sequence is underlined and contains a cysteine ​​at position 59 (double underlined), which is modified to formylglycine. The IDS was linked to the Fc polypeptide by a linker called GGGGS (SEQ ID NOs. 239). The N-terminus of the Fc polypeptide contained a portion of the IgG1 hinge region (DKTHTCPPCP, SEQ ID NOs. 113). Its CH2 domain sequence begins at position 541 in SEQ ID NOs. 115, 231, and 235.

[0321] The IDS-Fc fusion protein ETV:IDS35.21 used in the examples is a dimer formed by an IDS-Fc fusion polypeptide having one of the sequences of SEQ ID NOs: 115, 231, and 235, and a modified Fc polypeptide that binds to TfR, and having the modified Fc polypeptide having the sequence of SEQ ID NO: 116. The first 10 amino acids are part of the IgG1 hinge region. Its CH2 domain sequence starts at position 11 of SEQ ID NO: 116.

[0322] The IDS-Fc fusion protein ETV:IDS35.21.17.2 used in the examples is a dimer formed by an IDS-Fc fusion polypeptide having one of the sequences of SEQ ID NOs: 115, 231, and 235, and a modified Fc polypeptide that binds to TfR, and having the sequence of SEQ ID NO: 228. The first 10 amino acids are part of the IgG1 hinge region. Its CH2 domain sequence starts at position 11 of SEQ ID NO: 228.

[0323] The IDS-Fc fusion protein ETV:IDS35.23.2 used in the examples is a dimer formed by an IDS-Fc fusion polypeptide having one of the sequences of SEQ ID NOs: 115, 231, and 235, and a modified Fc polypeptide that binds to TfR, and having the sequence of SEQ ID NO: 229. The first 10 amino acids are part of the IgG1 hinge region. Its CH2 domain sequence starts at position 11 of SEQ ID NO: 229.

[0324] The IDS-Fc fusion protein ETV:IDS35.21.17 used in the examples is a dimer formed from an IDS-Fc fusion polypeptide having one of the sequences of SEQ ID NOs: 115, 231, and 235, and a modified Fc polypeptide that binds to TfR, having the sequence of SEQ ID NO: 151. The N-terminus of the modified Fc polypeptide may include a portion of the IgG1 hinge region (e.g., SEQ ID NO: 113).

[0325] Recombinant protein expression and purification To express recombinant IDS enzyme fused to the Fc region, ExpiCHO cells (Thermo Fisher Scientific) were transfected with the relevant DNA construct using the Expifectamine®CHO transfection kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The cells were grown in ExpiCHO® Expression Medium using an orbital shaker (Infors HT Multitron) at 37°C, 6% CO2, and 120 rpm. In short, 6 × 10⁶ DNA constructs were transfected into logarithmically growing ExpiCHO® cells. 6Cells were transfected with a DNA plasmid at a density of cells / ml, at a dose of 0.8 μg per 1 mL of culture volume. After transfection, the cells were returned to 37°C, and 18–22 hours after transfection, the feed solution as shown was added to the transfected culture medium. 120 hours after transfection, the supernatant of the transfected cell culture medium was collected by centrifugation at 3,500 rpm for 20 minutes. The clarified supernatant was filtered (through a 0.22 μM membrane) and stored at 4°C. Expression of an epitope-tagged IDS enzyme (used as a control) was performed with some modifications to the above procedure. Briefly, an IDS enzyme with a C-terminal hexahistidine tag (SEQ ID NO: 241) was expressed in ExpiCHO cells.

[0326] IDS-Fc fusion proteins with (or without) an engineered Fc region that binds to TfR were purified from the supernatant of cell culture medium using protein A affinity chromatography. The supernatant was passed through a protein A affinity column HiTrap MabSelect SuRe (GE Healthcare Life Sciences, using the Akta Pure System). Subsequently, the column was washed with more than 20 column volumes (CV) of PBS. The bound proteins were eluted using 100 mM citrate / NaOH buffer (pH 3.0) containing 150 mM NaCl. Immediately after elution, the fraction was neutralized with 1 M arginine-670 mM succinate buffer (pH 5.0, 1:5 dilution). The homogeneity of the IDS-Fc fusion proteins in the eluted fraction was evaluated by reduced SDS-PAGE and unreduced SDS-PAGE.

[0327] To purify the hexahistidine-tagged (SEQ ID NO: 241) IDS enzyme, the transfected supernatant was thoroughly dialyzed overnight in 15 L of 20 mM HEPES (pH 7.4) containing 100 mM NaCl. The dialyzed supernatant was then subjected to a HisTrap column (GE The enzyme was bound to a column (using Healthcare Life Sciences' Akta Pure System). After binding, the column was washed with 20 CV of PBS. The bound protein was eluted using PBS containing 500 mM imidazole. The homogeneity of the IDS enzyme in the eluted fraction was evaluated by reduced and unreduced SDS-PAGE. The pool fraction containing the IDS enzyme was diluted 1:10 with 50 mM Tris (pH 7.5) and further purified using Q Sepharose High Performance (GE Healthcare). After binding, the column was washed with 10 CV of 50 mM Tris (pH 7.5). The bound protein was eluted using a linear gradient to 50 mM Tris (pH 7.5) and 0.5 M NaCl and recovered in a 1 CV fraction. The fraction purity was evaluated by unreduced SDS-PAGE. As shown in Figure 1, purification yielded homogeneous IDS-Fc fusion protein and IDS enzyme tagged with hexahistidine (SEQ ID NO: 241).

