Fusion proteins comprising enzyme replacement therapy enzymes

Abstract

The present invention relates to fusion proteins comprising an enzyme replacement therapy enzyme, and provides fusion proteins comprising an enzyme replacement therapy enzyme and an Fc region, and methods of using such proteins to treat lysosomal storage disorders. Also provided herein are methods of delivering an agent across the blood-brain barrier.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201880068837.6, filed on October 1, 2018, entitled "Fusion Protein Containing Enzymes for Enzyme Replacement Therapy".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 566,898, filed October 2, 2017; U.S. Provisional Patent Application No. 62 / 583,276, filed November 8, 2017; U.S. Provisional Patent Application No. 62 / 626,365, filed February 5, 2018; U.S. Provisional Patent Application No. 62 / 678,183, filed May 30, 2018; and U.S. Provisional Patent Application No. 62 / 721,396, filed August 22, 2018, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0004] sequence list

[0005] This application contains a sequence list, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on September 28, 2018, and is named 102342-000350PC-1103949_SL.txt with a size of 580,464 bytes. Technical Field

[0006] This invention relates to fusion proteins comprising enzymes for enzyme replacement therapy, providing fusion proteins comprising enzymes for enzyme replacement therapy and an Fc region, and methods for using such proteins to treat lysosomal storage diseases. Methods for delivering agents across the blood-brain barrier are also provided herein. Background Technology

[0007] Lysosomal storage disorders (LSDs) are relatively rare inherited metabolic disorders caused by defects in lysosomal function. LSD is typically caused by a deficiency of a single enzyme involved in the breakdown of metabolic products in lysosomes. The accumulation of products due to the lack of enzyme activity affects various organ systems and can lead to severe symptoms and premature death. Most LSD cases also have significant neural components ranging from progressive neurodegeneration and severe cognitive impairment to epilepsy, behavioral, and psychiatric symptoms. Recombinant forms of the enzymes lacking in LSD can be used to treat these conditions, but such therapies may have little effect on the brain due to the difficulty in delivering the recombinant enzymes across the blood-brain barrier (BBB). Summary of the Invention

[0008] This article provides a fusion protein containing an enzyme replacement therapy (ERT) enzyme and a method for using said fusion protein to treat lysosomal storage disease (LSD).

[0009] In some respects, this document provides a protein comprising:

[0010] (a) a first Fc polypeptide, the first Fc polypeptide being linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and

[0011] (b) A second Fc polypeptide, wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide.

[0012] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide do not include the variable region sequence of the immunoglobulin heavy chain and / or light chain or its antigen-binding portion.

[0013] In some embodiments, the ERT enzyme is iduronate 2-sulfatase (IDS), an IDS variant, or a fragment thereof with catalytic activity. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 91, 92, 114, 230, and 234. In some embodiments, the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NO: 91, 92, 114, 230, and 234.

[0014] In some embodiments, the ERT enzyme is N-sulfoglucosamine sulfonylhydrolase (SGSH), an SGSH variant, or a fragment thereof with catalytic activity. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any of SEQ ID NO: 119 and 120. In some embodiments, the ERT enzyme comprises the amino acid sequence of any of SEQ ID NO: 119 and 120.

[0015] In some embodiments, the ERT enzyme is acid sphingomyelinase (ASM), an ASM variant, or a catalytically active fragment thereof. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 121, 122, and 123. In some embodiments, the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NO: 121, 122, and 123.

[0016] In some embodiments, the ERT enzyme is β-glucocerebrosidase (GBA), a GBA variant, or a fragment thereof with catalytic activity. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any of SEQ ID NO: 93 and 94. In some embodiments, the ERT enzyme comprises the amino acid sequence of any of SEQ ID NO: 93 and 94.

[0017] In some embodiments, the first Fc polypeptide is a fusion polypeptide linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof via peptide bonds or peptide linkers. In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the flexible peptide 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 an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof via a chemical cross-linking agent; for example, the fusion polypeptide does not include non-peptide bonds or non-peptide linkers.

[0018] In some embodiments, the fusion polypeptide comprises, from the N-terminus to the C-terminus: an ERT enzyme, an ERT enzyme variant or a fragment thereof with catalytic activity; a polypeptide linker; and a first Fc polypeptide.

[0019] In some embodiments, the second Fc polypeptide is linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof. In some embodiments, the second Fc polypeptide is a fusion polypeptide linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof via peptide bonds or peptide linkers. In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the flexible peptide 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 an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof via a chemical cross-linking agent; for example, the fusion polypeptide does not include non-peptide bonds or non-peptide linkers.

[0020] In some embodiments, the N-terminus of the first Fc polypeptide and / or the N-terminus of the second Fc polypeptide is linked to an ERT enzyme. In some embodiments, 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 another ERT enzyme.

[0021] In some embodiments, the C-terminus of the first Fc polypeptide and / or the C-terminus of the second Fc polypeptide is linked to an ERT enzyme. In some embodiments, 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 another ERT enzyme.

[0022] In some embodiments, 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 another ERT enzyme.

[0023] In some embodiments, the protein comprises a single ERT enzyme, and the N-terminus or C-terminus of the first Fc polypeptide is linked to the ERT enzyme. In some embodiments, the protein comprises two ERT enzymes (e.g., exactly two ERT enzymes). In some embodiments, the protein comprises exactly one or exactly two ERT enzymes, enzyme variants, or fragments thereof with catalytic activity.

[0024] In some embodiments, the first Fc polypeptide is a modified Fc polypeptide and / or the second Fc polypeptide is a modified Fc polypeptide.

[0025] In some embodiments, the first Fc polypeptide and the second Fc polypeptide each contain modifications that promote heterodimerization. In some embodiments, the Fc dimer is an Fc heterodimer. In some embodiments, according to EU designations, one of the Fc polypeptides has a T366W substitution and the other Fc polypeptide has T366S, L368A, and Y407V substitutions. In some embodiments, the first Fc polypeptide contains T366S, L368A, and Y407V substitutions and the second Fc polypeptide contains a T366W substitution. In some embodiments, the first Fc polypeptide is linked to an ERT enzyme IDS and contains the amino acid sequence of any one of SEQ ID NO: 117, 232, and 236. In some embodiments, the first Fc polypeptide contains a T366W substitution and the second Fc polypeptide contains T366S, L368A, and Y407V substitutions. In some embodiments, the first Fc polypeptide is linked to an ERT enzyme IDS and contains the amino acid sequence of any one of SEQ ID NO: 118, 233, and 237.

[0026] 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 includes modifications that reduce effector function. In some embodiments, the modification that reduces effector function is a substitution of Ala at position 234 and Ala at position 235 according to EU number. In some embodiments, the modification that reduces effector function further includes a substitution of Gly at position 329 according to EU number. In some embodiments, the first Fc polypeptide is linked to an ERT enzyme IDS and contains the amino acid sequence of any one of SEQ ID NO: 115, 231, and 235. In some embodiments, the first Fc polypeptide is linked to an ERT enzyme SGSH and contains the amino acid sequence of any one of SEQ ID NO: 149, 150, 152, and 153.

[0027] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises amino acid changes relative to the native Fc sequence that prolong the serum half-life. In some embodiments, the amino acid changes comprise substitutions for Tyr at position 252, Thr at position 254, and Glu at position 256 according to EU numbers. Alternatively, in other embodiments, the amino acid changes comprise substitutions for Leu at position 428 and Ser at position 434 according to EU numbers. Alternatively, in other embodiments, the amino acid changes comprise substitutions for either Ser or Ala at position 434 according to EU numbers.

[0028] In some implementations, the first Fc peptide and / or the second Fc peptide specifically bind to the transferrin receptor (TfR).

[0029] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises at least two substitutions at positions selected from the group consisting of the following according to EU designations: 384, 386, 387, 388, 389, 390, 413, 416, and 421. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises at least three, four, five, six, seven, eight, or nine substitutions at said positions.

[0030] In some embodiments, 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 according to EU designations. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises one, two, or three substitutions at positions including 414, 424, and 426 according to EU designations.

[0031] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises Trp at position 388. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises an aromatic amino acid at position 421. In some embodiments, the aromatic amino acid at position 421 is Trp or Phe.

[0032] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises at least one position selected from the following: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0033] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from the following: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0034] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises the following 11 positions: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0035] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide has a CH3 domain having at least 85%, at least 90%, or at least 95% identity with amino acids 111-217 of any of SEQ ID NO:34-38, 58 and 60-90, 151 and 156-229. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:156-229. In some embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues at EU index positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 corresponding to any of SEQ ID NO: 34-38, 58 and 60-90, 151 and 156-229 are not deleted or substituted.

[0036] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:157. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:169. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:181. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:193. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:205. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:217.

[0037] 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 other 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:169 or 229 (e.g., SEQ ID NO:229).

[0038] 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 other 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:169 or 229 (e.g., SEQ ID NO:229).

[0039] 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 other 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:169 or 229 (e.g., SEQ ID NO:229).

[0040] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide binds to the apical domain of the TfR. In some embodiments, protein binding to the TfR does not substantially inhibit transferrin binding to the TfR.

[0041] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide has at least 75% or at least 80%, 85%, 90%, 92%, or 95% amino acid sequence identity compared to the corresponding wild-type Fc polypeptide. In some embodiments, the corresponding wild-type Fc polypeptide is a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide.

[0042] In some embodiments, the uptake of ERT enzyme in the brain (e.g., using an appropriate animal model, such as those described herein) is greater than that of ERT enzyme in the absence of a first Fc polypeptide and / or a second Fc polypeptide, or in the absence of modifications to the first Fc polypeptide and / or the second Fc polypeptide that induce TfR binding. In some embodiments, the uptake of ERT enzyme in 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 that of ERT enzyme in the absence of a first Fc polypeptide and / or a second Fc polypeptide, or in the absence of modifications to the first Fc polypeptide and / or the second Fc polypeptide that induce TfR binding.

[0043] In some embodiments, the first Fc peptide is not modified to bind to the blood-brain barrier (BBB) ​​receptor and the second Fc peptide is modified to specifically bind to the TfR. In some embodiments, the first Fc peptide is modified to specifically bind to the TfR and the second Fc peptide is not modified to bind to the BBB receptor.

[0044] In some implementations, the protein does not include the variable region sequence of the immunoglobulin heavy chain and / or light chain or its antigen-binding portion.

[0045] In some aspects, this article provides a polypeptide comprising an Fc polypeptide linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof, wherein the Fc polypeptide contains one or more modifications that promote its heterodimerization with another Fc polypeptide.

[0046] In some embodiments, the ERT enzyme is an IDS, an IDS variant, or a catalytically active fragment thereof. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 91, 92, 114, 230, and 234. In some embodiments, the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NO: 91, 92, 114, 230, and 234.

[0047] In some embodiments, the ERT enzyme is SGSH, an SGSH variant, or a catalytically active fragment thereof. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any of SEQ ID NO: 119 and 120. In some embodiments, the ERT enzyme comprises the amino acid sequence of any of SEQ ID NO: 119 and 120.

[0048] In some embodiments, the ERT enzyme is ASM, an ASM variant, or a catalytically active fragment thereof. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 121, 122, and 123. In some embodiments, the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NO: 121, 122, and 123.

[0049] In some embodiments, the ERT enzyme is a GBA, a GBA variant, or a catalytically active fragment thereof. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any of SEQ ID NO: 93 and 94. In some embodiments, the ERT enzyme comprises the amino acid sequence of any of SEQ ID NO: 93 and 94.

[0050] 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 via peptide bonds or peptide linkers. In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the flexible peptide 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 via a chemical cross-linking agent; for example, the fusion polypeptide does not include non-peptide bonds or non-peptide linkers.

[0051] In some embodiments, the fusion polypeptide comprises, from the N-terminus to the C-terminus: an ERT enzyme, an ERT enzyme variant or a fragment thereof with catalytic activity; a polypeptide linker; and a first Fc polypeptide.

[0052] In some embodiments, according to the EU designation, the Fc polypeptide contains substitutions of T366S, L368A, and Y407V. In some embodiments, the polypeptide contains the amino acid sequence of any one of SEQ ID NO: 115, 117, 231, 232, 235, and 236. In some embodiments, the polypeptide contains the amino acid sequence of any one of SEQ ID NO: 149 and 150. In some embodiments, the Fc polypeptide contains the T366W substitution. In some embodiments, the polypeptide contains the amino acid sequence of any one of SEQ ID NO: 118, 233, and 237. In some embodiments, the polypeptide contains the amino acid sequence of any one of SEQ ID NO: 152-155. In some embodiments, the polypeptide also contains another Fc polypeptide. In some embodiments, the other Fc polypeptide contains the T366W substitution or contains substitutions of T366S, L368A, and Y407V and forms an Fc dimer with the ERT enzyme-Fc fusion polypeptide.

[0053] In some embodiments, the Fc peptide contains a native FcRn binding site. In some embodiments, the Fc peptide does not have effector function. In some embodiments, the Fc peptide includes 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 designation. In some embodiments, the modifications that reduce effector function also include a substitution of Gly at position 329 according to EU designation.

[0054] In some embodiments, the Fc polypeptide comprises amino acid changes relative to the native Fc sequence that prolong its serum half-life. In some embodiments, the amino acid changes comprise substitutions of Tyr at position 252, Thr at position 254, and Glu at position 256, according to EU numbers.

[0055] In some implementations, the Fc peptide specifically binds to TfR.

[0056] In some embodiments, the Fc polypeptide comprises at least two substitutions at positions selected from the group consisting of the EU designations: 384, 386, 387, 388, 389, 390, 413, 416, and 421. In some embodiments, the Fc polypeptide comprises at least three, four, five, six, seven, eight, or nine substitutions at said positions.

[0057] In some embodiments, the Fc polypeptide further comprises one, two, three, or four substitutions at positions including 380, 391, 392, and 415 according to EU designations. In some embodiments, the Fc polypeptide further comprises one, two, or three substitutions at positions including 414, 424, and 426 according to EU designations.

[0058] 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.

[0059] In some embodiments, the Fc polypeptide comprises at least one position selected from the following: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0060] In some embodiments, the Fc polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from the following: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0061] In some embodiments, the Fc polypeptide comprises the following 11 positions: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0062] In some embodiments, the Fc polypeptide has a CH3 domain having at least 85%, at least 90%, or at least 95% identity with amino acids 111-217 of any of SEQ ID NO:34-38, 58, and 60-90. In some embodiments, residues at at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the EU index positions corresponding to any of SEQ ID NO:34-38, 58, and 60-90 are not deleted or substituted.

[0063] In some embodiments, the Fc peptide binds to the apical domain of the TfR. In some embodiments, protein binding to the TfR does not substantially inhibit transferrin binding to the TfR.

[0064] In some embodiments, the Fc peptide has at least 75% or at least 80%, 85%, 90%, 92%, or 95% amino acid sequence identity compared to the corresponding wild-type Fc peptide. In some embodiments, the corresponding wild-type Fc peptide is a human IgG1, IgG2, IgG3, or IgG4 Fc peptide.

[0065] In some embodiments, the Fc polypeptide does not include the variable region sequence of the immunoglobulin heavy chain and / or light chain or its antigen-binding portion.

[0066] In some embodiments, this document provides a polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising an Fc polypeptide linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof, wherein the Fc polypeptide contains one or more modifications that promote heterodimerization with another Fc polypeptide. In some embodiments, this document provides a vector comprising said polynucleotide. In some embodiments, this document provides a host cell comprising said polynucleotide or said vector. In some embodiments, the host cell further comprises a polynucleotide containing a nucleic acid sequence encoding another Fc polypeptide. In some embodiments, this document provides a method for preparing the polypeptide described herein, the method comprising culturing a host cell under conditions that cause expression of the polypeptide encoded by the polynucleotide.

[0067] In some respects, this document provides a protein comprising:

[0068] (a) A first polypeptide chain comprising a modified Fc polypeptide specifically bound to TfR;

[0069] (b) a second polypeptide chain, wherein the first polypeptide chain comprises an Fc polypeptide, wherein the first and second polypeptide chains form an Fc dimer; and

[0070] (c) An ERT enzyme, an ERT enzyme variant or a catalytically active fragment thereof, wherein the ERT enzyme, ERT enzyme variant or a catalytically active fragment thereof is linked to the modified Fc polypeptide of (a) or the Fc polypeptide of (b).

[0071] In some embodiments, the ERT enzyme is an IDS, an IDS variant, or a catalytically active fragment thereof. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 91, 92, 114, 230, and 234. In some embodiments, the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NO: 91, 92, 114, 230, and 234.

[0072] In some embodiments, the ERT enzyme is SGSH, an SGSH variant, or a catalytically active fragment thereof. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any of SEQ ID NO: 119 and 120. In some embodiments, the ERT enzyme comprises the amino acid sequence of any of SEQ ID NO: 119 and 120.

[0073] In some embodiments, the ERT enzyme is ASM, an ASM variant, or a catalytically active fragment thereof. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 121, 122, and 123. In some embodiments, the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NO: 121, 122, and 123.

[0074] In some embodiments, the ERT enzyme is a GBA, a GBA variant, or a catalytically active fragment thereof. In some embodiments, the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any of SEQ ID NO: 93 and 94. In some embodiments, the ERT enzyme comprises the amino acid sequence of any of SEQ ID NO: 93 and 94.

[0075] In some embodiments, the ERT enzyme is linked to the modified Fc polypeptide in (a). In some embodiments, the ERT enzyme is linked to the Fc polypeptide in (b). In some embodiments, the Fc polypeptide in (b) is not modified to bind to the BBB receptor. In some embodiments, the Fc polypeptide in (b) is a modified Fc polypeptide that specifically binds to TfR.

[0076] In some embodiments, the ERT enzyme is linked (e.g., fused) to the modified Fc polypeptide of (a) or (b) via a peptide bond or via a 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 ERT enzyme is not linked to the modified Fc polypeptide of (a) or (b) via a chemical cross-linking agent; for example, the fusion polypeptide does not include a non-peptide bond or non-peptide linker.

[0077] In some embodiments, the ERT enzyme is linked to the N-terminus of (a) the modified Fc polypeptide or (b) the Fc polypeptide. In some embodiments, the ERT enzyme is linked to the C-terminus of (a) the modified Fc polypeptide or (b) the Fc polypeptide.

[0078] In some embodiments, the protein comprises two ERT enzymes. In some embodiments, one ERT enzyme is linked to the modified Fc polypeptide (a) and the other ERT enzyme is linked to the Fc polypeptide (b). In some embodiments, both ERT enzymes are linked to the N-terminus or both are linked to the C-terminus of the respective Fc polypeptides. In some embodiments, one ERT enzyme is linked to the N-terminus of the modified Fc polypeptide (a) and the other ERT enzyme is linked to the C-terminus of the Fc polypeptide (b). In some embodiments, one ERT enzyme is linked to the C-terminus of the modified Fc polypeptide (a) and the other ERT enzyme is linked to the N-terminus of the Fc polypeptide (b).

[0079] In some embodiments, the Fc peptides of (a) and (b) each contain modifications that promote heterodimerization. In some embodiments, according to EU designations, one of the Fc peptides has a T366W substitution and the other Fc peptide has T366S, L368A, and Y407V substitutions. In some embodiments, the modified Fc peptide of (a) contains a T366W substitution and the Fc peptide of (b) contains T366S, L368A, and Y407V substitutions. In some embodiments, the Fc peptide of (b) is linked to an ERT enzyme IDS and comprises the amino acid sequence of any one of SEQ ID NO: 117, 232, and 236. In some embodiments, the modified Fc peptide of (a) contains T366S, L368A, and Y407V substitutions and the Fc peptide of (b) contains a T366W substitution. In some embodiments, the Fc polypeptide of (b) is linked to the ERT enzyme IDS and contains the amino acid sequence of any one of SEQ ID NO: 118, 233 and 237.

[0080] In some embodiments, the modified Fc polypeptide of (a) and / or the Fc polypeptide of (b) contains 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) includes modifications that reduce effector function. In some embodiments, the modification that reduces effector function is a substitution of Ala at position 234 and Ala at position 235 according to EU numbering. 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 Fc polypeptide of (b) is linked to an ERT enzyme IDS and contains the amino acid sequence of any one of SEQ ID NO: 115, 231, and 235. In some embodiments, the Fc polypeptide of (b) is linked to an ERT enzyme SGSH and contains the amino acid sequence of any one of SEQ ID NO: 149, 150, 152, and 153.

[0081] In some embodiments, (a) the modified Fc polypeptide and / or (b) the Fc polypeptide comprises amino acid changes relative to the native Fc sequence that prolong the serum half-life. In some embodiments, the amino acid changes comprise substitutions of Tyr at position 252, Thr at position 254, and Glu at position 256 according to EU numbers.

[0082] In some embodiments, the modified Fc polypeptide comprises at least two substitutions at positions selected from the group consisting of the following according to EU numbers: 384, 386, 387, 388, 389, 390, 413, 416, and 421. In some embodiments, the modified Fc polypeptide comprises at least three, four, five, six, seven, eight, or nine substitutions at said positions.

[0083] In some embodiments, the modified Fc polypeptide further comprises one, two, three, or four substitutions at positions including 380, 391, 392, and 415 according to EU numbers. In some embodiments, the modified Fc polypeptide further comprises one, two, or three substitutions at positions including 414, 424, and 426 according to EU numbers.

[0084] 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.

[0085] In some embodiments, the modified Fc polypeptide contains at least one position selected from the following: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0086] In some embodiments, the modified Fc polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from the following: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0087] In some embodiments, the modified Fc polypeptide comprises the following 11 positions: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0088] In some embodiments, the modified Fc polypeptide has a CH3 domain having at least 85%, at least 90%, or at least 95% identity with amino acids 111-217 of any of SEQ ID NO:34-38, 58 and 60-90, 151 and 156-229. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:156-229. In some embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues at EU index positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 corresponding to any of SEQ ID NO: 34-38, 58 and 60-90, 151 and 156-229 are not deleted or substituted.

[0089] In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:157. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:169. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:181. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:193. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:205. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:217.

[0090] 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 other embodiments, 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.

[0091] 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 other embodiments, 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.

[0092] 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 other embodiments, 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.

[0093] In some embodiments, the modified Fc peptide binds to the apical domain of the TfR. In some embodiments, protein binding to the TfR does not substantially inhibit transferrin binding to the TfR.

[0094] In some embodiments, the modified Fc polypeptide has at least 75% or at least 80%, 85%, 90%, 92%, or 95% amino acid sequence identity compared to the corresponding wild-type Fc polypeptide. In some embodiments, the corresponding wild-type Fc polypeptide is a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide.

[0095] In some embodiments, the uptake of ERT enzyme in the brain (e.g., using appropriate animal models, such as those described herein) is greater than ERT enzyme uptake in the absence of Fc peptides or in the absence of modifications to the Fc peptides that induce TfR binding. In some embodiments, the uptake of ERT enzyme in 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 ERT enzyme uptake in the absence of Fc peptides or in the absence of modifications to the Fc peptides that induce TfR binding.

[0096] In some aspects, this document provides a method for treating LSD, the method comprising administering a protein or peptide as described above to a patient in need. In some embodiments, the method reduces the accumulation of toxic metabolites in the patient, for example, reducing toxic metabolites in the patient's brain and / or cerebrospinal fluid (CSF).

[0097] In a related aspect, this article provides a method for reducing the accumulation of toxic metabolites in a patient with LSD, the method comprising administering a protein or peptide as described above to the patient. In some embodiments, the method reduces the accumulation of toxic metabolites in the patient's brain and / or CSF.

[0098] In some embodiments, LSD is Hunter syndrome, and the ERT enzyme is IDS. In some embodiments, the toxic metabolites comprise disaccharides derived from heparan sulfate and / or disaccharides derived from dermatan sulfate.

[0099] In some embodiments, LSD is Sanfilippo syndrome A, and the ERT enzyme is SGSH. In some embodiments, the toxic metabolite comprises an oligosaccharide (e.g., a hexasaccharide) derived from heparan sulfate.

[0100] In some embodiments, LSD is Niemann-Pick disease, and the ERT enzyme is ASM. In some embodiments, the toxic metabolite comprises sphingomyelin.

[0101] In some embodiments, LSD is Gaucher's disease or Parkinson's disease, and the ERT enzyme is GBA. In some embodiments, the toxic metabolite comprises glucosylceramide.

[0102] 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 said protein or peptide. Illustrative analyses for measuring ERT enzyme activity and substrate accumulation are described herein.

[0103] In some respects, this article provides a pharmaceutical composition comprising a protein or polypeptide as described above and a pharmaceutically acceptable carrier.

[0104] In some aspects, this paper provides a method for monitoring substrate accumulation to assess IDS activity, the method comprising:

[0105] (a) Microbubbles in a cell or fluid sample from a subject who has been given a protein or peptide as described above are broken up to obtain a glycosaminoglycan (GAG) solution to be analyzed.

[0106] (b) Digesting a GAG solution with at least one heparinase (e.g., heparinase I, heparinase II and heparinase III) and chondroitinase B to obtain a GAG-derived disaccharide.

[0107] (c) Analysis of GAG-derived disaccharides by mass spectrometry (e.g., LC-MS / MS); and

[0108] (d) Determine the levels of heparan sulfate-derived disaccharides and / or dermatan sulfate-derived disaccharides, wherein a decrease in the levels of heparan sulfate-derived disaccharides and / or dermatan sulfate-derived disaccharides compared with controls lacking IDS activity indicates an increase in IDS activity in the sample compared with the control.

[0109] In some embodiments, the step of disrupting cells or microbubbles 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.

[0110] In some embodiments, the levels of heparan sulfate-derived disaccharides and / or dermatan sulfate-derived disaccharides 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 heparan sulfate-derived disaccharides and / or dermatan sulfate-derived disaccharides in controls lacking IDS activity. In some embodiments, the control is a cell or tissue sample of the same tissue type obtained from the subject prior to administration of the protein or peptide. In some embodiments, the control is a cell or tissue sample of the same tissue type known to lack IDS activity. In some implementations, the protein or peptide 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.

[0111] In other respects, this paper provides a method for monitoring substrate accumulation to assess SGSH activity, the method comprising:

[0112] (a) Microbubbles in a cell or fluid sample from a subject who has been given a protein or peptide as described above are broken up to obtain a glycosaminoglycan (GAG) solution to be analyzed.

[0113] (b) Digesting a GAG solution with at least one heparinase to obtain a GAG-derived disaccharide;

[0114] (c) Analysis of GAG-derived disaccharides by mass spectrometry (e.g., LC-MS / MS); and

[0115] (d) Determine the level of heparan sulfate-derived disaccharides, where a decrease in the level of heparan sulfate-derived disaccharides compared with a control lacking SGSH activity indicates an increase in SGSH activity in the sample compared with the control.

[0116] 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 St. Philippian syndrome A.

[0117] In some embodiments, the level of heparan sulfate-derived disaccharide 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 disaccharide in a control lacking SGSH activity. In some embodiments, the control is a cell or tissue sample of the same tissue type obtained from the subject prior to administration of the protein or peptide. In some embodiments, the control is a cell or tissue sample of the same tissue type known to lack SGSH activity. In some embodiments, the protein or peptide 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-fold compared to the SGSH activity in the control.

[0118] In other aspects, this document provides a method for delivering an agent across the mammalian brain's blood-brain barrier (BBB), the method comprising exposing the BBB to a protein that binds to a TfR with an affinity of about 50 nM to about 250 nM, wherein the protein is bonded to the agent and the bonded agent is delivered across the BBB. In some embodiments, the maximum concentration (C) of the agent in the mammalian brain is... 最大 The effect is improved. In some embodiments, the agent can be used to treat LSD.

[0119] In other respects, this document provides a method for treating LSD, the method 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 an agent for treating LSD, thereby exposing the mammalian brain to the agent. In some embodiments, the agent, compared to a reference protein linked to a TfR with a weaker affinity, causes the C0 of the agent in the brain to be reduced. 最大 There is some improvement. In some implementations, the reference protein binds to TfR with an affinity of about 600 nM or less.

[0120] 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 apical domain of the TfR.

[0121] In some embodiments, the protein binds to the TfR with an affinity of about 100 nM to about 200 nM. In some embodiments, the protein binds to the TfR with an affinity of about 110 nM to about 150 nM.

[0122] In some embodiments, the therapeutically effective concentration of the agent is a concentration capable of treating one or more symptoms of LSD in mammals. In some embodiments, the agent is a protein replacement therapy. In some embodiments, the agent or protein replacement therapy is an enzyme.

[0123] In some embodiments, when the enzyme is linked to said protein, the accumulation of toxic metabolites in the brain of mammals with LSD is reduced to a greater extent compared to 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 comprise heparin sulfate-derived disaccharides and / or dermatan sulfate-derived disaccharides. In some embodiments, the enzyme is SGSH and the LSD is San Philippe 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.

[0124] 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.

[0125] In some embodiments, the protein is a modified Fc polypeptide containing a non-natural binding site capable of binding TfR. In some embodiments, the protein comprises an antibody variable region that specifically binds to TfR. In some embodiments, the protein comprises an antibody fragment. In some embodiments, the protein comprises Fab or scFv.

[0126] In some implementations, the protein linked to the agent is administered as part of a pharmaceutically acceptable carrier. Attached Figure Description

[0127] Figure 1 The purification and analysis of the IDS-Fc fusion protein, which includes an Fc polypeptide linked to iduronic acid-2-sulfatase (IDS) and a modified Fc polypeptide bound to transferrin receptor (TfR), are shown.

[0128] Figure 2 It shows the Figure 1 The binding affinity analysis of the IDS-Fc fusion protein confirmed that the fusion protein binds to TfR.

[0129] Figure 3 Provide display Figure 1 Data on the in vitro IDS activity of the IDS-Fc fusion protein analyzed in this study.

[0130] Figure 4This study provides data demonstrating that the levels of heparan sulfate-derived disaccharides in IDS-deficient knockout cells were increased as assessed by LC-MS / MS analysis, and that IDS expression in IDS knockout (KO) cells rescued the knockout phenotype.

[0131] Figures 5A to 5D : Figure 5A This indicates that IDS-Fc fusion proteins containing the same TfR-binding Fc polypeptide (i.e., CH3C.35.21.17), acting as either an N-terminal or C-terminal monoenzyme, reverse the accumulation of heparan sulfate and dermatan sulfate in IDS KO cells. Figure 5B This indicates that the N-terminal single enzyme (“ETV:IDS 35.21.17”) has similar cellular efficacy to IDS. Figure 5C This shows the accumulation of S in fibroblasts from MPS II patients treated with IDS-Fc fusion protein (“ETV:IDS”) or IDS. 35 - The dose-dependent reduction of sulfate-labeled proteins; n=8. Figure 5D This illustrates the effects of increased doses of IDS-Fc fusion protein (“ETV:IDS”) or IDS on S in MPS II patient fibroblasts with or without 5 mM M6P. 35 Assessment of M6PR-dependent clearance of tagged proteins; n=3. Figures 5C to 5D “ETV:IDS” = ETV:IDS 35.23.2; The graph shows the mean ± SEM of the repeated experiments.

[0132] Figure 6 Provides data on the levels of heparan sulfate and dermatan sulfate over time in the serum of wild-type (WT) mice administered the agent or IDS KO mice administered IDS or the IDS-Fc fusion protein (“ETV:IDS”). “ETV:IDS” = ETV:IDS 35.21.