[0328] Example 2. Characterization of IDS fusion proteins IDS-Fc fusion proteins with a modified TfR binding site bind to human TfR. To determine whether an IDS-Fc fusion protein with an engineered TfR binding site affects the ability of the modified Fc domain to interact with human TfRs, the affinity of this protein to human TfRs was evaluated using a Biacore® surface plasmon resonance assay. Anti-human Fab was immobilized on a Biacore® Series S CM5 sensor chip (GE Healthcare Human Fab Capture Kit). 5 μg / mL of IDS-Fc fusion protein was captured on each flow cell for 1 minute, and 3-fold serial dilutions of human apical domain TfRs were injected at a flow rate of 30 μL / min. Each sample was analyzed after 3 minutes of binding and 3 minutes of dissociation. After each injection, the chip was regenerated with 10 mM glycine-HCl (pH 2.1). The binding response was corrected by subtracting RU obtained from flow cells containing unrelated IgG at similar densities. Using Biacore® T200 Evaluation Software v3.1, steady-state affinity was obtained by fitting the equilibrium binding response to concentration. As shown in Figure 2, analysis using Biacore® revealed that the IDS-Fc fusion protein, which has a modified TfR binding site in its Fc region, binds to human TfR. Analysis using Biacore® also revealed that the IDS-Fc fusion protein ETV:IDS35.21 binds to human TfR with an affinity of approximately 200 nM.

[0329] IDS-Fc fusion proteins with manipulated TfR binding sites exhibit activity in vitro, intracellular, and in vivo. The in vitro and intracellular activity of the manipulated TfR-binding IDS-Fc fusion protein was evaluated, and it was shown that IDS retained its enzymatic activity when fused to a human IgG fragment. In vitro activity was measured by a two-step fluorescence enzyme assay using an artificial substrate. Specifically, 20 μL of 1 mM 4-methylumbelliferyl α-idopyranosideuronic acid 2-sodium sulfate basic (Carbosynth Limited, #EM03201) was diluted with assay buffer (100 mM sodium acetate, 10 mM lead acetate, 0.05% Triton X-100, pH 5.0) and mixed with 10 μL of 0.2 nM IDS. The first reaction mixture was incubated at 37°C for 4 hours and stopped in 60 μL of 0.2 M phosphate-citrate buffer (pH 5.0). Next, the second reaction was carried out in the presence of 15 μg of cell lysate obtained from HEK293T cells transiently transfected with human α-idulonidase (IDUA). The reaction was incubated at 37°C for 16 hours and stopped by adding 100 μL of 0.5 M sodium carbonate buffer (pH 10.5). Subsequently, the fluorescence of the reaction solution (excitation at 365 nm and emission at 450 nm) was measured. To calculate the amount of product, a calibration curve using 4-methylumbelliferone was fitted by linear regression and confirmed to be less than 10% of the total substrate cleavage. The specific activity (amount of product (nmol) / min / IDS 1 nmol) was calculated by dividing the amount of product by the reaction time and the molar amount of IDS.

[0330] In vitro enzyme activity assays demonstrated that the IDS-Fc fusion protein was active, and that fusion of the Fc region with IDS did not impair the enzyme activity (Figure 3).

[0331] Using CRISPR / CAS9, IDS knockout (KO) cells were generated to obtain a cell line for testing the intracellular activity of the engineered IDS-Fc fusion protein. HEK293T cells (ATCC) were transfected with the CRISPR / CAS9 pCas-Guide-EF1a-GFP vector (Origene), which contains a guide sequence targeting the latter half of exon 1 of human IDS. Single-cell clones were analyzed for the presence of indels in the IDS genome sequence using the Guide-it Mutation Detection Kit (Clontech) according to the manufacturer's instructions. To identify IDS KO cells, the lysates of indel-positive clone cells were analyzed using the in vitro IDS enzyme assay described above. In short, the in vitro activity assay was performed using 12.5 μg, 25 μg, 50 μg, and 100 μg of cell lysates in lead acetate assay buffer (pH 5.0, 100 mM sodium acetate, 10 mM lead acetate, 0.02% sodium azide), as previously described (Vozyni et al., J. Inherit. Metab. Dis., 24:675-80 (2001)). The reaction was initiated by mixing 10 μL of standardized cell lysates (in water) with 1 mM substrate in 20 μL of lead acetate buffer. The first reaction mixture was incubated at 37°C for 4 hours and stopped with 60 μL of 0.2 M phosphate-citrate buffer (pH 5.0). Next, the second reaction was carried out by adding 10 μg / 10 μL of cell lysate obtained from HEK293T cells transiently transfected with human α-idulonidase (IDUA). This reaction was allowed to proceed for 24 hours at 37°C and stopped by adding 100 μL of 0.5 M sodium carbonate buffer (pH 10.3). Subsequently, the fluorescence of the reaction solution (excitation at 365 nm and emission at 450 nm) was measured. The IDS activity of HEK293T CRISPR clones was compared with recombinant IDS (used as an assay standard), HEK wild-type (WT) lysate, and HEK cell lysate overexpressing IDS. After mini-Topo (ThermoFisher) cloning, clones with enzyme activity levels corresponding to the background signal were sequenced and identified as KO clones.Subsequent cell assays utilized three identified unique IDS knockout clones and three independent WT HEK293T cell batches.

[0332] To test the intracellular activity of naked IDS enzymes or IDS-Fc fusion proteins, LC-MS / MS-based glycomics assays were performed to monitor the accumulation of substrates (heparan sulfate and dermatan sulfate) as indicators of IDS activity. Substrate accumulation was monitored before and after adding IDS or IDS-Fc fusion proteins to the cell culture medium. Measurements were performed using knockout cells. Briefly, cells were washed three times with PBS, pelletized, and frozen. The cell pellets were sonicated in disaccharide digestion buffer (111 mM NH4OAc, 11 mM CaOAc, pH 7.0). Protein concentration was measured using a BCA assay (Pierce). Total protein (100 μg) was added to 100 μL of digestion buffer containing 2 mM DTT, 1.25 mIU heparinase I (Galen), 1.25 mIU heparinase II (Galen), 1.25 mIU heparinase III (Galen), and 6.25 mIU chondroitinase B (Galen). After 3 hours at 30°C, digestion of heparan sulfate and dermatan sulfate was complete, and then 20 ng of internal standard (4UA-2S-GlcNCOEt-6S HD009 [Galen]) was added to each sample. The enzyme was inactivated by adding 6 μL of 250 mM EDTA, and the sample was boiled at 95°C for 10 minutes. Subsequently, the sample was centrifuged at 16,000 × G for 5 minutes at room temperature. The supernatant was transferred to an Amicon Ultra 30KD (Millipore) centrifuge filter and centrifuged at 14,000 × G for 15 minutes. The disaccharide was concentrated in the flow-through, resuspended in a mixture of assay buffer:acetonitrile [1:1, v / v], and then transferred to a mass spectrometry vial for further analysis.