[0133] Figure 7Data are provided to illustrate the following: Total sGAG levels (referred to as “total sGAG levels”) of disaccharides DOS0, DOA0, and DOA4 in peripheral tissues of IDS KO mice were assessed seven days after a single intravenous injection of 40 mg / kg IDS-Fc fusion protein (“ETV:IDS”) or 5.3 mg / kg IDS, compared with vector-treated IDS KO and wild-type mice; IDS KO group n=8 and wild-type group n=3. Data are presented as mean ± SEM. p-values: one-way ANOVA plus Dunnett multiple comparison test; ** p < 0.01 and **** p < 0.0001. “ETV:IDS” = ETV:IDS 35.21.

[0134] Figure 8 Provides instructions for human TfR knock-in (TfR knock-in) following peripheral administration of the IDS-Fc fusion protein ETV:IDS 35.21 or the control IDS-Fc fusion protein lacking a mutation conferring TfR binding (“IDS:Fc”). ms / hu Data on the concentration of IDS-Fc fusion protein in the brains of KI mice.

[0135] Figures 9A to 9B : Figure 9A Instructions are provided regarding TfR following peripheral administration of the IDS-Fc fusion protein ETV:IDS 35.21.17.2 or ETV:IDS 35.23.2 or a control IDS-Fc fusion protein lacking a mutation conferring TfR binding. ms / hu Data on the concentration of IDS-Fc fusion protein in the brains of KI mice. Figure 9B Instructions are provided for TfR following a single intravenous injection of a dose of 50 mg / kg. ms / hu Data on liver concentrations of the IDS-Fc fusion protein ETV:IDS 35.21 or IDS:Fc in KI mice; n=4–5. Plotted to show mean ± SEM.

[0136] Figures 10A to 10C This indicates that ETV:IDS enables IDS KO x TfR ms / hu GAGs were reduced in the brain and peripheral tissues of KI mice. As described in Example 2, IDS KO x TfR ms / hu KI mice were administered a single intravenous injection of 40 mg / kg ETV:IDS or 14.2 mg / kg IDS, or a four-week dose. KO x TfR was measured after each single dose with IDS. ms / hu In KI mice, serum ( Figure 10A ) and organizations Figure 10B IDS concentration in the sample. Tissue PK is shown at 2 h post-dose; n=4. Graphs show mean ± SEM and p-values: unpaired t-test analysis. Figure 10C The measurement of IDS KO x TfR after a single dose or multiple doses of ETV:IDS or IDS is shown. ms / hu The levels of disaccharides DOSO, DOA0, and DOA4 (“total sGAG levels”) in the brain, CSF, and peripheral tissues of KI mice were compared with those in vector-treated and wild-type mice; each IDS KO xTfR ms / hu The KI group had n=8 and the wild-type group had n=5. The graph shows the mean ± SEM and p-values: one-way ANOVA with Durner's multiple comparison test; ** p < 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

[0137] Figure 11 The purification and analysis of the ASM-Fc fusion protein, which contains the Fc region linked to two acidic sphingomyelinase (ASM) enzymes, are shown.

[0138] Figure 12 Provide display Figure 11 Data on the in vitro ASM activity of the ASM-Fc fusion protein analyzed in this study.

[0139] Figure 13 Providing analysis using imaging-based methods to demonstrate, for example Figure 11 The data analyzed showed that the ASM-Fc fusion protein reduced sphingomyelin accumulation in ASM KO cells.

[0140] Figure 14 The analysis provided using LC-MS / MS indicates that, for example Figure 11 The data analyzed showed that the ASM-Fc fusion protein reduced sphingomyelin accumulation in ASMKO cells.

[0141] Figure 15 Data are provided demonstrating the in vitro N-sulfoglucosamine sulfonylhydrolase (SGSH) activity of the SGSH-Fc fusion protein described in Example 6.

[0142] Figure 16 Provide data on the increased levels of heparan sulfate-derived disaccharides in SGSH-deficient knockout (KO) cells as assessed by LC-MS / MS analysis. n=3–4 independent cell lines; data are presented as mean ± sem.

[0143] Figure 17 Provide explanation Figure 15The analysis of data showed that the SGSH-Fc fusion protein reversed the accumulation of heparan sulfate in SGSH KO cells.

[0144] Figure 18 The hTfR affinity of engineered TfR-binding peptides and their interaction with TfR were demonstrated. ms / hu Relationship between brain exposure over time in KI mice. Points represent TfR. ms / hu Cumulative brain exposure (AUC) over time for different engineered TfR-binding peptide affinity variants in KI mice following a single 50 mg / kg dose. Brain concentrations of the peptide (as measured by huIgG1) were calculated at different days post-dose (range 1–10 days). Data represent a pooled sum of three independent studies, n = 4–5 mice per group per study.

[0145] Figure 19 The hTfR affinity of engineered TfR-binding peptides and their interaction with TfR were demonstrated. ms / hu Relationship between maximum brain concentrations in KI mice. Points represent the maximum brain concentrations of different peptide affinity variants measured on day 1 after a single 50 mg / kg dose. Data represent a pooled sum of three independent studies, n = 4–5 mice per group per study.

[0146] Figure 20 The hTfR affinity of engineered TfR-binding peptides and their interaction with TfR were demonstrated. ms / hu Relationship between the ratio of peptide brain concentration to plasma concentration in KI mice. Points represent the ratio of maximum brain concentration to plasma concentration of different peptide affinity variants measured on day 1 after a single 50 mg / kg dose. Data represent a pooled sum of three independent studies, n = 4–5 mice per group per study.

[0147] Figure 21A and Figure 21B The TfR was demonstrated following a single systemic injection of 50 mg / kg of an anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 peptide fusion. ms / hu Plasma from knock-in (KI) mice Figure 21A ) and brain lysis products ( Figure 21B The concentration of huIgG1 in the sample (mean ± SEM, n = 5 samples per group).

[0148] Figure 21CDemonstrating TfR following a single systemic injection of 50 mg / kg of an anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 peptide fusion. ms / hu Endogenous mouse Aβ concentration in brain lysis products of KI mice (mean ± SEM, n = 5 mice per group).

[0149] Figure 21D Demonstrating the effect of a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 peptide fusions on TfR ms / hu Western blot quantification of actin-normalized brain TfR proteins in brain lysates from KI mice (mean ± SEM, n = 5 per group). Detailed Implementation

[0150] I. Introduction

[0151] We have developed fusion proteins comprising enzyme replacement therapy (ERT) enzymes linked to Fc polypeptides. These proteins can be used to treat lysosomal storage diseases (LSD). In some cases, the protein comprises a dimerized Fc polypeptide, wherein one of the Fc polypeptide monomers is linked to the ERT enzyme. The Fc polypeptide can increase the enzyme's half-life and, in some cases, can be modified to confer additional functional properties to the protein. Fusion proteins that facilitate the delivery of ERT enzymes across the blood-brain barrier (BBB) ​​are also described herein. These proteins comprise an Fc polypeptide and a modified Fc polypeptide, said Fc polypeptide and the modified Fc polypeptide forming a dimer; and an ERT enzyme linked to the Fc region and / or the modified Fc region. The modified Fc region can specifically bind to a BBB receptor, such as a transferrin receptor (TfR). In some embodiments, the ERT enzyme is an iduronate 2-sulfatase (IDS) or a catalytically active variant or fragment of wild-type IDS (e.g., wild-type human IDS). In other embodiments, the ERT enzyme is an N-sulfoglucosamine sulfonylhydrolase (SGSH), acid sphingomyelinase (ASM), β-glucocerebrosidase (GBA), or a catalytically active variant or fragment of wild-type SGSH, ASM, or GBA (e.g., wild-type human SGSH, ASM, or GBA).

[0152] We have also developed methods for delivering therapeutic agents bound to TfR-binding peptides and proteins across the body's border (BBB) ​​to treat diseases. We found that the TfR binding affinity required to deliver therapeutic agents across the BBB depends on the target of the therapeutic agent and the mechanism of action driving its efficacy in treating the disease. Specifically, we found that using peptides and proteins with strong TfR affinity yields larger C... 最大 However, it is relatively quick to remove.

[0153] For some therapies, such as protein replacement therapy using ERT enzymes like IDS (e.g., for treating Hunter syndrome) that can be used to treat LSD, and others, high brain C levels of the therapeutic agent need to be achieved within the administration window. 最大 This is because higher extracellular concentrations lead to increased intracellular protein concentrations. After delivery into the cell, the intracellular half-life of the delivered protein is longer compared to plasma retention time. Furthermore, proteins with high C... 最大 It can be beneficial for enzyme replacement because high enzyme concentrations can promote an increased substrate turnover rate induced by the enzyme. To improve brain C 最大 Peptides and proteins with a TfR affinity range of 50-250 nM are particularly suitable.

[0154] II. Definition

[0155] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “described” include plural indicators. Thus, for example, references to “a polypeptide” may include two or more such molecules, etc.

[0156] As used herein, the terms “about” and “approximately”, when used to modify a quantity specified in a numerical value or range, indicate that the numerical value, as well as reasonable deviations from the value known to those skilled in the art (e.g., ±20%, ±10%, or ±5%), are within the intended meaning of the stated value.

[0157] "Enzyme replacement therapy enzyme" or "ERT enzyme" refers to an enzyme lacking in lysosomal storage diseases. "ERT enzyme variant" refers to a functional variant of the wild-type ERT enzyme or a fragment thereof, including allelic variants and splice variants, wherein, for example, when analyzed under identical conditions, the ERT enzyme variant 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 ERT enzyme or a fragment thereof. "Catalytically active fragment" of an ERT enzyme refers to a portion of the full-length ERT enzyme or a variant thereof, wherein, for example, when analyzed under identical conditions, the catalytically active fragment 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 full-length ERT enzyme or a variant thereof.

[0158] As used herein, “iduronate sulfatase,” “iduronate-2-sulfatase,” or “IDS” refers to iduronate-2-sulfatase (EC 3.1.6.13), an enzyme involved in the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. IDS deficiency is associated with mucopolysaccharidosis II (also known as Hunter syndrome). As used herein as a component of a protein containing an Fc polypeptide, the term “IDS” refers to a catalytically active functional variant of wild-type IDS or a fragment thereof, including allelic variants and splice variants. The sequence of human IDS isotype I is the human sequence designated as the canonical sequence, available under UniProt entry P22304, and encoded by the human IDS gene at Xq28. The full-length sequence is provided as SEQ ID NO:91. As used herein, a “mature” IDS sequence refers to a polypeptide chain lacking the signal and propeptide sequences of the naturally occurring full-length polypeptide chain. The amino acid sequence of a mature human IDS polypeptide is provided in SEQ ID NO:92, corresponding to amino acids 34-550 of the full-length human sequence. As used herein, a “truncated” IDS sequence refers to a catalytically active fragment of a naturally occurring full-length polypeptide chain. An exemplary truncated human IDS polypeptide amino acid sequence is provided in SEQ ID NO:114, corresponding to amino acids 26-550 of the full-length human sequence. The structure of human IDS has been adequately characterized. Illustrative structures are available under PDB accession code 5FQL. The structure is also described in Nat. Comm. 8:15786 doi: 10.1038 / ncomms15786, 2017. Non-human primate IDS sequences, including those of chimpanzees (UniProt entry K7BKV4) and rhesus monkeys (UniProt entry H9FTX2), have also been described. Mouse IDS sequences are available under UniProt entry Q08890. IDS variants, for example, 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 IDS or its fragments when analyzed under the same conditions. Catalytically active IDS fragments, for example, 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 full-length IDS or its variants when analyzed under the same conditions.

[0159] As used herein, “sulfoglucosamine sulfonylhydrolase,” “N-sulfoglucosamine sulfonylhydrolase,” or “SGSH” refers to N-sulfoglucosamine sulfonylhydrolase (EC 3.10.1.1), an enzyme involved in the lysosomal degradation of heparan sulfate. Mutations in this gene are associated with St. Philip's syndrome A, a type of lysosomal storage disease, mucopolysaccharidosis III, caused by impaired degradation of heparan sulfate. As used herein as a component of a protein containing an Fc polypeptide, the term “SGSH” refers to catalytically active and encompasses functional variants of wild-type SGSH or fragments thereof, including allelic variants and splice variants. The sequence of human SGSH is available under UniProt entry P51688 and is encoded by the human SGSH gene at 17q25.3. The full-length sequence is provided as SEQ ID NO:119. As used herein, a “mature” SGSH sequence refers to a polypeptide chain lacking the signal sequence of the naturally occurring full-length polypeptide chain. The amino acid sequence of a mature human SGSH polypeptide is provided as SEQ ID NO:120, corresponding to amino acids 21-502 of the full-length human sequence. As used herein, a “truncated” SGSH sequence refers to a catalytically active fragment of the naturally occurring full-length polypeptide chain. The structure of human SGSH has been adequately characterized. Illustrative structures are available under PDB accession code 4MHX. Non-human primate SGSH sequences, including chimpanzee (UniProt entry K7C218), have also been described. Mouse SGSH sequences are available under UniProt entry Q9EQ08. SGSH variants, for example, 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 fragments thereof when analyzed under the same conditions. Catalytically active SGSH fragments, for example, have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length SGSH or its variants when analyzed under the same conditions.

[0160] As used herein, “acidic sphingomyelinase,” “sphingomyelin phosphodiesterase,” or “ASM” 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 type B). As used herein as a component of a protein containing an Fc polypeptide, the term “ASM” refers to catalytically active and encompasses functional variants of wild-type ASM or fragments thereof, including allelic variants and splice variants. The sequence of human ASM isotype 1 is the human sequence designated as the canonical sequence, available under UniProt entry P17405, and encoded by the human SMPD1 gene at 11p15.4. The full-length sequence is provided as SEQ ID NO:121. As used herein, a “mature” ASM sequence refers to a polypeptide chain lacking the signal sequence of the naturally occurring full-length polypeptide chain. The amino acid sequence of a mature human ASM polypeptide is provided in SEQ ID NO:122, corresponding to amino acids 47-629 of the full-length human sequence. As used herein, a “truncated” ASM sequence refers to a catalytically active fragment of the naturally occurring full-length polypeptide chain. An exemplary truncated human ASM polypeptide amino acid sequence is provided in SEQ ID NO:123, corresponding to amino acids 47-620 of the full-length human sequence. The structure of human ASM has been adequately characterized. Illustrative structures are available under PDB accession code 5I81. Non-human primate ASM sequences, including chimpanzee (UniProt entry H2Q319), have also been described. Mouse ASM sequences are available under UniProt entry Q04519. ASM variants, for example, 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 ASM or fragments thereof when analyzed under the same conditions. Catalytically active ASM fragments, for example, have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length ASM or its variants when analyzed under the same conditions.

[0161] “β-glucocerebrosidase” or “GBA” is also known as glucosylceramidinase (EC 3.2.1.45). As used herein, the term refers to a lysosomal enzyme possessing glucosylceramidinase activity and catalyzing the breakdown of glucosylceramide into ceramide and glucose. GBA deficiency is associated with Gaucher disease and Parkinson's disease. As used herein as a component of proteins containing Fc polypeptides, the term “GBA” refers to catalytically active and encompasses functional variants of wild-type GBA or fragments thereof, including allelic variants and splice variants. The sequence of the human GBA long isotype is designated as the canonical sequence, available under UniProt entry P04062-1, and encoded by the human GBA gene at 1q22. The full-length sequence is provided as SEQ ID NO:93. As used herein, a “mature” GBA sequence refers to a polypeptide chain lacking the signal and propeptide sequences of the naturally occurring full-length polypeptide chain. The amino acid sequence of the mature human GBA polypeptide is provided in SEQ ID NO:94, which corresponds to amino acids 40-536 of the full-length human sequence. As used herein, a “truncated” GBA sequence refers to a catalytically active fragment of the naturally occurring full-length polypeptide chain. The structure of human GBA has been well characterized. Nearly 20 crystal structures of GBA are available. Non-human primate GBA sequences, including those of chimpanzees (UniProt entry Q9BDT0) and orangutans (UniProt entry Q5R8E3), have also been described. Mouse GBA sequences are available under UniProt entry P17439. GBA variants, for example, 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 fragments thereof when analyzed under the same conditions. Catalytically active GBA fragments, for example, have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length GBA or its variants when analyzed under the same conditions.