[0333] Disaccharides produced by enzymatic digestion of heparan sulfate and dermatan sulfate were analyzed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) combined with electrospray mass spectrometry (Sciex 6500 + QTRAP, Sciex, Framingham, MA, USA). For each analysis, 10 μL of sample was injected into an ACQUITY UPLC BEH Amide 1.7 μm (2.1 × 150 mM) column (Waters Corporation, Milford, Massachusetts, 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. Mobile phase B consisted of acetonitrile containing 0.1% formic acid. The gradient was programmed as follows: B85% for 0.0-1.0 minutes, B85% to B50% for 1.0-5.0 minutes, B50% to B85% for 5.0-6.0 minutes, and B85% held for 6-8.0 minutes. Ionization by electrospray was performed in negative ion mode with the following settings: curtain gas: 30, collision gas: moderate, ion spray voltage: -4500, temperature: 450, ion source gas 1: 50, ion source gas 2: 60. Data was acquired using Analyst 1.6.3 (Sciex) in multiple reaction monitoring mode (MRM) with a dwell time of 25 milliseconds, collision energy: -30, declustering potential: -80, entrance potential: -10, and collision cell exit potential: -10.MRM transitions (D0A0 for m / z378.1>87.0, D0a0 for m / z378.1>175.0, D0S0 for m / z416.1>138.0, D0a4 for m / z458.1>300.0, D0A6, D2A0, D0a6, D2a0 for m / z458.1>97.0, D0S6 for m / z496.0>416.1) GAGs were detected as [M--H] using D2S0, D2a4, D2a6, D0a10, D2A6 (m / z 538.0 > 458.0), D0S6 (m / z 575.95 > 97.0), and 4UA-2S-GlcNCOEt-6S (m / z 472.0 (fragment ion) > 97.0) as internal standards (IS). GAGs were identified based on their retention time and agreement of MRM transitions to a commercially available reference standard (Iduron Ltd, Manchester, UK). Quantification was performed using MultiQuant 3.0.2 (Sciex) by area ratio to IS. GAGs were normalized to the total protein amount. Protein concentrations were measured using a BCA assay (Pierce).

[0334] As reflected in the amount of disaccharides observed after digestion of heparan sulfate and dermatan sulfate, significant substrate accumulation was observed in IDS KO cells compared to control cell lines, and its effects could be rescued by adding recombinant IDS to these cells (Figure 4). This validated that the intracellular activity of IDS and IDS-Fc fusion proteins can be evaluated using LC-MS / MS-based assays.

[0335] Using this assay, when cells were treated with an IDS-Fc fusion protein containing either the same TfR-binding Fc polypeptide (i.e., CH3C.35.21.17) as an N-terminal monozyme (i.e., ETV:IDS35.21.17) or a C-terminal monozyme, the levels of heparan sulfate and dermatan sulfate-derived disaccharides were reduced to levels observed in wild-type cells (Figure 5A). In addition, the activity of its N-terminal monozyme was comparable to that of IDS (Figure 5B). Considering these results, these data indicate that the IDS-Fc fusion protein can maintain enzymatic activity while reducing substrate accumulation in IDS knockout cells.

[0336] As previously described (Lu et al., Bioconjugate Chemistry, 21:151-156 (2010)), newly synthesized GAGs 35 Incorporate S 35 Using the S-pulse-chase assay, intracellular activity of the IDS-Fc fusion protein was investigated in fibroblasts derived from MPS II patients and healthy controls. Fibroblasts from MPS II patients lacked detectable IDS activity, resulting in approximately 10-fold substrate accumulation. 35 S signal accumulation is 2.5 times higher (Figure 5C). Similar to IDS KO cells, IDS-Fc fusion proteins such as ETV:IDS35.23.2 are highly effective in MPS II patient-derived cells as well. 35 Cellular EC to reduce the accumulation of labeled proteins 50 The levels were low picomolar levels (Figure 5C). Furthermore, the intracellular activity of IDS-Fc fusion proteins such as ETV:IDS35.23.2 was shown to be M6PR-dependent. In fibroblasts from treated MPS II patients, excess M6P was observed. 35 This is because the removal of the S-labeled protein was inhibited (Figure 5D). Considering these results, these data indicate that the IDS-Fc fusion protein can retain the M6PR-dependent transport and intracellular activity of IDS.

[0337] To measure heparan sulfate and dermatan sulfate disaccharides in vivo, LC-MS / MS-based glycomic assays were adapted for tissue and fluid-based analysis. Briefly, all tissues and fluids were frozen immediately after collection and stored at -80°C. Samples underwent five freeze-thaw cycles and were processed as described above for cellular analysis. Significant accumulation of heparan sulfate and dermatan sulfate disaccharides was observed in all analyzed tissues and fluids from male IDS KO mice compared to male wild-type littermates (Table 1). This assay is used in in vivo efficacy testing of fusion proteins. IDS KO mice were obtained from The Jackson Laboratories (JAX strain 024744). [Table 1]

[0338] Using this method, levels of heparan sulfate and dermatan sulfate disaccharides were evaluated in serum from wild-type (WT) mice administered with the vehicle and from IDS KO mice administered with IDS or IDS-Fc fusion protein (i.e., ETV:IDS35.21). Baseline measurements before administration showed significant accumulation of heparan sulfate and dermatan sulfate disaccharides in serum from IDS KO mice compared to WT mice. After administration of the IDS-Fc fusion protein, levels of heparan sulfate and dermatan sulfate disaccharides were significantly reduced in IDS KO serum, comparable to the levels observed in serum from IDS KO mice administered with IDS (Figure 6). These data demonstrate that the IDS-Fc fusion protein is active in vivo and can reduce substrate accumulation in IDS KO mice. Based on these data, distribution and pharmacodynamic (PD) responses were evaluated in the tissues of IDS KO mice 7 days after a single administration of IDS-Fc fusion protein. IDS KO mice were intravenously administered either 40 mg / kg of IDS-Fc fusion protein or 5.3 mg / kg of IDS (25% molar equivalent) as a positive control, and GAG levels were assessed. Distribution of both molecules in peripheral tissues was confirmed 2 hours after administration. A significant decrease in substrates was observed in the liver, spleen, and lungs of IDS KO mice 7 days after administration of IDS-Fc fusion protein (Figure 7).