[0162] As used herein, “transferrin receptor” or “TfR” refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is described in SEQ ID NO:96. Transferrin receptor protein 1 sequences in other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; bovine, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term “transferrin receptor” also encompasses allelic variants of exemplary reference sequences (e.g., human sequences) encoded by genes at chromosomal loci of transferrin receptor protein 1. The full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and a apical domain. The apical domain sequence of human transferrin receptor 1 is described in SEQ ID NO:238.

[0163] As used herein, "fusion protein" or "[ERT enzyme]-Fc fusion protein" refers to a dimer protein comprising a first Fc polypeptide (i.e., "[ERT]-Fc fusion polypeptide") linked (e.g., fused) to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and a second Fc polypeptide forming an Fc dimer with the first Fc polypeptide. The second Fc polypeptide may also be linked (e.g., fused) to an 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 via peptide bonds or via peptide linkers. The first Fc polypeptide and / or the second Fc polypeptide may be modified Fc polypeptides containing one or more modifications that promote heterodimerization with another Fc polypeptide. The first Fc polypeptide and / or the second Fc polypeptide may be modified Fc polypeptides containing one or more modifications that enable them to bind to the transferrin receptor. The first Fc polypeptide and / or the second Fc polypeptide may be modified Fc polypeptides, wherein the modified Fc polypeptides contain one or more modifications that reduce effector function. The first Fc polypeptide and / or the second Fc polypeptide may be modified Fc polypeptides, wherein the modified Fc polypeptides contain one or more modifications that prolong serum half-life.

[0164] As used herein, "fusion polypeptide" or "[ERT enzyme]-Fc fusion polypeptide" refers to an Fc polypeptide that is linked (e.g., fused) to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof. The Fc polypeptide may be linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof via peptide bonds or peptide linkers. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that promote its heterodimerization with another Fc polypeptide. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that enable it to bind to the transferrin receptor. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that reduce effector function. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that prolong serum half-life.

[0165] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by Ig folding as a domain. Fc polypeptides contain a constant region sequence including at least a CH2 domain and / or a CH3 domain and may contain at least a portion of a hinge region. Generally, Fc polypeptides do not contain a variable region.

[0166] "Modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, such as substitution, deletion or insertion, compared to the wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the native Fc polypeptide.

[0167] The term "FcRn" refers to the neonatal Fc receptor. Binding of Fc peptides to FcRn reduces Fc peptide clearance and increases serum half-life. Human FcRn protein is a heterodimer composed of a protein approximately 50 kDa in size that resembles a major histocompatibility (MHC) class I protein and a β2-microglobulin approximately 15 kDa in size.

[0168] As used herein, "FcRn binding site" refers to the region in an Fc polypeptide that binds to FcRn. In human IgG, using EU index numbers, FcRn binding sites include T250, L251, M252, I253, S254, R255, T256, T307, E380, M428, H433, N434, H435, and Y436. These positions correspond to positions 20 to 26, 77, 150, 198, and 203 to 206 of SEQ ID NO:1.

[0169] As used in this article, "natural FcRn binding site" refers to a region in an Fc polypeptide that binds to FcRn and has the same amino acid sequence as the region in a naturally occurring Fc polypeptide that binds to FcRn.

[0170] As used herein, the terms “CH3 domain” and “CH2 domain” refer to immunoglobulin constant region domain polypeptides. For the purposes of this application, a CH3 domain polypeptide refers to the amino acid segment from approximately position 341 to approximately position 447 according to EU numbering, and a CH2 domain polypeptide refers to the amino acid segment from approximately position 231 to approximately position 340 according to EU numbering, excluding the hinge region sequence. CH2 and CH3 domain polypeptides may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, whereby the CH2 domain is numbered 1-110 and the CH3 domain is numbered 1-107 according to IMGT scientific charts (IMGT website). The CH2 and CH3 domains are part of the immunoglobulin Fc region. The Fc region refers to the amino acid segment from approximately position 231 to approximately position 447 according to EU numbering, but as used herein, may include at least a portion of the antibody hinge region. The illustrative hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO:95).

[0171] The terms “wild type,” “natural,” and “naturally existing” for CH3 or CH2 domains are used in this paper to refer to domains having a sequence that exists in nature.

[0172] As used herein, the term "mutant" for mutant polypeptides or mutant polynucleotides may be used interchangeably with "variant." A variant of a given wild-type CH3 or CH2 domain reference sequence may include naturally occurring allelic variants. A "non-natural" CH3 or CH2 domain refers to a variant or mutant domain of a naturally occurring CH3 or CH2 domain polynucleotide or polypeptide that is not present in cells in nature and is produced through genetic modification, for example, using genetic engineering or mutagenesis techniques. A "variant" includes any domain containing at least one amino acid mutation relative to the wild type. Mutations may include substitutions, insertions, and deletions.

[0173] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid simulants that function in a manner similar to naturally occurring amino acids.

[0174] Naturally occurring amino acids are those encoded by the genetic code, as well as those subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimics" are compounds whose structure differs from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0175] Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0176] Amino acids can be referred to in this article by either the commonly known three-letter symbol or by the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0177] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L-amino acids and D-amino acids.

[0178] As used herein, the term "protein" refers to a polypeptide or a dimer (i.e., both) or a polymer (i.e., three or more) of a single-chain polypeptide. A single-chain polypeptide of a protein can be linked by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.

[0179] The terms “conservative substitution,” “conservative mutation,” or “variant of conserved modification” refer to a change that causes an amino acid to be replaced by another amino acid that can be classified as having similar characteristics. Examples of conserved amino acid group categories defined in this way may include: “charged / polar group,” including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); “aromatic group,” including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and “aliphatic group,” including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Within each group, subgroups can also be identified. For example, the charged or polar amino acid group can be further divided into subgroups including: a "positively charged subgroup" containing Lys, Arg, and His; a "negatively charged subgroup" containing Glu and Asp; and a "polar subgroup" containing Asn and Gln. In another example, the aromatic or cyclic group can be further divided into subgroups including: a "nitrogen-cyclic subgroup" containing Pro, His, and Trp; and a "phenyl subgroup" containing Phe and Tyr. In yet another example, the aliphatic group can be further divided into subgroups such as: a "liphatic nonpolar subgroup" containing Val, Leu, Gly, and Ala; and a "liphatic slightly polar subgroup" containing Met, Ser, Thr, and Cys. Examples of conserved mutation categories include amino acid substitutions within the above subgroups, such as, but not limited to: Lys substitution for Arg or vice versa, to maintain a positive charge; Glu substitution for Asp or vice versa, to maintain a negative charge; Ser substitution for Thr or vice versa, to maintain free -OH; and Gln substitution for Asn or vice versa, to maintain free -NH2. In some embodiments, hydrophobic amino acids are substituted, for example, naturally occurring hydrophobic amino acids at the active site to retain hydrophobicity.

[0180] In the context of two or more polypeptide sequences, the term "identity" or "identity percentage" refers to the identity of two or more sequences or subsequences when compared and aligned on a comparison window or designated region to obtain maximum correspondence, as measured by using sequence comparison algorithms or by manual alignment and visual inspection, that is, the same in a designated region or has a specified percentage of the same amino acid residues, such as 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 greater.

[0181] For peptide sequence comparisons, typically an amino acid sequence serves as a reference sequence for comparison with candidate sequences. Alignment can be performed using various methods available to those skilled in the art (e.g., visual alignment) or using publicly available software with known algorithms to achieve optimal alignment. Such programs include BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to achieve optimal alignment can be determined by those skilled in the art. For sequence comparisons of peptide sequences used for the purposes of this application, the BLASTP algorithm for aligning two protein sequences, Standard Protein BLAST, is used with default parameters.

[0182] When the terms "corresponds to," "reference...determined," or "reference...number" are used in the context of identifying a given amino acid residue in a polypeptide sequence, it refers to the position of the residue in the reference sequence when the given amino acid sequence is best aligned and compared with a reference sequence. Thus, for example, when an amino acid residue in the Fc polypeptide to be modified is aligned with an amino acid in SEQ ID NO:1 during best alignment with SEQ ID NO:1, the residue "corresponds to" an amino acid in SEQ ID NO:1. The polypeptide aligned to the reference sequence does not need to be the same length as the reference sequence.

[0183] As used herein, “binding affinity” refers to the strength of the non-covalent interaction between two molecules (e.g., a single binding site on a polypeptide and its bound target, such as the transferrin receptor). Therefore, by way of example, unless otherwise indicated or clearly understood from the context, the term may refer to a 1:1 interaction between a polypeptide and its target. This can be measured by measuring the equilibrium dissociation constant (K0). D To quantify binding affinity, the equilibrium dissociation constant refers to the dissociation rate constant (k). d ,time -1 Divide by the association rate constant (k) a ,time -1 M -1 K can be determined by measuring the kinetics of complex formation and dissociation. D For example, using surface plasmon resonance (SPR) methods, such as the Biacore™ system; kinetic exclusion analysis, such as KinExA ® ; and BioLayer intervention (e.g., using ForteBio) ® Octet ®(Platform). As used herein, “binding affinity” includes not only formal binding affinity, such as those reflecting a 1:1 interaction between a peptide and its target, but also calculated KB, which reflects affinity binding. D 'The apparent affinity to be achieved.'

[0184] As used herein, when referring to engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies as described herein, the terms "specific binding" or "selective binding" to a target, such as TfR, refer to a binding reaction by which the engineered TfR-binding peptide, TfR-binding peptide, or TfR-binding antibody binds to the target with greater affinity, greater affinity, and / or greater persistence compared to binding to a structurally dissimilar target. In a typical embodiment, when analyzed under the same affinity assay conditions, the engineered TfR-binding peptide, TfR-binding peptide, or TfR-binding antibody exhibits at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for a specific target (e.g., TfR) compared to an unrelated target. As used herein, the terms "specifically binds to a specific target (e.g., TfR)," "specifically binds to a specific target (e.g., TfR)," or "is specific to a specific target (e.g., TfR)" can be exemplified, for example, by molecules that exhibit, for example, a 10% specificity to the target to which they bind. -4 M or smaller (e.g., 10) -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 The equilibrium dissociation constant K of M) D In some embodiments, engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies specifically bind to epitopes on TfR that are conserved across species (e.g., structurally conserved across species), such as between non-human primates and humans (e.g., structurally conserved between non-human primates and humans). In some embodiments, engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies may exclusively bind to human TfR.

[0185] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is derived from germline variable (V) genes, diversity (D) genes, or linkage (J) genes (and not from constant (Cμ and Cδ) gene segments) and imparts specificity to the antibody's binding to the antigen. Typically, an antibody variable region comprises four conserved "framework" regions interspersed with three hypervariable "complementarity-determining regions."

[0186] The terms “antigen-binding portion” and “antigen-binding fragment” are used interchangeably herein and refer to one or more fragments in an antibody that retain the ability to specifically bind to an antigen via a variable region. Examples of antigen-binding fragments include, but are not limited to, Fab fragments (monovalent fragments consisting of VL, VH, CL, and CH1 domains), F(ab')2 fragments (bivalent fragments containing two Fab fragments linked by disulfide bonds located in the hinge region), single-chain Fv (scFv), disulfide-linked Fv (dsFv), complementarity-determining regions (CDRs), VL (light chain variable region), and VH (heavy chain variable region).

[0187] The terms “treatment” and “treating” are generally used in this document to refer to achieving the desired pharmacological and / or physiological effect. “Treatment” can refer to any marker of successful treatment or improvement of a lysosomal storage disease (e.g., Hunter syndrome, St. Philippian syndrome A, Niemann-Pick disease, Gaucher's disease, or Parkinson's disease), including any objective or subjective parameters such as elimination, remission, improved patient survival, increased survival time or survival rate, reduction of symptoms or making the condition more tolerable for the patient, slowing the rate of degeneration or decline, or improving the patient's physical or mental health. Treatment or improvement of symptoms can be based on objective or subjective parameters. The effects of treatment can be compared with untreated individuals or groups of individuals, or with the same patient before or at different times during treatment.

[0188] As used interchangeably herein, the terms “subject / individual” and “patient” refer to mammals, including but not limited to humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cattle, pigs, horses, and other mammal species. In one implementation, the patient is a human.

[0189] The term “pharmaceuticalally acceptable excipient” refers to an inactive pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, buffers, carriers, or preservatives.

[0190] As used herein, a "therapeutic dose," "therapeutic effective dose," or "therapeutic effective concentration" refers to the amount or concentration of an agent that can treat signs or symptoms of a disease (e.g., LSD) in a subject (e.g., a mammal).

[0191] The term "administration" refers to a method of delivering an agent, compound, or composition to a desired biological site of action. These methods include, but are not limited to, surface 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.

[0192] III. Enzyme Replacement Therapy (ERT)

[0193] Lysosomal storage disorders (LSDs) are inherited metabolic disorders characterized by the accumulation of undigested or partially digested macromolecules, ultimately resulting in cellular dysfunction and clinical abnormalities. Traditionally, LSDs have been defined as lysosomal dysfunction, often categorized according to the substrates involved and including sphingolipidose, oligosaccharidose, mucolipidose, mucopolysaccharidose, lipoprotein storage disorders, neuronal ceroid lipofuscinose, and other lysosomal storage disorders. Recently, the classification of these disorders has been expanded to include deficiencies or defects in other proteins that cause the accumulation of macromolecules, such as proteins essential for the normal post-translational modifications of lysosomal enzymes or proteins important for proper lysosomal transport.

[0194] In some aspects, the fusion protein described herein comprises: (i) an Fc polypeptide, which may contain modifications (e.g., one or more modifications that promote heterodimerization) or may be a wild-type Fc polypeptide; and an ERT enzyme; and (ii) an Fc polypeptide, which may contain modifications (e.g., one or more modifications that promote heterodimerization) or may be a wild-type Fc polypeptide; and optionally an ERT enzyme. In some embodiments, one or both Fc polypeptides may contain modifications that induce binding to blood-brain barrier (BBB) ​​receptors, such as transferrin receptors (TfR). The ERT enzyme may be any enzyme lacking in LSD. The ERT enzyme incorporated into the fusion protein is catalytically active, i.e., it retains the enzyme activity lacking in LSD. In some embodiments, the ERT enzyme is iduronate 2-sulfatase (IDS), which is lacking in Hunter syndrome. In some embodiments, the ERT enzyme is N-sulfoglucosamine sulfonylhydrolase (SGSH), which is deficient in San Philippe syndrome. In some embodiments, the ERT enzyme is acid sphingomyelinase (ASM), which is deficient in Niemann-Pick disease. In some embodiments, the ERT enzyme is β-glucocerebrosidase (GBA), which is deficient in Gaucher's disease and Parkinson's disease.

[0195] In some embodiments, the fusion protein comprising an ERT enzyme and optionally a modified Fc polypeptide (e.g., a TfR-binding Fc polypeptide) that binds to a BBB receptor comprises a catalytically active fragment or variant of wild-type IDS. In some embodiments, the IDS enzyme is a variant or catalytically active fragment of an IDS protein comprising the amino acid sequence of any one 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 greater of the wild-type IDS enzyme activity.

[0196] In some embodiments, the fusion protein comprising an ERT enzyme and optionally a modified Fc polypeptide (e.g., a TfR-binding Fc polypeptide) that binds to a BBB receptor 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 an SGSH protein comprising the amino acid sequence of any one of SEQ ID NO: 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 greater of the wild-type SGSH enzyme activity.