[0339] To determine whether brain delivery was improved in the TfR-binding IDS-Fc fusion protein compared to the control IDS-Fc fusion protein, human TfR knock-in (TfR ms / hu KI) Mice were administered 50 mg / kg of either the TfR-binding IDS-Fc fusion protein ETV:IDS35.21 or a control IDS-Fc fusion protein ("IDS:Fc") that does not have mutations that lead to TfR binding. Four hours after administration, the concentration of IDS-Fc fusion protein in the brain was measured using the sandwich ELISA-based assay described in Example 3 below. ms / huKI mice were generated using CRISPR / Cas9 technology as described in International Publication No. 2018 / 152285. The human Tfrc apical domain was expressed within the mouse Tfrc gene, and the resulting chimeric TfR was expressed in vivo under the control of an endogenous promoter. Significantly higher levels of the IDS-Fc fusion protein ETV:IDS35.21 were detected in the brain compared to the control IDS-Fc fusion protein, with a mean brain concentration of 23.7 nM for ETV:IDS35.21 (Figure 8). ms / hu Using KI mice, we evaluated the brain uptake of two additional TfR-binding IDS-Fc fusion proteins, ETV:IDS35.21.17.2 and ETV:IDS35.23.2. ms / hu KI mice were administered 50 mg / kg of either ETV:IDS35.21.17.2, ETV:IDS35.23.2, or the control IDS-Fc fusion protein ("IDS:Fc"), and the concentration of IDS-Fc fusion protein in the brain was measured 2 and 8 hours after administration using a sandwich ELISA-based assay. Administration of TfR-bound IDS-Fc fusion protein resulted in a 5-fold increase in brain uptake at 2 hours and a 10- to 20-fold increase in brain concentration at 8 hours compared to the control IDS-Fc fusion protein (Figure 9A). Since serum PK and hepatic accumulation of intact fusion proteins were similar for both ETV:IDS35.21 and IDS:Fc (Figure 9B), it is indicated that the distribution phase of plasma clearance is mainly determined by the IDS portion. Brain levels of TfR-binding IDS-Fc fusion protein remained high for 8 hours and decreased slightly due to peripheral clearance. Considering these factors, these data suggest that the interaction between TfR-binding IDS-Fc fusion protein and TfR significantly improves brain exposure while largely maintaining peripheral distribution.

[0340] Intravenous administration of ETV:IDS reduces GAG levels in the brain. In addition to the above, to investigate whether the improvement in brain exposure observed with the TfR-binding IDS-Fc fusion protein (hereinafter referred to as ETV:IDS) prepared according to Example 1 corresponds to a corresponding reduction in substrate accumulation in the brain, an IDS-deficient mouse model was created in which the human TfR apical domain was knocked into mouse TfR (hereinafter referred to as IDS KO×TfR). ms / hu (Known as KI mouse). In short, TfR ms / hu KI male mice were crossed with female IDS heterozygous mice to induce TfR ms / hu IDS knockout mice were generated from a KI homozygous background. All mice used in this study were male and were fed a 12-hour light-dark cycle with ad libitum access to feed (LabDiet JL irradiated 6F) and water.

[0341] IDS KO×TfR ms / hu KI mice were administered ETV:IDS or IDS intravenously as a single dose or once weekly for 4 weeks at doses of equivalent activity (747 μmol of product / min / kg, or 40 mg / kg for ETV:IDS and 14.2 mg / kg for IDS), and their pharmacokinetic and pharmacodynamic responses were evaluated. Specifically, 2-month-old IDS KO×TfR mice were administered intravenously (iv) once (n=8) or once weekly for 4 weeks (n=8) using physiological saline, IDS (14.2 mg / kg body weight), or ETV:IDS (40 mg / kg body weight). ms / hu Using KI mice, IDS KO × TfR ms / hu In KI mice, the effects of peripheral administration of ETV:IDS on GAGs in the brain and tissues were investigated. Two-month-old littermates TfR mice were given either a single dose of saline (n=5) or weekly injections for four weeks (n=5). ms / hu KI mice were used as controls. Serum samples were collected at various time points by submandibular blood sampling from mice administered IDS or ETV:IDS while they were still alive. All mice were euthanized either 7 days after the first dose or 7 days after the last dose at week 4. Urine, serum, CSF, liver, kidneys, spleen, lungs, heart, and right hemisphere of the brain were dissected and rapidly frozen with dry ice.

[0342] When evaluated using the ELISA-based assay described in Example 3 below, which detects the concentration of IDS, ETV:IDS showed a serum clearance profile similar to that of IDS after a single administration, providing further support that peripheral clearance is primarily determined by this enzyme (Figure 10A). Brain levels of ETV:IDS were significantly higher than those of IDS two hours after administration, with mean concentrations of 8.4 nM for ETV:IDS and 1.6 nM for IDS. Liver and spleen levels of ETV:IDS were also significantly higher than those of IDS (Figure 10B).