[0197] In some embodiments, the fusion protein comprising an ERT enzyme and optionally a modified Fc polypeptide (e.g., a TfR-binding Fc polypeptide) that binds to a BBB receptor 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 an ASM protein comprising the amino acid sequence of any one of SEQ ID NO: 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 greater of the wild-type ASM enzyme activity.

[0198] In some embodiments, the fusion protein comprising an ERT enzyme and optionally a modified Fc polypeptide (e.g., a TfR-binding Fc polypeptide) that binds to a BBB receptor 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 a GBA protein comprising the amino acid sequence of any one of SEQ ID NO: 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 greater of the wild-type GBA enzyme activity.

[0199] In some embodiments, the ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) or its catalytically active variant or fragment present in the fusion protein described herein retains at least 25% of its activity compared to its activity when not linked to an Fc peptide or a TfR-binding Fc peptide. In some embodiments, the ERT enzyme or its catalytically active 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 linked to an Fc peptide or a TfR-binding Fc peptide. In some embodiments, the ERT enzyme or its catalytically active variant or fragment retains at least 80%, 85%, 90%, or 95% of its activity compared to its activity when not linked to an Fc peptide or a TfR-binding Fc peptide. In some embodiments, fusion to an Fc peptide does not reduce the activity of the ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) or its catalytically active variant or fragment. In some implementations, fusion into the TfR-binding Fc peptide does not reduce the activity of the ERT enzyme.

[0200] IV. Fc peptide modification for blood-brain barrier (BBB) ​​receptor binding

[0201] In some aspects, this document provides fusion proteins capable of transporting across the blood-brain barrier (BBB). Such proteins comprise a modified Fc polypeptide that binds to a BBB receptor. The BBB receptor is expressed on BBB endothelium as well as other cell and tissue types. In some embodiments, the BBB receptor is a transferrin receptor (TfR).

[0202] In this document, EU index numbers are used to number the specified amino acid residues in various Fc modifications, including those that introduce modified Fc peptides that bind to BBB receptors (e.g., TfR). Any Fc peptide (e.g., IgG1, IgG2, IgG3, or IgG4 Fc peptides) may have modifications, such as amino acid substitutions, at one or more positions as described herein.

[0203] The modified Fc polypeptide present in the fusion protein described herein (e.g., enhancing heterodimerization and / or BBB receptor binding) 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 fragments thereof (e.g., fragments of at least 50 amino acids, at least 100 amino acids, or longer). In some embodiments, the native Fc amino acid sequence is the Fc region sequence of SEQ ID NO:1. In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 1-110 of SEQ ID NO:1 or with amino acids 111-217 of SEQ ID NO:1 or fragments thereof (e.g., fragments of at least 50 amino acids, at least 100 amino acids, or longer).

[0204] In some embodiments, the modified Fc polypeptide (e.g., for enhanced heterodimerization and / or BBB receptor binding) comprises at least 50 amino acids corresponding to the amino acid sequence of the native Fc region, or at least 60, 65, 70, 75, 80, 85, 90, or 95 or more, or at least 100 or more amino acids. In some embodiments, the modified Fc polypeptide comprises at least 25 consecutive amino acids corresponding to the amino acid sequence of the native Fc region (such as SEQ ID NO:1), or at least 30, 35, 40, or 45 consecutive amino acids, or 50 consecutive amino acids, or at least 60, 65, 70, 75, 80, 85, 90, or 95 or more, or at least 100 or more consecutive amino acids.

[0205] In some implementations, the domain modified to obtain BBB receptor binding activity is a human Ig CH3 domain, such as the IgG1 CH3 domain. The CH3 domain can belong to any IgG subtype, i.e., derived from IgG1, IgG2, IgG3, or IgG4. In the context of IgG1 antibodies, the CH3 domain refers to the amino acid segment numbered from approximately position 341 to approximately position 447 according to the EU numbering procedure.

[0206] In some implementations, the domain modified to obtain BBB receptor binding activity is a human Ig CH2 domain, such as an IgG CH2 domain. The CH2 domain can belong to any IgG subtype, i.e., derived from IgG1, IgG2, IgG3, or IgG4. In the context of IgG1 antibodies, the CH2 domain refers to the amino acid segment numbered from approximately position 231 to approximately position 340 according to the EU numbering procedure.

[0207] 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 at amino acid positions comprising 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 according to the EU numbering procedure; and in some embodiments, at least four, five, six, seven, eight, nine, or ten substitutions.

[0208] 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 at amino acid positions comprising 274, 276, 283, 285, 286, 287, 288, 289, and 290 according to the EU numbering procedure; and in some embodiments, at least four, five, six, seven, eight, or nine substitutions.

[0209] 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 at amino acid positions comprising 268, 269, 270, 271, 272, 292, 293, 294, 296, and 300 according to the EU numbering procedure; and in some embodiments, at least four, five, six, seven, eight, nine, or ten substitutions.

[0210] 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 at amino acid positions comprising 272, 274, 276, 322, 324, 326, 329, 330, and 331 according to the EU numbering procedure; and in some embodiments, at least four, five, six, seven, eight, or nine substitutions.

[0211] 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 at amino acid positions including 345, 346, 347, 349, 437, 438, 439, and 440, according to the EU numbering procedure; and in some embodiments, at least four, five, six, or seven substitutions.

[0212] 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 at amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering procedure; and in some embodiments, at least four, five, six, seven, eight, or nine substitutions.

[0213] FcRn binding site

[0214] In some aspects, the modified (e.g., BBB receptor-binding) Fc peptide or the Fc peptide present in the fusion protein described herein and not specifically bound to the BBB receptor may also include an FcRn binding site. In some embodiments, the FcRn binding site is located within the Fc peptide or a fragment thereof.

[0215] In some embodiments, the FcRn binding site comprises a natural FcRn binding site. In some embodiments, the amino acid sequence of the FcRn binding site does not contain any amino acid changes relative to the natural FcRn binding site. In some embodiments, the natural FcRn binding site is an IgG binding site, such as a human IgG binding site. In some embodiments, the FcRn binding site contains modifications that alter FcRn binding.

[0216] In some embodiments, the FcRn binding site has one or more mutated (e.g., substituted) amino acid residues, wherein the one or more mutations increase the serum half-life or substantially decrease the serum half-life (i.e., reduce the serum half-life by no more than 25% compared to the corresponding modified Fc peptide having wild-type residues at the mutated site when analyzed under the same conditions). In some embodiments, the FcRn binding site has one or more amino acid residues substituted at positions 250-256, 307, 380, 428, and 433-436 according to the EU numbering sequence.

[0217] In some embodiments, one or more residues located at or near the FcRn binding site are mutated relative to the natural human IgG sequence to prolong the serum half-life of the modified peptide. In some embodiments, the mutation is introduced into one, two, or all three of positions 252, 254, and 256. In some embodiments, the mutation is M252Y, S254T, and T256E. In some embodiments, the modified Fc peptide further comprises the mutations M252Y, S254T, and T256E. In some embodiments, the modified Fc peptide comprises substitutions at one, two, or all three of positions T307, E380, and N434 according to the EU numbering procedure. In some embodiments, the mutation is T307Q and N434A. In some embodiments, the modified Fc peptide comprises the mutations T307A, E380A, and N434A. In some embodiments, the modified Fc peptide comprises substitutions at positions T250 and M428 according to the EU numbering procedure. In some embodiments, the modified Fc peptide comprises the mutations T250Q and / or M428L. In some embodiments, the modified Fc peptide comprises substitutions at positions M428 and N434 according to the EU numbering procedure. In some embodiments, the modified Fc peptide comprises the mutations M428L and N434S. In some embodiments, the modified Fc peptide comprises the N434S or N434A mutation.

[0218] V. Transferrin receptor-binding Fc polypeptide

[0219] This section describes the generation of the modified Fc peptide that binds to the transferrin receptor (TfR) and is capable of crossing the blood-brain barrier (BBB).

[0220] TfR-binding Fc peptides containing mutations in the CH3 domain

[0221] In some embodiments, the modified Fc polypeptide that specifically binds to TfR contains a substitution in the CH3 domain. In some embodiments, the modified Fc polypeptide contains a human Ig CH3 domain, such as an IgG CH3 domain, which is modified to obtain TfR binding activity. The CH3 domain can belong to any IgG subtype, i.e., derived from IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH3 domain refers to the amino acid segment numbered from approximately position 341 to approximately position 447 according to the EU numbering procedure.

[0222] In some embodiments, the modified Fc polypeptide that specifically binds to TfR binds to the apical domain of TfR and can bind to TfR without blocking or otherwise inhibiting transferrin binding to TfR. In some embodiments, transferrin binding to TfR is not substantially inhibited. In some embodiments, transferrin binding to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, transferrin binding to TfR is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0223] In some embodiments, the modified Fc polypeptide that specifically binds to TfR comprises 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 procedure. Illustrative substitutions that may 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, such as 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.

[0224] In some embodiments, at least one of the following positions is substituted: 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, such as 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 conserved substitutions, such as amino acids in the same charge grouping, hydrophobic grouping, side-chain ring structure grouping (e.g., aromatic amino acids), or size grouping and / or polar or nonpolar grouping of designated amino acids at one or more positions in the set. Thus, for example, Ile may be present at positions 384, 386, and / or position 413. In some embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Glu. In other embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Asp. In some embodiments, two, three, four, five, six, seven, or all eight of positions 384, 386, 387, 388, 389, 413, 416, and 421 have amino acid substitutions as specified in this paragraph.

[0225] In some embodiments, the modified Fc polypeptide, as described in the preceding two paragraphs, contains a natural 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 contains one, two, three, or four substitutions at positions 380, 391, 392, and 415 according to the EU numbering procedure. 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.

[0226] In some embodiments, the modified Fc polypeptide comprises two, three, four, five, six, seven, eight, nine, ten, or eleven molecules selected from the following positions: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, Val, or Asn at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and / or Phe at position 421. In some embodiments, the modified Fc polypeptide comprises all eleven of the following positions: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, Val, or Asn at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and / or Phe at position 421.

[0227] In some embodiments, the modified Fc polypeptide includes 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 also includes Ser, Thr, Gln, or Phe at position 391. In some embodiments, the modified Fc polypeptide also includes 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.

[0228] In other embodiments, the modified Fc polypeptide comprises 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 comprises native Asn at position 390. In some embodiments, the modified Fc polypeptide also comprises Trp, Tyr, Leu, or Gln at position 380; and / or Glu at position 415. In some embodiments, the modified Fc polypeptide also comprises Trp at position 380 and / or Glu at position 415.

[0229] In additional embodiments, the modified Fc polypeptide further comprises one, two, or three substitutions at positions including 414, 424, and 426 according to the EU numbering procedure. 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.

[0230] In some embodiments, the modified Fc polypeptide comprises one or more of the following substitutions: Trp at position 380 according to the EU numbering procedure; 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.

[0231] In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 111-217 of any of SEQ ID NO:4-90, 97-100, and 105-108 (e.g., SEQ ID NO:34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO:4-90, 97-100, and 105-108 (e.g., SEQ ID NO:34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide comprises amino acids at EU index positions 384-390 and / or 413-421 of any of SEQ ID NO: 4-90, 97-100, and 105-108 (e.g., SEQ ID NO: 34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide comprises amino acids at EU index positions 380-390 and / or 413-421 of any of SEQ ID NO: 4-90, 97-100, and 105-108 (e.g., SEQ ID NO: 34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide comprises amino acids at EU index positions 380-392 and / or 413-426 of any of SEQ ID NO: 4-90, 97-100, and 105-108 (e.g., SEQ ID NO: 34-38, 58, and 60-90).

[0232] 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 of SEQ ID NO:4-90, 97-100, and 105-108 (e.g., SEQ ID NO:34-38, 58, and 60-90), and further comprises at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the following positions according to the EU index number: Trp, Tyr, Leu, Gln, or Glu at position 380; 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; and an aromatic amino acid at position 388. For example, Trp; Val, Ser, or Ala at position 389; Ser or Asn at position 390; Ser, Thr, Gln, or Phe at position 391; Gln, Phe, or His at position 392; acidic amino acids, Ala, Ser, Leu, Thr, or Pro at position 413; Lys, Arg, Gly, or Pro at position 414; Glu or Ser at position 415; Thr or acidic amino acids at position 416; Trp, Tyr, His, or Phe at position 421; Ser, Thr, Glu, or Lys at position 424; and Ser, Trp, or Gly at position 426.

[0233] In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO:34-38, 58, and 60-90. In other embodiments, the modified Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO:34-38, 58, and 60-90, but one, two, or three amino acids are substituted.

[0234] In some embodiments, the modified Fc peptide contains additional mutations, such as those described in Section VI below, including but not limited to mortar mutations (e.g., T366W as numbered with reference to EU numbers), mortar mutations (e.g., T366S, L368A, and Y407V as numbered with reference to EU numbers), mutations that regulate effector function (e.g., L234A, L235A, and / or P329G as numbered with reference to EU numbers (e.g., L234A and L235A)), and / or mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E as numbered with reference to EU numbers, or (ii) N434S as numbered according to the EU numbering procedure with or without M428L). As an example, SEQ ID NO:156-229 provides 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).

[0235] In some embodiments, the modified Fc polypeptide comprises a club-shaped mutation (e.g., T366W, as designated by reference to EU) and has at least 85%, at least 90%, or at least 95% sequence identity with any of SEQ ID NO: 156, 168, 180, 192, 204, and 216. In some embodiments, the modified Fc polypeptide comprises the sequence of any of SEQ ID NO: 156, 168, 180, 192, 204, and 216.

[0236] In some embodiments, the modified Fc polypeptide comprises a club-shaped mutation (e.g., T366W, as designated by reference to EU numbers) and a mutation regulating effector function (e.g., L234A, L235A, and / or P329G, as designated by reference to EU numbers; e.g., L234A and L235A)), and has at least 85%, at least 90%, or at least 95% sequence identity with any of the sequences in SEQ ID NO: 157, 158, 169, 170, 181, 182, 193, 194, 205, 206, 217, 218, 228, and 229. In some embodiments, the modified Fc polypeptide comprises the sequence in any of SEQ ID NO: 157, 158, 169, 170, 181, 182, 193, 194, 205, 206, 217, and 218.

[0237] In some embodiments, the modified Fc polypeptide comprises a club-shaped mutation (e.g., T366W, as numbered with reference to EU numbers) and a mutation that increases serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E, as numbered with reference to EU numbers, or (ii) N434S, with or without M428L, as numbered according to the EU numbering procedure), and has at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO: 159, 171, 183, 195, 207, and 219. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NO: 159, 171, 183, 195, 207, and 219.

[0238] In some embodiments, the modified Fc polypeptide comprises a club-shaped mutation (e.g., T366W as referred to EU numbering), a mutation regulating effector function (e.g., L234A, L235A and / or P329G as referred to EU numbering, e.g., L234A and L235A)), and a mutation increasing serum stability or serum half-life (e.g., (i) M252Y, S254T and T256E as referred to EU numbering, or (ii) N434S as referred to according to EU numbering procedure with or without M428L), and has at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO: 160, 161, 172, 173, 184, 185, 196, 197, 208, 209, 220 and 221. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NO: 160, 161, 172, 173, 184, 185, 196, 197, 208, 209, 220, and 221.

[0239] In some embodiments, the modified Fc polypeptide comprises a morbidity mutation (e.g., T366S, L368A, and Y407V, numbered according to EU references) and has at least 85%, at least 90%, or at least 95% sequence identity with any of SEQ ID NO: 162, 174, 186, 198, 210, and 222. In some embodiments, the modified Fc polypeptide comprises the sequence of any of SEQ ID NO: 162, 174, 186, 198, 210, and 222.