[0343] To determine if ETV:IDS reduces substrate levels in the brain, IDS knockout × TfR was performed after a single dose of the enzyme or four weekly doses. ms / hu In KI mice, GAG levels were evaluated as described in Example 2. IDS slightly reduced brain GAG levels at an early stage, but did not significantly reduce GAG ​​after 4 weeks of treatment (Figure 10C). However, ETV:IDS reduced brain GAG levels by approximately 58% after a single dose and by 71% after 4 weeks of treatment (Figure 10C). This resulted in an approximately 75% decrease in CSF GAG after a single dose, and this decrease persisted after 4 weeks of treatment (Figure 10C). Both molecules effectively reduced liver and spleen GAG levels after 1 week, and the response was sustained by repeated administration (Figure 10C), indicating that binding to TfR did not adversely affect the pharmacodynamic response in these tissues. Considering these factors, these data demonstrate that ETV:IDS significantly improves brain exposure to the enzyme while steadily reducing substrate accumulation in both the peripheral and central nervous system (CNS).

[0344] Example 3. Pharmacokinetic characterization of IDS fusion proteins This example describes the pharmacokinetic (PK) characterization of the manipulated IDS-Fc fusion protein in mouse plasma. To determine the plasma half-life and clearance of TfR-conjugated IDS-Fc fusion protein, 7-8 week old male C57BL / 6 mice were administered 10 mg / kg of two IDS-Fc fusion protein molecules (N-terminal monozyme and C-terminal monozyme) by tail vein injection. The concentration of IDS-Fc fusion protein remaining in the plasma was measured over a 24-hour period using an ELISA-based assay. In short, the concentration of IDS-Fc fusion protein in mouse plasma was quantified using a sandwich ELISA. An anti-Fc capture antibody (Abcam#ab124055) was coated at 3 μg / mL on a 384-well MaxiSorp® plate (Thermo Scientific#464718). The plate was blocked with 5% BSA and then incubated with plasma diluted at either 1:1,000 or 1:10,000. Next, polyclonal anti-IDS detection antibody (R&D Systems #AF2449) was added at 0.5 μg / mL, followed by anti-goat HRP antibody. The plates were color-developed using TMB substrate, stopped with sulfuric acid, and the absorbance at 450 nm was measured using a BioTek plate reader. Calibration curves were individually constructed from 200 to 0.1 ng / mL in a 4-fold dilution series and fitted using 4-parameter logistic regression.

[0345] This assay revealed that the terminal phase half-life of the IDS-Fc fusion protein in plasma was 7.7–10 hours (Table 2). No unexpected PK-related problems were observed with the IDS-Fc fusion protein in vivo. [Table 2]

[0346] Example 4. Construction of a fusion protein containing acid sphingomyelinase (ASM) Design and cloning ASM-Fc fusion proteins were designed as dimers of fusion polypeptides ("ASM-Fc fusion polypeptides") in which a mature human ASM enzyme is fused to a human IgG1 fragment containing an Fc region. In some embodiments, the ASM-Fc fusion polypeptides contain a modified Fc region with mutations that result in binding to the transferrin receptor (TfR). Specifically, ASM-Fc fusion polypeptides were constructed in which the ASM fragment is fused to the N-terminus of the human IgG1 Fc region. In some cases, a linker was placed between the ASM and the IgG1 fragment to alleviate any steric hindrance between the two fragments. In all constructs, amino acids (UniProtKB) at positions 1-46 of the native ASM signal sequence were used. ASM secretion was enhanced by removing ID-P17405 and replacing it with a secretory signal derived from amino acids 1-20 of the κ chain V-III (UniProtKB ID-P01661). In addition, in the fusion protein, ASM was truncated at its C-terminus (terminated at amino acid Q620) to prevent any unwanted cleavage between ASM and its human IgG1 Fc region. Subsequently, a fragment of the human IgG1 Fc region (UniProtKB ID-P01857) was positioned in frame with the C-terminus of ASM (starting at amino acid E99), and the cysteine ​​at position 103 was mutated to serine. In some embodiments, the IgG1 fragment included additional mutations to promote heterodimerization of the two Fc regions. In addition, the ASM-Fc fusion protein was constructed to contain one or two molecules of ASM. As a control, we designed an ASM-hexahistidine (SEQ ID NO: 241) fusion protein consisting of amino acids from positions 1 to 628 of ASM, which have been truncated to remove the C-terminal cysteine ​​and enhance enzyme activity, and fused to a hexahistidine tag (SEQ ID NO: 241) at the C-terminus.

[0347] Recombinant protein expression and purification To express recombinant ASM enzyme fused to the Fc region, follow the manufacturer's (Thermo Fisher Scientific) instructions and inject 6 × 10⁶ cells into ExpiCHO-S cells (Thermo Fisher).6 The cells were transfected with the Expifectamine CHO / plasmid DNA complex at a density of cells / ml. After transfection, the cells were incubated in an orbital shaker (Infors HT Multitron) at 32°C in a humidified atmosphere of 6-8% CO2. One day after transfection, the Expifectamine enhancer and Expifectamine feed solution were added to the culture. After 48-72 hours of expression, the supernatant was collected by centrifugation. The clarified supernatant was then supplemented with an EDTA-free protease inhibitor (Roche) and stored at -80°C.

[0348] For the purification of the ASM-Fc fusion protein, 200 μM zinc acetate (Sigma Aldrich) was added to the clarified culture medium supernatant. The supernatant was then processed using a protein A affinity column, HiTrap MabSelect SuRe (GE Healthcare). The samples were run through a spectroscopy system (GE Healthcare Life Sciences) and washed with 200 mM arginine and 137 mM succinate buffer (pH 5.0, arginine-succinate buffer). The fusion protein was eluted with 100 mM QB citrate buffer (pH 3.0) with 200 μM zinc acetate added. Immediately after elution, arginine-succinate buffer was added to adjust the pH. Protein aggregates were separated from the ASM-Fc fusion protein by size exclusion chromatography (SEC) using a Superdex 200 increase 10 / 300 GL column (GE Healthcare Life Sciences). The SEC mobile phase was retained in arginine-succinate buffer (pH 5.0) with 200 μM zinc acetate added. All chromatography steps were performed using an Akta Pure System or Akta Avant system (GE Healthcare Life Sciences). Fraction purity was evaluated by non-reducing SDS-PAGE. As shown in Figure 11, a homogeneous ASM-Fc fusion protein was obtained by purification.