[0240] In some embodiments, the modified Fc polypeptide comprises a morbidity mutation (e.g., T366S, L368A, and Y407V, as designated by EU reference) and a mutation regulating effector function (e.g., L234A, L235A, and / or P329G, as designated by EU reference, e.g., L234A and L235A)), and has at least 85%, at least 90%, or at least 95% sequence identity with any of SEQ ID NO: 163, 164, 175, 176, 187, 188, 199, 200, 211, 212, 223, and 224. In some embodiments, the modified Fc polypeptide comprises the sequence of any of SEQ ID NO: 163, 164, 175, 176, 187, 188, 199, 200, 211, 212, 223, and 224.

[0241] In some embodiments, the modified Fc polypeptide comprises a morbidity mutation (e.g., T366S, L368A, and Y407V, numbered according to EU numbers) and a mutation that increases serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E, numbered according to EU numbers, or (ii) N434S, numbered according to EU numbering procedures, with or without M428L), and has at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO: 165, 177, 189, 201, 213, and 225. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NO: 165, 177, 189, 201, 213, and 225.

[0242] In some embodiments, the modified Fc polypeptide comprises a morbidity mutation (e.g., T366S, L368A, and Y407V as numbered with reference to EU numbers), a mutation regulating effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) as numbered with reference to EU numbers), and a mutation increasing serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E as numbered with reference to EU numbers, or (ii) N434S as numbered according to EU numbering procedures with or without M428L), and has at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO: 166, 167, 178, 179, 190, 191, 202, 203, 214, 215, 226, and 227. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NO: 166, 167, 178, 179, 190, 191, 202, 203, 214, 215, 226, and 227.

[0243] In some embodiments, the modified Fc polypeptide that specifically binds to TfR comprises 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 sequence. Illustrative modified Fc polypeptides are provided in SEQ ID NO: 124-128. In some embodiments, the modified Fc polypeptide comprises Gly at position 437; Phe at position 438; and / or Asp at position 440. In some embodiments, Glu is present at position 440. In some embodiments, the modified Fc polypeptide includes at least one substitution at the following positions: Phe or Ile at position 345; Asp, Glu, Gly, Ala, or Lys at position 346; Tyr, Met, Leu, Ile, or Asp at position 347; Thr or Ala at position 349; Gly at position 437; Phe at position 438; His, Tyr, Ser, or Phe at position 439; or Asp at position 440. In some embodiments, two, three, four, five, six, seven, or all eight of positions 345, 346, 347, 349, 437, 438, 439, and 440 have the substitutions specified in this paragraph. In some embodiments, the modified Fc polypeptide may contain conserved substitutions, such as amino acids in groups of the same charge, hydrophobic groups, side-chain ring structures (e.g., aromatic amino acids), or size groups and / or polar or nonpolar groups of designated amino acids at one or more positions in a set.

[0244] In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 111-217 of any of SEQ ID NO:124-128. In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:124-128. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:124-128. In other embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:124-128, but one, two, or three amino acids are substituted.

[0245] TfR-binding Fc peptides containing mutations in the CH2 domain

[0246] In some embodiments, the modified Fc peptide that specifically binds to TfR contains a substitution in the CH2 domain. In some embodiments, the modified Fc peptide comprises a human Ig CH2 domain, such as an IgG CH2 domain, which is modified to obtain TfR binding activity. The CH2 domain may belong to any IgG subtype, i.e., from IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH2 domain refers to the amino acid segment numbered from approximately position 231 to approximately position 340 according to the EU numbering procedure.

[0247] In some embodiments, the modified Fc polypeptide that specifically binds to TfR binds to the apical domain of TfR and can bind to TfR without blocking or otherwise inhibiting transferrin binding to TfR. In some embodiments, transferrin binding to TfR is not substantially inhibited. In some embodiments, transferrin binding to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, transferrin binding to TfR is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0248] In some embodiments, the modified Fc polypeptide that specifically binds to TfR comprises 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 sequence. Illustrative modified Fc polypeptides are provided in SEQ ID NO: 129-133. In some embodiments, the modified Fc polypeptide comprises Glu at position 287 and / or Trp at position 288. In some embodiments, the modified Fc polypeptide contains at least one substitution at the following positions: Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp at position 274; Ile, Val, Asp, Glu, Thr, Ala, or Tyr at position 276; Asp, Pro, Met, Leu, Ala, Asn, or Phe at position 283; Arg, Ser, Ala, or Gly at position 285; Tyr, Trp, Arg, or Val at position 286; Glu at position 287; Trp or Tyr at position 288; Gln, Tyr, His, Ile, Phe, Val, or Asp at position 289; or Leu, Trp, Arg, Asn, Tyr, or Val at position 290. In some embodiments, two, three, four, five, six, seven, eight, or all nine of positions 274, 276, 283, 285, 286, 287, 288, and 290 have the substitutions specified in the paragraphs above. In some embodiments, the modified Fc polypeptide may contain conserved substitutions, such as amino acids in groups of the same charge, hydrophobic groups, side-chain ring structures (e.g., aromatic amino acids), or size groups and / or polar or nonpolar groups of designated amino acids at one or more positions in the set.

[0249] In some embodiments, the modified Fc peptide comprises 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 comprises 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.

[0250] In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 1-110 of any of SEQ ID NO:129-133. In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:129-133. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:129-133. In other embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:129-133, but one, two, or three amino acids are substituted.

[0251] In some embodiments, the modified Fc polypeptide that specifically binds to TfR comprises 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 sequence. Illustrative modified Fc polypeptides are provided in SEQ ID NO: 134-138. In some embodiments, the modified Fc polypeptide comprises Pro at position 270, Glu at position 295, and / or Tyr at position 297. In some embodiments, the modified Fc polypeptide contains at least one substitution at the following positions: Pro, Phe, Ala, Met, or Asp at position 266; Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr at position 267; Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu at position 268; Pro, Val, Ala, Thr, or Asp at position 269; Pro, Val, or Phe at position 270; Trp, Gln, Thr, or Glu at position 271; Glu, Val, Thr, Leu, or Trp at position 295; Tyr, His, Val, or Asp at position 297; Thr, His, Gln, Arg, Asn, or Val at position 298; or Tyr, Asn, Asp, Ser, or Pro at position 299. In some embodiments, two, three, four, five, six, seven, eight, nine, or all ten of positions 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 have the substitutions specified in the preceding paragraph. In some embodiments, the modified Fc polypeptide may contain conserved substitutions, such as amino acids in the same charge grouping, hydrophobic grouping, side-chain ring structure grouping (e.g., aromatic amino acids), or size grouping and / or polar or nonpolar grouping of designated amino acids at one or more positions in the set.

[0252] In some embodiments, the modified Fc polypeptide comprises 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.

[0253] In some embodiments, the modified Fc polypeptide comprises 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.

[0254] In some embodiments, the modified Fc polypeptide comprises 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.

[0255] In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 1-110 of any of SEQ ID NO:134-138. In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:134-138. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:134-138. In other embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:134-138, but one, two, or three amino acids are substituted.

[0256] In some embodiments, the modified Fc polypeptide that specifically binds to TfR comprises 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 sequence. Illustrative modified Fc polypeptides are provided in SEQ ID NO: 139-143. In some embodiments, the modified Fc polypeptide includes at least one substitution at the following positions: Val or Asp at position 268; Pro, Met, or Asp at position 269; Pro or Trp at position 270; Arg, Trp, Glu, or Thr at position 271; Met, Tyr, or Trp at position 272; Leu or Trp at position 292; Thr, Val, Ile, or Lys at position 293; Ser, Lys, Ala, or Leu at position 294; His, Leu, or Pro at position 296; or Val or Trp at position 300. In some embodiments, two, three, four, five, six, seven, eight, nine, or all ten of positions 268, 269, 270, 271, 272, 292, 293, 294, and 300 have the substitutions specified in the preceding paragraph. In some embodiments, the modified Fc polypeptide may contain conserved substitutions, such as amino acids in groups of the same charge, hydrophobic groups, side-chain ring structures (e.g., aromatic amino acids), or size groups and / or polar or nonpolar groups of designated amino acids at one or more positions in a set.

[0257] In some embodiments, the modified Fc peptide comprises 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.

[0258] In some embodiments, the modified Fc polypeptide comprises 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.

[0259] In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 1-110 of any of SEQ ID NO:139-143. In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:139-143. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:139-143. In other embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:139-143, but one, two, or three amino acids are substituted.

[0260] In some embodiments, the modified Fc polypeptide that specifically binds to TfR has 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 sequence. Illustrative modified Fc polypeptides are provided in SEQ ID NO: 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 at the following positions: Trp, Val, Ile, or Ala at position 272; Trp or Gly at position 274; Tyr, Arg, or Glu at position 276; Ser, Arg, or Gln at position 322; Val, Ser, or Phe at position 324; Ile, Ser, or Trp at position 326; Trp, Thr, Ser, Arg, or Asp at position 329; Trp at position 330; or Ser, Lys, Arg, or Val at position 331. In some embodiments, two, three, four, five, six, seven, eight, or all nine of positions 272, 274, 276, 322, 324, 326, 329, 330, and 331 have the substitutions specified in the preceding paragraph. In some embodiments, the modified Fc polypeptide may contain conserved substitutions, such as amino acids in groups of the same charge, hydrophobic groups, side-chain ring structures (e.g., aromatic amino acids), or size groups and / or polar or nonpolar groups of designated amino acids at one or more positions in a set.

[0261] In some embodiments, the modified Fc polypeptide comprises two, three, four, five, six, seven, eight, or nine positions selected from the following: position 272 is Trp, Val, Ile, or Ala; position 274 is Trp or Gly; position 276 is Tyr, Arg, or Glu; position 322 is Ser, Arg, or Gln; position 324 is Val, Ser, or Phe; position 326 is Ile, Ser, or Trp; position 329 is Trp, Thr, Ser, Arg, or Asp; position 330 is Trp; and position 331 is 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.

[0262] In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 1-110 of any of SEQ ID NO:144-148. In some embodiments, the modified Fc polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:144-148. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:144-148. In other embodiments, the modified Fc polypeptide comprises the amino acid sequence of any of SEQ ID NO:144-148, but one, two, or three amino acids are substituted.

[0263] VI. Additional Fc peptide mutations

[0264] In some aspects, the fusion protein described herein comprises two Fc polypeptides, each of which may contain independently selected modifications or may be a wild-type Fc polypeptide, such as the human IgG1 Fc polypeptide. In some embodiments, one or both Fc polypeptides contain one or more modifications that enable them to bind to a blood-brain barrier (BBB) ​​receptor, such as the transferrin receptor (TfR). Non-limiting examples of other mutations that may be introduced into one or both Fc polypeptides include mutations for purposes such as increasing serum stability or serum half-life, regulating effector function, affecting glycosylation, reducing immunogenicity in humans, and / or providing mordant and valence heterodimerization of the Fc polypeptide.

[0265] In some embodiments, the Fc polypeptide present in the fusion protein independently has 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).

[0266] In some embodiments, the Fc peptide present in the fusion protein includes club and mortar mutations to promote heterodimer formation and prevent homodimer formation. Typically, modifications introduce a protrusion (“club”) at the interface of the first peptide and a corresponding cavity (“mortar”) at the interface of the second peptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and thus prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain at the interface of the first peptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of the same or similar size as the protrusion is formed at the interface of the second peptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). In some embodiments, such additional mutations are located in the Fc peptide at a position that does not negatively affect peptide binding to a BBB receptor (e.g., TfR).

[0267] In one illustrative embodiment of the mortar and pestle dimerization method, one of the Fc peptides present in the fusion protein contains tryptophan at position 366 (according to EU procedural designation) as a substitute for native threonine. The other Fc peptide in the dimer has valine at position 407 (according to EU procedural designation) as a substitute for native tyrosine. The other Fc peptide may also contain substitutions in which native threonine at position 366 (according to EU procedural designation) is replaced by serine and native leucine at position 368 (according to EU procedural designation) is replaced by alanine. Thus, one of the Fc peptides of the fusion protein described herein has the T366W mortar mutation and the other Fc peptide has the Y407V mutation, typically accompanied by the T366S and L368A mortar mutations.

[0268] In some embodiments, modifications may be introduced to enhance serum half-life. For example, in some embodiments, one or both Fc peptides present in the fusion protein described herein may contain tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, as designated according to the EU numbering procedure. Thus, one or both Fc peptides may have M252Y, S254T, and T256E substitutions. Alternatively, one or both Fc peptides may have M428L and N434S substitutions, as designated according to the EU numbering procedure. Alternatively, one or both Fc peptides may have N434S or N434A substitutions.

[0269] In some embodiments, one or both Fc peptides present in the fusion protein described herein may contain modifications that reduce effector function, i.e., have a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on effector cells that mediate effector function. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phage activity (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector function may vary depending on the antibody class. For example, natural human IgG1 and IgG3 antibodies may induce ADCC and CDC activity upon binding to an appropriate Fc receptor present on immune system cells; and natural human IgG1, IgG2, IgG3, and IgG4 may induce ADCP function upon binding to an appropriate Fc receptor present on immune cells.

[0270] In some implementations, one or both Fc peptides present in the fusion protein described herein may also be engineered to contain other modifications for heterodimerization, such as electrostatic engineering or hydrophobic patch modification of naturally charged contact residues within the CH3-CH3 interface.

[0271] In some implementations, one or both Fc peptides present in the fusion protein described herein may include additional modifications to regulate effector function.

[0272] In some embodiments, one or both Fc peptides present in the fusion protein described herein may contain modifications that reduce or eliminate effector function. Illustrative Fc peptide mutations that reduce effector function include, but are not limited to, substitutions at positions 234 and 235 in the CH2 domain, for example, according to the EU numbering procedure. For instance, in some embodiments, one or both Fc peptides may contain alanine residues at positions 234 and 235. Thus, one or both Fc peptides may have L234A and L235A (LALA) substitutions.

[0273] Additional Fc peptide mutations that regulate effector function include, but are not limited to, mutations at position 329 in which proline is replaced by glycine or arginine, or amino acid residues large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and tryptophan residues Trp 87 and Trp 110 of FcγRIII. Additional illustrative substitutions include S228P, E233P, L235E, N297A, N297D, and P331S according to the EU numbering protocol. Multiple substitutions may also be present, such as L234A and L235A of the human IgG1 Fc region according to the EU numbering scheme; L234A, L235A, and P329G of the human IgG1 Fc region; S228P and L235E of the human IgG4 Fc region; L234A and G237A of the human IgG1 Fc region; L234A, L235A, and G237A of the human IgG1 Fc region; V234A and G237A of the human IgG2 Fc region; L235A, G237A, and E318A of the human IgG4 Fc region; and S228P and L236E of the human IgG4 Fc region. In some embodiments, one or both Fc peptides may have one or more amino acid substitutions that regulate ADCC, such as substitutions at positions 298, 333, and / or 334 according to the EU numbering scheme.

[0274] Illustrative Fc peptides containing additional mutations

[0275] As a non-limiting example, one or both Fc peptides present in the fusion proteins described herein may contain additional mutations, including clasp mutations (e.g., T366W as numbered according to the EU numbering process), mortar mutations (e.g., T366S, L368A, and Y407V as numbered according to the EU numbering process), mutations that regulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) as numbered according to the EU numbering process), and / or mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E as numbered with reference to the EU numbering process, or (ii) N434S as numbered according to the EU numbering process, with or without M428L).

[0276] In some embodiments, the Fc polypeptide may have a club-shaped mutation (e.g., T366W according to EU numbering procedures) and have at least 85%, at least 90%, or at least 95% identity with the sequences of any one of SEQ ID NO:1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having the sequence of any one of SEQ ID NO:1, 4-90, and 124-148 may be modified to have a club-shaped mutation.

[0277] In some embodiments, the Fc peptide may have a club-shaped mutation (e.g., T366W according to EU numbering procedures), a mutation for regulatory effector function (e.g., L234A, L235A and / or P329G according to EU numbering procedures, e.g., L234A and L235A)), and have at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO:1, 4-90, and 124-148. In some embodiments, the Fc peptide having the sequence of any one of SEQ ID NO:1, 4-90, and 124-148 may be modified to have a club-shaped mutation and a mutation for regulatory effector function.

[0278] In some embodiments, the Fc peptide may have a club-shaped mutation (e.g., T366W according to the EU numbering procedure), a mutation that increases serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E as numbered according to EU numbers, or (ii) N434S with or without M428L as numbered according to the EU numbering procedure), and have at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO:1, 4-90, and 124-148. In some embodiments, the Fc peptide having the sequence of any one of SEQ ID NO:1, 4-90, and 124-148 may be modified to have a club-shaped mutation and a mutation that increases serum stability or serum half-life.

[0279] In some embodiments, the Fc peptide may have a pestle mutation (e.g., T366W according to EU numbering procedures), a mutation regulating effector function (e.g., L234A, L235A and / or P329G (e.g., L234A and L235A) according to EU numbering procedures), or a mutation increasing serum stability or serum half-life (e.g., (i) M252Y, S254T and T256E as numbered with reference to EU numbers, or (ii) N434S with or without M428L according to EU numbering procedures), and have at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO:1, 4-90, and 124-148. In some embodiments, the Fc peptide having the sequence of any one of SEQ ID NO:1, 4-90, and 124-148 may be modified to have a pestle mutation, a mutation regulating effector function, or a mutation increasing serum stability or serum half-life.

[0280] In some embodiments, the Fc polypeptide may have a mortis mutation (e.g., T366S, L368A, and Y407V according to the EU numbering procedure) and have at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO:1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having the sequence of any one of SEQ ID NO:1, 4-90, and 124-148 may be modified to have a mortis mutation.

[0281] In some embodiments, the Fc peptide may have a mortise mutation (e.g., T366S, L368A, and Y407V according to EU numbering procedures), a regulatory effector mutation (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) according to EU numbering procedures), and have at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO:1, 4-90, and 124-148. In some embodiments, the Fc peptide having the sequence of any one of SEQ ID NO:1, 4-90, and 124-148 may be modified to have a mortise mutation and a regulatory effector mutation.

[0282] In some embodiments, the Fc peptide may have a mortise mutation (e.g., T366S, L368A, and Y407V as numbered according to the EU numbering procedure), a mutation that increases serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E as numbered according to the EU numbering procedure, or (ii) N434S as numbered according to the EU numbering procedure with or without M428L), and have at least 85%, at least 90%, or at least 95% identity with the sequence of any of SEQ ID NO:1, 4-90, and 124-148. In some embodiments, the Fc peptide having the sequence of any of SEQ ID NO:1, 4-90, and 124-148 may be modified to have a mortise mutation and a mutation that increases serum stability or serum half-life.

[0283] In some embodiments, the Fc peptide may have mutations such as T366S, L368A, and Y407V (as per EU numbering procedures), mutations that regulate effector function (as per L234A, L235A, and / or P329G (as per EU numbering procedures)), mutations that increase serum stability or serum half-life (as per (i) M252Y, S254T, and T256E (as per EU numbering procedures), or (ii) N434S (as per EU numbering procedures) with or without M428L), and have at least 85%, at least 90%, or at least 95% identity with the sequence of any one of SEQ ID NO: 1, 4-90, and 124-148. In some embodiments, Fc peptides having the sequence of any one of SEQ ID NO:1, 4-90, and 124-148 may be modified to have acetabular mutations, mutations with regulatory effects, and mutations that increase serum stability or serum half-life.

[0284] VII. Illustrative fusion proteins containing ERT enzymes

[0285] In some aspects, the fusion protein described herein comprises a first Fc polypeptide linked to an enzyme replacement therapy (ERT) enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and a second Fc polypeptide forming an Fc dimer with the first Fc polypeptide. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide does not include the variable region sequence of the immunoglobulin heavy chain and / or light chain, or its antigen-binding portion. 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 contains one or more modifications that promote its heterodimerization with another Fc polypeptide. In some embodiments, the modified Fc polypeptide contains one or more modifications that reduce effector function. In some embodiments, the modified Fc polypeptide contains one or more modifications that prolong serum half-life. In some embodiments, the modified Fc polypeptide contains one or more modifications that enable it to bind to a blood-brain barrier (BBB) ​​receptor (e.g., transferrin receptor (TfR)).

[0286] In other aspects, the fusion protein described herein comprises a first polypeptide chain containing a modified Fc polypeptide that specifically binds to a BBB receptor (e.g., TfR); and a second polypeptide chain containing an Fc polypeptide dimerized with the modified Fc polypeptide to form an Fc dimer. An ERT enzyme may be linked to either the first or second polypeptide chain. In some embodiments, the ERT enzyme is IDS, SGSH, ASM, or GBA. In some embodiments, the ERT enzyme is linked to the second polypeptide chain. In some embodiments, the protein comprises two ERT enzymes, each linked to one of the 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 in the first polypeptide chain. In some embodiments, the Fc polypeptide does not specifically bind to a BBB receptor.

[0287] In some embodiments, the fusion protein described herein comprises a first polypeptide chain containing a modified Fc polypeptide that specifically binds to a TfR; and a second polypeptide chain containing an Fc polypeptide, wherein the modified Fc polypeptide dimerizes with the Fc polypeptide to form an Fc dimer. In some embodiments, the ERT enzyme is IDS, SGSH, ASM, or GBA. In some embodiments, the ERT enzyme is linked to the first polypeptide chain. In some embodiments, the ERT enzyme is linked to the second polypeptide chain. In some embodiments, the Fc polypeptide does not specifically bind to a BBB receptor, such as TfR.

[0288] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a modified Fc polypeptide bound to TfR and comprising a T366W (valve) substitution; and a second polypeptide chain comprising an Fc polypeptide comprising T366S, L368A, and Y407V (valve) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprises L234A and L235A (LALA) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprises M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprises L234A and L235A (LALA) substitutions and M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc peptide and / or the Fc peptide contains human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0289] In some embodiments, the modified Fc polypeptide comprises the IL-1, LALA, and YTE mutations as specified in any of SEQ ID NO:97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, and has at least 85%, at least 90%, or at least 95% identity with the corresponding sequences; or comprises the sequence of any of SEQ ID NO:97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221. In some embodiments, the Fc polypeptide comprises the α, LALA, and YTE mutations as specified in any of SEQ ID NO:101-104 and has at least 85%, at least 90%, or at least 95% identity with the corresponding sequences; or comprises a sequence of any of SEQ ID NO:101-104. In some embodiments, the modified Fc polypeptide comprises any of SEQ ID NO:97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, and the Fc polypeptide comprises any of SEQ ID NO:101-104. In some embodiments, the modified Fc polypeptide and / or the N-terminus of the 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 of SEQ ID NO:116, 228, and 229, or contains the sequence of any of SEQ ID NO:116, 228, and 229.

[0290] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a modified Fc polypeptide bound to TfR and comprising T366S, L368A, and Y407V (valve) substitutions; and a second polypeptide chain comprising an Fc polypeptide comprising T366W (valve) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprises L234A and L235A (LALA) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprises M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprises L234A and L235A (LALA) substitutions and M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc peptide and / or the Fc peptide contains human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0291] In some embodiments, the modified Fc polypeptide comprises the α, LALA, and YTE mutations as specified in any of SEQ ID NO: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, and has at least 85%, at least 90%, or at least 95% identity with the corresponding sequences; or comprises the sequence of any of SEQ ID NO: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227. In some embodiments, the Fc polypeptide comprises the IL-1, LALA, and YTE mutations as specified in any of SEQ ID NO:109-112 and has at least 85%, at least 90%, or at least 95% identity with the corresponding sequences; or comprises a sequence of any of SEQ ID NO:109-112. In some embodiments, the modified Fc polypeptide comprises any of SEQ ID NO:105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, and the Fc polypeptide comprises any of SEQ ID NO:109-112. In some embodiments, the modified Fc polypeptide and / or the N-terminus of the Fc polypeptide includes a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO:113).

[0292] In some embodiments, an ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) present in the fusion protein described herein is linked to a polypeptide chain comprising an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO:101-104, or comprising a sequence of any of SEQ ID NO:101-104 (e.g., in the form of a fusion polypeptide). In some embodiments, the ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) is linked to the Fc polypeptide and / or a hinge region or a portion thereof via a linker such as a flexible linker (e.g., DKTHTCPPCP; SEQ ID NO:113). In some embodiments, the ERT enzyme comprises an IDS sequence having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO:114, 230, and 234, or comprises a sequence of any of SEQ ID NO: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 NO: 115, 117, 231, 232, 235, and 236, or contains the sequence of any one of SEQ ID NO: 115, 117, 231, 232, 235, and 236. In some embodiments, the ERT enzyme contains an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NO: 120, or contains 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 NO: 149 and 150, or contains the sequence of any one of SEQ ID NO: 149 and 150. In some embodiments, the fusion protein comprises a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO:97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, or comprises a sequence of any of SEQ ID NO: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 the modified 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 of SEQ ID NO:116, 228, and 229, or contains the sequence of any of SEQ ID NO:116, 228, and 229.

[0293] In some embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:115; and a modified Fc polypeptide comprising the sequence of either SEQ ID NO:205 or 228. In other embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:115; and a modified Fc polypeptide comprising the sequence of either SEQ ID NO:169 or 229.

[0294] In some embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:231; and a modified Fc polypeptide comprising the sequence of either SEQ ID NO:205 or 228. In other embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:231; and a modified Fc polypeptide comprising the sequence of either SEQ ID NO:169 or 229.

[0295] In some embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:235; and a modified Fc polypeptide comprising the sequence of either SEQ ID NO:205 or 228. In other embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:235; and a modified Fc polypeptide comprising the sequence of either SEQ ID NO:169 or 229.

[0296] In some embodiments, an ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) present in the fusion protein described herein is linked to a polypeptide chain comprising an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO:109-112, or comprising a sequence of any of SEQ ID NO:109-112 (e.g., in the form of a fusion polypeptide). In some embodiments, the ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) is linked to the Fc polypeptide via a linker such as a flexible linker, and / or a hinge region or a portion thereof (e.g., DKTHTCPPCP; SEQ ID NO:113). In some embodiments, the ERT enzyme comprises an IDS sequence having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO:114, 230, and 234, or comprises a sequence of any of SEQ ID NO: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 of SEQ ID NO:118, 233, and 237, or contains the sequence of any of SEQ ID NO:118, 233, and 237. In some embodiments, the ERT enzyme contains an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO:120, or contains 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 of SEQ ID NO:152 and 153, or contains the sequence of any of SEQ ID NO:152 and 153. In some embodiments, the fusion protein comprises a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO:105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, or comprises a sequence of any of SEQ ID NO: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 the modified Fc polypeptide includes a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO:113).

[0297] In some embodiments, an ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) present in the fusion protein described herein is linked to a polypeptide chain comprising a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, or comprising a sequence (e.g., in the form of a fusion polypeptide) of any of SEQ ID NO: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221. In some embodiments, an ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) is linked to the modified Fc polypeptide via a linker such as a flexible linker, and / or a hinge region or a portion thereof (e.g., DKTHTCPPCP; SEQ ID NO: 113). In some embodiments, the ERT enzyme comprises an IDS sequence having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO: 114, 230, and 234, or comprises a sequence of any of SEQ ID NO: 114, 230, and 234. In some embodiments, the ERT enzyme comprises an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO: 120, or comprises 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 any of SEQ ID NO: 154 and 155, or comprises a sequence of any of SEQ ID NO: 154 and 155. In some embodiments, the fusion protein comprises an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO:101-104, 149, and 150, or comprises a sequence of any of SEQ ID NO:101-104, 149, and 150. In some embodiments, the modified Fc polypeptide and / or the N-terminus of the Fc polypeptide includes a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO:113).

[0298] In some embodiments, an ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) present in the fusion protein described herein is linked to a polypeptide chain comprising a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, or comprising a sequence (e.g., in the form of a fusion polypeptide) of any of SEQ ID NO: 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 the modified Fc polypeptide and / or a hinge region or a portion thereof (e.g., DKTHTCPPCP; SEQ ID NO: 113) via a linker such as a flexible linker. In some embodiments, the ERT enzyme comprises an IDS sequence having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO: 114, 230, and 234, or comprises a sequence of any of SEQ ID NO: 114, 230, and 234. In some embodiments, the ERT enzyme comprises an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO: 120, or comprises a sequence of SEQ ID NO: 120. In some embodiments, the fusion protein comprises an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any of SEQ ID NO: 109-112, or comprises a sequence of any of SEQ ID NO: 109-112. In some embodiments, the fusion protein comprises an SGSH sequence linked to the Fc peptide, said SGSH sequence having at least 85%, at least 90%, or at least 95% identity with either SEQ ID NO: 152 or 153, or comprising a sequence of either SEQ ID NO: 152 or 153. In some embodiments, the modified Fc peptide and / or the N-terminus of the Fc peptide includes a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 113).

[0299] VIII. Measurement of binding dynamics, affinity, brain concentration, and brain exposure

[0300] The fusion proteins and other compositions described herein can have a wide range of binding affinities. For example, in some embodiments, the protein has an affinity for blood-brain barrier (BBB) ​​receptors (e.g., transferrin receptor (TfR)) ranging from 1 pM to 10 μM. In some embodiments, the affinity for TfR is ranging from 1 nM to 5 μM or from 10 nM to 1 μM. In some embodiments, the affinity for TfR is ranging from about 50 nM to about 250 nM.

[0301] In some embodiments, the affinity of TfR-binding peptides can be measured in a monovalent form. In other embodiments, the affinity can be measured in a divalent form, for example, as a dimer comprising a peptide-Fab fusion protein.

[0302] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity to analyze binding to BBB receptors (e.g., TfR) are known in the art. These methods include, but are not limited to, solid-state binding assays (e.g., ELISA), immunoprecipitation, surface plasmon resonance (SPR) (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet® (FortéBio, Inc., Menlo Park, CA)), and Western ink dot assays. 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 following embodiment section. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, kinetic exclusion assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some implementations, BioLayer interferometry analysis is used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0303] The following Example 13 describes a non-limiting example of a method for determining binding affinity (e.g., for TfR), in which Biacore is used. ™The instrument determines affinity via surface plasmon resonance (SPR). In this method, the engineered TfR-binding peptide, TfR-binding peptide, or TfR-binding antibody of interest is captured onto a sensor chip, and a serially diluted TfR is injected onto the sensor chip at a specified flow rate (e.g., 30 μL / min) and temperature (e.g., room temperature). The sample is analyzed using specified association and dissociation times (e.g., 45 and 180 seconds, respectively), followed by sensor chip regeneration. The binding reaction is corrected by subtracting the measured reaction from a control (e.g., using unrelated IgG of similar density), and steady-state affinity can then be determined by fitting an equilibrium reaction for concentration using software.

[0304] For example, a human transferrin receptor (hTfR) knock-in mouse model can be used to measure the concentration of engineered TfR-binding peptides, TfR-binding peptides, TfR-binding antibodies, or agents (e.g., linked to engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies) in the brain and / or plasma. Such models can be used, for example, to measure and / or compare the highest brain concentration (C0). 最大 ) and / or brain exposure, for example, to determine C 最大 Whether it increases and / or prolongs brain exposure. Human apical TfR (TfR) is described below in Example 12. ms / hu Creation of a mouse knock-in model. To create a suitable model, a CRISPR / Cas9 system can be used to generate mice expressing the human Tfrc apical domain within the mouse Tfrc gene (e.g., where in vivo expression is controlled by an endogenous promoter). Specifically, Cas9, a single guide RNA, and donor DNA (e.g., a human apical domain coding sequence optimized for expression codons in mice) can be introduced into mouse embryos (e.g., via pronuclear injection). The embryos can then be transferred to pseudopregnant females. The founder males of the offspring from the receiving females can be bred with wild-type females to produce F1 heterozygous mice. Homozygous mice can then be subsequently generated from the F1 heterozygous mice.