[0349] Example 5. Characterization of ASM fusion protein The ASM-Fc fusion protein is active both in vitro and in cells. To demonstrate that ASM retains its enzymatic activity when fused to human IgG heavy chains, the in vitro and intracellular activity of ASM-Fc fusion proteins was evaluated. The in vitro activity of recombinant ASM enzyme or recombinant ASM-Fc fusion protein was measured using a synthetic chromogenic analog of sphingomyelin. Specifically, 2.5 mM 2-(N-hexadecanoylamino)-4-nitrophenyl phosphorylcholine (EMD Millipore) and 0.75 nM ASM were mixed in 100 mM sodium acetate buffer (pH 5.3, final concentration in 100 μL reaction volume). The reaction mixture was incubated at 37°C for 16 hours and stopped by adding an equal volume of 0.2 M NaOH. Subsequently, the absorbance of the reaction solution was measured at 410 nm. To calculate the amount of product, a calibration curve using p-nitrophenol was fitted by linear regression and confirmed to be less than 10% of the total substrate cleavage. The specific activity (product amount (nmol) / min / IDS 1nm) was calculated by dividing the product amount by the reaction time and the molar amount of IDS. This in vitro enzyme activity assay demonstrated that the ASM-Fc fusion protein was active and that its enzyme activity was not impaired by the fusion of the Fc region to ASM (Figure 12).

[0350] Using CRISPR / CAS9, ASM knockout cells were generated to obtain a cell line for testing the intracellular activity of the ASM-Fc fusion protein. HEK293T cells (ATCC) were transfected with the CRISPR / CAS9 pCas-Guide-EF1a-GFP vector (Origene), which contains a guide sequence targeting the latter half of exon 2 of human SMPD1. Single-cell clones were analyzed for the presence of indels in the ASM genome sequence using the Guide-it Mutation Detection Kit (Clontech) according to the manufacturer's instructions. In vitro ASM enzyme assays were performed on indel-positive clone cell lysates using the ASM chromogenic substrate 2-N-hexadecanoylamino-4-nitrophenyl phosphorylcholine (EMD Millipore). Briefly, the in vitro activity assays were performed using cell lysates of 12.5 μg, 25 μg, 50 μg, and 100 μg in 100 mM sodium acetate buffer (pH 5.3). The reaction was initiated by adding a 2.5 mM substrate and stopped after 20 hours by adding 0.2 M NaOH. ASM activity in HEK293T CRISPR clones was compared with recombinant ASM (used as an assay baseline), HEK wild-type (WT) lysates, and HEK cells overexpressing ASM. After mini-Topo (ThermoFisher) cloning, clones with enzyme activity levels corresponding to the background signal were sequenced and identified as KO clones. Subsequent cell assays used the three identified unique ASM KO clones and three independent WT HEK293T cell batches.

[0351] To test the intracellular activity of naked ASM enzyme or ASM-Fc fusion protein, two intracellular assays were performed in ASM KO cells to monitor substrate (sphingomyelin) accumulation, both as a baseline and after treatment with ASM or ASM-Fc fusion. First, an imaging-based assay was performed to monitor the accumulation of BODIPY-conjugated C5-sphingomyelin in ASM KO cells. Briefly, HEK293T WT cells and ASM KO cells were seeded at low density in DMEM supplemented with 10% FBS (Gibco) on a PDL-coated 96-well plate (Perkin Elmer). Four hours after seeding, recombinant ASM enzyme, ASM-Fc fusion protein, or control buffer was added to each well, and incubated at 37°C for 48 hours. The medium was removed and replaced with fresh medium containing 1 μM BODIPY-C5-sphingomyelin (Thermo Fisher Scientific), and incubated at 37°C for 16 hours. Next, the cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with nuclear staining (DAPI, Thermo Fisher) and cytoplasmic staining (far-infrared cell mask, Thermo Fisher Scientific). Images were acquired using a confocal microscope, Opera Phenix (Perkin Elmer), at 63x objective magnification, with multiple fields per well and three wells per condition. Image analysis was performed on a single-cell basis using Harmony software (Perkin Elmer) to detect and analyze the average total intensity, number of fluorescence spots, and fluorescence spot intensity of BODIPY-C5-sphingomyelin. Subsequently, these single-cell-based values ​​were averaged to obtain per-well values, and these values ​​were used to analyze the effect of genotype and / or treatment on BODIPY-C5-sphingomyelin accumulation. ASM KO cells showed a significant accumulation of BODIPY-C5-sphingomyelin compared to control cell lines, and this effect could be rescued by adding recombinant ASM enzyme and ASM-Fc fusion protein (Figure 13).

[0352] To further confirm that the ASM-Fc fusion protein retains its activity within cells, an LC-MS / MS-based assay was developed to monitor the accumulation of endogenous sphingomyelin in ASM KO cells. HEK293T WT cells and ASM KO cells were cultured and treated with enzymes as described above. After 68 hours from seeding, with or without treatment with ASM or ASM-Fc fusion protein, the cells were thoroughly washed with PBS, and lipids were extracted with a water:methanol [1:1, v / v] mixture with the addition of appropriate internal standards. Lipids were extracted using methyl-tert-butyl ether (MTBE), vortexed, and centrifuged at 10,000 × g at 4°C for 10 minutes. Subsequently, the upper MTBE fraction containing lipids was evaporated to dryness under a gentle stream of nitrogen. The lipids were resuspended in a mixture of isopropanol:acetonitrile:water [2:1:1, v / v / v] and then transferred to a mass spectrometry vial for further analysis.

[0353] Lipid analysis was performed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) combined with electrospray mass spectrometry (Sciex 6500 + QTRAP, Sciex, Framingham, MA, USA). For each analysis, 5 μL of sample was injected into a BEH C18 1.7 μm (2.1 × 100 mm) column (Waters Corporation, Milford, Massachusetts, USA) at a flow rate of 0.25 mL / min at 55°C. Mobile phase A consisted of 60:40 acetonitrile / water (v / v) containing 10 mM ammonium formate + 0.1% formic acid. Mobile phase B consisted of 90:10 isopropanol / acetonitrile (v / v) containing 10 mM ammonium formate + 0.1% formic acid. The gradient was programmed as follows: B45%→B99% at 0.0-8.0 mins, B99% at 8.0-10.0 mins, B45% at 10.0-10.1 mins, and B45% at 10.1-12.0 mins. Ionization by electrospray was performed in positive ion mode with the following settings: curtain gas: 20, collision gas: moderate, ion spray voltage: 5200, temperature: 250, ion source gas 1: 50, ion source gas 2: 60. Data was acquired using Analyst1.6.(Sciex) in multiple reaction monitoring mode (MRM) with collision energy: 40, declustering potential: 80, entrance potential: 10, and collision cell exit potential: 12.5. MRM transition (m / z 538.5 > 264.3 Cer Cer for d18:1 / 16:0, m / z566.6>264.3 Cer for d18:1 / 18:0, m / z594.6>264.3 Cer for d18:1 / 20:0, m / z622.6>264.3 Cer for d18:1 / 22:0, m / z650.6>264.3 Using Cer d18:1 / 24:0 (m / z 648.6>264.3) and Cer d18:1 / 24:1 (m / z 552.4>264.3) (with Cer d18:1 / 17:0 used as internal standards), ceramide (Cer) was converted to [M-H2O+H] +It was detected as [M+H] using MRM transitions (SM d18:1 / 16:0 for m / z703.7>184.1, SM d18:1 / 18:0 for m / z731.7>184.1, SM d18:1 / 20:0 for m / z759.7>184.1, SM d18:1 / 22:0 for m / z787.7>184.1, SM d18:1 / 24:0 for m / z815.7>184.1, SM d18:1 / 24:1 for m / z813.7>184.1, and SM d18:1 / 18:1(d9) for m / z738.7>184.1). + Lipids were detected as follows. Lipids were identified based on their retention time and the MRM characteristics of a commercially available reference standard (Avanti Polar Lipids, Birmingham, AL, USA). Quantification was performed using MultiQuant3.02 (Sciex). Lipids were normalized to the total protein amount. Protein concentrations were measured using a BCA assay (Pierce). LC-MS / MS analysis showed that the ASM-Fc fusion protein could reduce the level of endogenous sphingomyelin in ASM KO cells to the level seen in wild-type cells (Figure 14). Furthermore, in all assays, the ASM-Fc fusion protein showed a sphingomyelin-reducing effect comparable to that of the naked ASM enzyme (Table 3). [Table 3]

[0354] Considering these factors, these data indicate that ASM-Fc fusion proteins maintain their activity and can rescue substrate accumulation in ASM-deficient cells.

[0355] Example 6. Construction of a fusion protein containing N-sulfoglucosamine sulfohydrose (SGSH). Design and cloning We designed SGSH-Fc fusion proteins containing (i) a fusion polypeptide in which a mature human SGSH enzyme is fused to a human IgG1 fragment containing an Fc region ("SGSH-Fc fusion polypeptide"), and (ii) a modified human IgG1 fragment containing a mutation in the Fc region that leads to binding to the transferrin receptor (TfR) ("modified Fc polypeptide"). Specifically, we created SGSH-Fc fusion polypeptides by fusioning the SGSH fragment to either the N-terminus or C-terminus of the human IgG1 Fc region. In some cases, a linker was placed between the SGSH and the IgG1 fragment to alleviate any steric hindrance between the two fragments. In all constructs, a signal peptide derived from amino acids 1-20 of the κ chain V-III (UniProtKB ID-P01661) was inserted upstream of the fusion to promote secretion and truncate the SGSH to consist of amino acids R21-L502 (UniProtKB ID-P51688). The human IgG1 Fc region fragment used corresponds to amino acids D104-K330 of the sequence in UniProtKB ID P01857 (positions 221-447 in EU index number, including the 10 amino acids at its hinge (positions 221-230)). In some embodiments, to produce a heterodimer fusion protein ("monozyme") having one SGSH enzyme, a second Fc polypeptide derived from human IgG1 residues D104-K330, which contains a mutation in its Fc region resulting in binding to TfR but does not contain SGSH fusion, was cotransfected with an SGSH-Fc fusion polypeptide. In another embodiment, to produce a heterodimer fusion protein ("byzyme") having two SGSH enzymes, a second Fc polypeptide derived from human IgG1 residues D104-K330, containing a mutation in its Fc region that results in binding to TfR, was cotransfected with an SGSH-Fc fusion polypeptide. In some constructs, additional mutations were included in the IgG1 fragment to promote heterodimerization of the two Fc regions. A control SGSH-Fc fusion protein without the mutation resulting in TfR binding was similarly designed and constructed.As an additional control, SGSH (amino acids R21-L502) with a C-terminal hexahistidine tag (SEQ ID NO: 241) was prepared to facilitate detection and purification.

[0356] The SGSH-Fc fusion protein containing a TfR linkage used in the examples is a dimer formed by an SGSH-Fc fusion polypeptide and a modified Fc polypeptide that binds to TfR, wherein the modified Fc polypeptide is either not fused to SGSH ("monozyme") or is fused to a second SGSH molecule ("byzyme").

[0357] The SGSH-Fc fusion polypeptide, which contains a mature human SGSH sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence having a whole mutation and an LALA mutation, has the sequence of SEQ ID NO: 149. The SGSH enzyme is linked to the Fc polypeptide by a linker called GGGGS (SEQ ID NO: 239), and the N-terminus of the Fc polypeptide includes a portion of the IgG1 hinge region (DKTHTCPPCP, SEQ ID NO: 113).

[0358] An SGSH-Fc fusion polypeptide containing a mature human SGSH sequence fused to the C-terminus of an IgG1 Fc polypeptide sequence having a whole mutation and an LALA mutation has the sequence of SEQ ID NO: 150. The SGSH enzyme is linked to the Fc polypeptide by a linker called GGGGS (SEQ ID NO: 239), and the N-terminus of the Fc polypeptide may contain a portion of the IgG1 hinge region (e.g., SEQ ID NO: 113).

[0359] A modified Fc polypeptide that binds to TfR and contains the sequence of clone CH3C.35.21.17 (SEQ ID NO: 58) along with knob mutations and LALA mutations has the sequence of SEQ ID NO: 151. The N-terminus of the modified Fc polypeptide may include a portion of the IgG1 hinge region (e.g., SEQ ID NO: 113).

[0360] An "N-terminal monozyme" containing one SGSH molecule at the N-terminus of an Fc polypeptide was formed between SEQ ID NOs. 149 and SEQ ID NOs. 151. A "C-terminal monozyme" containing one SGSH molecule at the C-terminus of an Fc polypeptide was formed between SEQ ID NOs. 150 and SEQ ID NOs. 151.

[0361] A modified Fc polypeptide that binds to TfR, and which includes a mature human SGSH sequence fused to the N-terminus of the sequence of clone CH3C.35.21.17 (SEQ ID NO: 58) along with knob mutations and LALA mutations, has the sequence of SEQ ID NO: 154. The SGSH enzyme is linked to the modified Fc polypeptide by a linker called GGGGS (SEQ ID NO: 239), and a portion of the IgG1 hinge region (SEQ ID NO: 113) is included at the N-terminus of the modified Fc polypeptide.

[0362] An "N-terminal byzyme" containing a first SGSH molecule at the N-terminus of the Fc polypeptide and a second SGSH molecule at the N-terminus of the modified Fc polypeptide was formed between the sequences 149 and 154.

[0363] 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 (Thermo Fisher Scientific) according to the manufacturer's instructions. The cells were grown in an orbital shaker (Infors HT Multitron) in ExpiCHO® Expression Medium at 37°C, 6% CO2, and 120 rpm. In short, 6 × 10⁶ DNA constructs were transfected into logarithmically growing ExpiCHO® cells. 6Cells were transfected with a DNA plasmid at a density of cells / ml, at a dose of 0.8 μg per 1 mL of culture volume. After transfection, the cells were returned to 37°C, and 18–22 hours after transfection, the feed solution as shown was added to the transfected culture medium. 120 hours after transfection, the supernatant of the transfected cell culture medium was collected by centrifugation at 3,500 rpm for 20 minutes. The clarified supernatant was filtered (through a 0.22 μM membrane) and stored at 4°C. Expression of epitope-tagged SGSH enzyme (used as a control) was performed with some modifications to the above procedure. Briefly, SGSH enzyme with a C-terminal hexahistidine tag (SEQ ID NO: 241) was expressed in ExpiCHO cells.

[0364] Using protein A affinity chromatography, SGSH-Fc fusion proteins with (or without) an engineered Fc region that binds to TfR were purified from the supernatant of cell culture medium. The supernatant was passed through a protein A affinity column, HiTrap MabSelect SuRe (GE Healthcare Life Sciences, Akta Pure System). Subsequently, the column was washed with more than 20 column volumes (CV) of PBS. The bound proteins were eluted using 100 mM citrate / NaOH buffer (pH 3.0) containing 150 mM NaCl. Immediately after elution, the fraction was neutralized with 1 M arginine-670 mM succinate buffer (pH 5.0, 1:5 dilution). The homogeneity of the SGSH-Fc fusion proteins in the eluted fraction was evaluated by reduced and unreduced SDS-PAGE.

[0365] To purify hexahistidine-tagged (SEQ ID NO: 241) SGSH, the transfected supernatant was thoroughly dialyzed overnight in 15 L of 20 mM HEPES (pH 7.4) containing 100 mM NaCl. Before purification, 20 mM imidazole was added to the dialyzed supernatant. The dialyzed supernatant was conjugated to a HisTrap column (GE Healthcare Life Sciences, Akta Pure System). After conjugation, the column was washed with 20 CV of PBS. The conjugated protein was eluted using PBS containing 500 mM imidazole. The homogeneity of the SGSH enzyme in the eluted fraction was evaluated by reduced and unreduced SDS-PAGE. The pooled fraction containing SGSH can be further purified by diluting it 1:10 with 50 mM Tris (pH 7.5) and using Q Sepharose High Performance (GE Healthcare). After binding, the column is washed with 10 CV of 50 mM Tris (pH 7.5). The bound protein is eluted using a linear gradient to 50 mM Tris (pH 7.5) and 0.5 M NaCl, and recovered in a 1 CV fraction. The fraction purity is evaluated by non-reducing SDS-PAGE. Purification yields homogeneous SGSH-Fc fusion protein and SGSH with a hexahistidine tag (SEQ ID NO: 241).

[0366] Example 7. Characterization of SGSH fusion protein SGSH-Fc fusion proteins with a modified TfR binding site bind to human TfR. To determine whether an SGSH-Fc fusion protein with an engineered TfR binding site affects the ability of the modified Fc domain to interact with human TfRs, the affinity of this protein to human TfRs can be evaluated using a Biacore® surface plasmon resonance assay. Anti-human Fab is immobilized on a Biacore® Series S CM5 sensor chip (GE Healthcare Human Fab Capture Kit). 5 μg / mL of SGSH-Fc fusion protein is captured on each flow cell for 1 minute, and a 3-fold serial dilution of human apical domain TfRs is injected at a flow rate of 30 μL / min. Each sample is analyzed for 3 minutes of binding and 3 minutes of dissociation. After each injection, the chip is regenerated with 10 mM glycine-HCl (pH 2.1). The binding response is corrected by subtracting the RU obtained from a flow cell containing unrelated IgG at a similar density. Using Biacore® T200 Evaluation Software v3.1, steady-state affinity is obtained by fitting the equilibrium binding response to concentration. Analysis using Biacore® reveals that SGSH-Fc fu...

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[Claim 1] The invention as shown in the drawings.

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