[0305] For the assessment of brain and / or plasma concentrations or exposure to engineered TfR-binding peptides, TfR-binding peptides, TfR-binding antibodies, or agents (e.g., conjugated to engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies), the results can be introduced into mouse models (e.g., TfR...). ms / huThe engineered TfR-binding peptide, TfR-binding peptide, or TfR-binding antibody (e.g., conjugated to the agent) is administered. Plasma samples can be obtained from mice after a suitable time period, followed by perfusion of the vascular system with a suitable solution. After perfusion, the brain (or a portion thereof) can be extracted and homogenized and dissolved. The concentration of the agent in the plasma and / or brain lysate can then be determined using standard methods known to those skilled in the art. As a non-limiting example, an ELISA-based assay, such as the ELISA-based assay described in Example 3 below, can be used to measure the concentration. In simple terms, a sandwich ELISA can be used to quantify the concentration of the agent, engineered TfR-binding peptide, TfR-binding peptide, or TfR-binding antibody (e.g., in plasma or lysate). The 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., about 0.5 μg / mL), followed by a second antibody, such as an anti-goat-HRP antibody. The plate is then developed (e.g., using a TMB substrate), the reaction is stopped (e.g., with sulfuric acid), and the absorbance is measured at an appropriate wavelength (e.g., 450 nm) on a plate reader (e.g., a BioTek plate reader). A standard curve can be generated using an appropriate (e.g., 4-fold) dilution series and fitted using an algorithm such as four-parameter logistic regression.

[0306] A standard curve can be generated by administering a series of doses to a knock-in mouse model. By administering agents linked to different engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies (e.g., with different TfR affinities) or agents linked to a reference peptide or protein (e.g., with a weaker affinity for TfR compared to the peptide or protein of interest) to the knock-in mouse model, the C-values ​​of the engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies in the brain exposed to said agents and / or said agents in the brain can be evaluated. 最大 The impact of the values ​​is compared.

[0307] IX. ERT enzymes linked to Fc polypeptides

[0308] In some embodiments, the fusion protein described herein comprises two Fc peptides as described herein, and one or both of the Fc peptides may further comprise a partial or complete hinge region. The hinge region may be derived from any immunoglobulin subclass or isotype. An illustrative immunoglobulin hinge is an IgG hinge region, such as an IgG1 hinge region, for example, 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 peptide.

[0309] In some embodiments, the Fc polypeptide is linked to the ERT enzyme via a linker (e.g., a peptide linker). In some embodiments, the Fc polypeptide is linked to the ERT enzyme via a peptide bond or via a peptide linker, for example, as a fusion polypeptide. The peptide linker may be configured such that it allows the ERT enzyme to rotate relative to the Fc polypeptide to which it is linked; and / or is resistant to digestion by a protease. The peptide linker may contain native amino acids, non-natural amino acids, or combinations thereof. In some embodiments, the peptide linker may be a flexible linker, for example containing amino acids such as Gly, Asn, Ser, Thr, Ala, etc. Such linkers are designed using known parameters and may have any length and contain any number of repeating units of any length (e.g., repeating units of Gly and Ser residues). For example, the linker may have repeats, 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, for example, it may be cleaved by an enzyme present in the central nervous system.

[0310] In some embodiments, the ERT enzyme is linked to the N-terminus of the Fc polypeptide via, for example, a Gly4-Ser adapter (SEQ ID NO: 239) or a (Gly4-Ser)2 adapter (SEQ ID NO: 240). In some embodiments, the Fc polypeptide may include a hinge sequence or a portion of a hinge sequence at the N-terminus, which is connected to the adapter or directly to the ERT enzyme.

[0311] In some embodiments, the ERT enzyme is linked to the C-terminus of the Fc peptide, for example, via a Gly4-Ser adapter (SEQ ID NO: 239) or a (Gly4-Ser)2 adapter (SEQ ID NO: 240). In some embodiments, the C-terminus of the Fc peptide is directly linked to the ERT enzyme.

[0312] In some embodiments, the ERT enzyme is linked to the Fc peptide via a chemical cross-linking agent. Well-known chemical cross-linking reagents and methods can be used to generate such conjugates. For example, a large number of chemical cross-linking agents are known to those skilled in the art and can be used to cross-link peptides with agents of interest. For example, the cross-linking agent is a heterobifunctional cross-linking agent, which can be used for stepwise bonding of molecules. Heterobifunctional cross-linking agents provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrence of unwanted side reactions, such as homologous protein polymers. Various heterobifunctional crosslinking agents are known in the art, including N-hydroxysuccinimide (NHS) or its water-soluble analogues, such as N-hydroxysulfonylsuccinimide (sulfon-NHS), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), m-maleimidemethylbenzoyl-N-hydroxysuccinimide ester (MBS); aminobenzoic acid N-succinimide (4-iodoacetyl) ester (SIAB), 4-(p-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), and 4-(p-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (MBS); Crosslinking agents containing an N-hydroxysuccinimide moiety are available in the form of N-hydroxysulfonylsuccinimide analogs, which typically exhibit greater water solubility. Furthermore, crosslinking agents containing disulfide bonds within the linker chain can be synthesized alternatively as alkyl derivatives to reduce the amount of in vivo linker cleavage. Besides heterobifunctional crosslinkers, many other crosslinkers exist, including homobifunctional and photoreactive crosslinkers. Examples include disuccinimide octanoate (DSS), bismaleimide hexane (BMH), and dimethylpimelimidate. 2HCl (DMP) is an example of a usable homobifunctional crosslinker, and bis-[B-(4-azidosalicylic acid)ethyl]disulfide (BASED) and N-succinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of usable photoreactive crosslinkers.

[0313] X. Assessment of protein activity

[0314] Various analyses can be used to evaluate the activity of fusion proteins containing ERT enzymes such as IDS, SGSH, ASM, or GBA as described herein, including analyses that measure in vitro activity using artificial substrates, such as those described in the Examples section. An illustrative protocol for measuring in vitro IDS activity is provided in Example 2. An illustrative protocol for measuring in vitro ASM activity is provided in Example 5. Illustrative protocols for measuring in vitro SGSH activity are provided in Examples 7 and 8.

[0315] In some applications, IDS activity is assessed by analyzing the amounts of glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate that accumulate due to IDS deficiency in samples such as cell samples, tissue samples, or fluid samples (e.g., CSF or urine). The amounts of heparan sulfate and dermatan sulfate are determined by digesting the GAGs present in the sample with heparinase and chondroitinase. The resulting disaccharides can then be analyzed by mass spectrometry (e.g., LC-MS / MS). Samples containing high levels of accumulated heparan sulfate and dermatan sulfate will have increased amounts of heparan sulfate- and dermatan sulfate-derived disaccharides. Therefore, the level of disaccharides is inversely proportional to IDS enzyme activity.

[0316] Mass spectrometry (e.g., LC-MS / MS) analysis can be performed on any sample containing GAG accumulation, including cellular samples, tissue samples, and fluid samples. Such samples can be evaluated to monitor the activity of the IDS-containing protein described herein, for example, when administered to cells in vitro or, in some embodiments, to a subject in vivo. The subject may be an animal, such as a rodent, like a mouse, or a non-human primate. In some embodiments, the subject is a human patient, such as a patient with Hunter syndrome undergoing IDS therapy, wherein the analysis is used to monitor IDS activity in the patient. In some embodiments, the human patient is undergoing therapy with the fusion protein described herein.

[0317] For cellular samples, such as cell or tissue samples, the analysis involves disrupting the cells and rupturing the microvesicles. Disruption of cells or microvesicles can be achieved by using freeze-thaw and / or sonication to obtain an extract containing GAG (e.g., a cell extract). The GAG ​​is then treated with heparinase (e.g., any heparinase described herein) and chondroitinase, which break down heparan sulfate and dermatan sulfate GAG. After digestion, a supernatant containing GAG disaccharide is obtained, and the disaccharide product is analyzed by mass spectrometry (e.g., LC-MS / MS). An illustrative protocol is provided in Example 2.

[0318] In some embodiments, cell samples to be analyzed for IDS activity are washed and frozen. Cell spherules are sonicated in a disaccharide digestion buffer. The desired amount of total protein from the sonicated sample is then added to a digestion buffer containing heparinase I, heparinase II, heparinase III, and chondroitin B, the latter being specific for dermatan sulfate. After digestion, for example at 30°C for approximately 3 hours, the enzymes are deactivated with EDTA and by boiling. The sample is then centrifuged, for example at 16,000 x G, and the supernatant is transferred to a centrifuge filter and centrifuged at approximately 14,000 x G. The disaccharide is then resuspended in a 1:1 v / v ratio analysis buffer:acetonitrile mixture and further analyzed, for example as described in Example 2, by liquid chromatography coupled with electrospray mass spectrometry. GAG-derived disaccharide products can be identified based on retention time compared to commercially available reference standards. Illustrative disaccharides derived from heparan sulfate include D0SO and D2SO (according to Lawrence et al., Nat. Methods, 5:291-292 (2008)).

[0319] In other respects, SGSH activity is assessed by analyzing the amount of heparan sulfate glycosaminoglycans (GAGs) that accumulate in samples such as cell or tissue samples due to SGSH deficiency. The amount of heparan sulfate is determined by digesting the GAGs present in the sample with a heparinase (such as any heparinase described herein). The resulting disaccharides are then analyzed by mass spectrometry (e.g., LC-MS / MS). Samples containing high levels of heparan sulfate accumulation will have an increased amount of heparan sulfate-derived disaccharides. Therefore, the level of disaccharides is inversely proportional to SGSH enzyme activity.

[0320] Mass spectrometry (e.g., LC-MS / MS) analysis can be performed on any sample containing GAG accumulation, including cellular, tissue, and fluid samples. Such samples can be evaluated to monitor the activity of SGSH-containing proteins described herein, for example, administered to cells in vitro or, in some embodiments, to a subject in vivo. Subjects can be animals, such as rodents, like mice, or non-human primates. In some embodiments, the subject is a human patient, such as a patient with St. Philippian syndrome A who is undergoing SGSH therapy, wherein the analysis is used to monitor SGSH activity in the patient. In some embodiments, the human patient is undergoing treatment with the fusion protein described herein.

[0321] For cellular samples, such as cell or tissue samples, the analysis includes disrupting the cells and / or rupturing the microvesicles. Disrupting the cells or microvesicles can be achieved using freeze-thaw and / or sonication to obtain an extract containing GAG (e.g., a cell extract). The GAG ​​is then treated with a heparinase (e.g., any heparinase described herein), which breaks down heparan sulfate GAG. After digestion, a supernatant containing GAG disaccharides is obtained and the disaccharide product is analyzed by mass spectrometry (e.g., LC-MS / MS). An illustrative protocol is provided in Example 7.

[0322] In some embodiments, cell samples to be analyzed for SGSH activity are washed and frozen. Cell spherules are sonicated in a disaccharide digestion buffer. The desired amount of total protein from the sonicated sample is then added to a digestion buffer containing heparinase I, heparinase II, and / or heparinase III. After digestion, for example at 30°C for approximately 3 hours, the enzymes are deactivated with EDTA and by boiling. The sample is then centrifuged, for example at 16,000 x G, and the supernatant is transferred to a centrifuge filter and centrifuged at approximately 14,000 x G. The disaccharide is then resuspended in a 1:1 v / v ratio analysis buffer:acetonitrile mixture and further analyzed, for example as described in Example 7, by liquid chromatography coupled with electrospray mass spectrometry. GAG-derived disaccharide products can be identified based on retention time compared to commercially available reference standards. Illustrative disaccharides derived from heparan sulfate include D0SO and D2SO (according to Lawrence et al., Nat. Methods, 5:291-292 (2008)).

[0323] In some implementations, tissue samples are evaluated. The tissue samples can be analyzed using the analyses described above, except that typically multiple freeze-thaw cycles (e.g., 2, 3, 4, 5 or more) are included prior to the sonication step to ensure microbubble rupture.

[0324] The samples being evaluated, including those from the brain, liver, kidneys, lungs, spleen, plasma, serum, cerebrospinal fluid (CSF), and urine, can be analyzed as described herein. In some embodiments, CSF samples from patients receiving the enzyme-Fc fusion protein described herein (e.g., IDS-Fc or SGSH-Fc fusion protein) can be evaluated.

[0325] XI. Nucleic acids, vectors, and host cells

[0326] Typically, recombinant methods are used to prepare the polypeptide chains contained in fusion proteins as described herein. Therefore, in some aspects, this disclosure provides isolated nucleic acids containing nucleic acid sequences encoding any of the polypeptide chains comprising the Fc polypeptides described herein; and a host cell incorporating said nucleic acid for replicating the nucleic acid encoding the polypeptide and / or expressing the polypeptide. In some embodiments, the host cell is a eukaryotic cell, such as a human cell.

[0327] In another embodiment, a polynucleotide comprising a nucleotide sequence encoding the polypeptide chain described herein is provided. The polynucleotide may be single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA. In a particular embodiment, the polynucleotide is cDNA. In some embodiments, the polynucleotide is RNA.

[0328] In some embodiments, the polynucleotide is included within the nucleic acid construct. In some embodiments, the construct is a reproducible vector. In some embodiments, the vector is selected from plasmids, viral vectors, phage particles, yeast chromosome vectors, and non-attachment mammalian vectors.

[0329] In some embodiments, a polynucleotide is operatively linked to one or more regulatory nucleotide sequences in the expression construct. In a range of embodiments, the nucleic acid expression construct is suitable for use as a surface expression library. In some embodiments, the library is suitable for surface expression in yeast. In some embodiments, the library is suitable for surface expression in bacteriophages. In another series of embodiments, the nucleic acid expression construct is suitable for expressing a peptide in a system that allows for the separation of the peptide in milligrams or grams. In some embodiments, the system is a mammalian cell expression system. In some embodiments, the system is a yeast cell expression system.

[0330] Expression media used to prepare recombinant peptides include plasmids and other vectors. Suitable vectors, for example, include plasmids of the following types: 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*). Vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg are examples of mammalian expression vectors suitable for transfecting eukaryotic cells. Alternatively, viral derivatives such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived types, and p205) can be used for transient expression of peptides in eukaryotic cells. In some implementations, it may be necessary to express recombinant peptides using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors. Additional expression systems include adenovirus, adeno-associated virus, and other viral expression systems.

[0331] The vector can be transformed into any suitable host cell. In some embodiments, the host cell (e.g., bacterial or yeast cells) may be suitable for use as a surface expression library. In some cells, the vector is expressed in the host cell to express a relatively large amount of the polypeptide. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cells are mammalian cells, such as Chinese hamster ovary (CHO) cells, young hamster kidney (BHK) cells, NSO cells, YO cells, HEK293 cells, COS cells, Vero cells, or HeLa cells.

[0332] Host cells transfected with expression vectors encoding one or more Fc polypeptide chains as described herein can be cultured under appropriate conditions to allow the expression of one or more polypeptides. The polypeptides can be secreted or isolated from cell mixtures and culture media containing the polypeptides. Alternatively, the polypeptides can be retained in the cytoplasm or membrane fractions, and the cells can be harvested, lysed, and the polypeptides isolated using desired methods.

[0333] XII. Treatment Methods

[0334] The fusion proteins or agents (e.g., therapeutic agents) of engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies described herein can be therapeutically used to treat LSD. In some embodiments, patients with Hunter syndrome are treated using fusion proteins or agents containing IDS that are engineered to contain IDS. In some embodiments, patients with St. Philippian syndrome A are treated using fusion proteins or agents containing SGSH that are engineered to contain SGSH. In some embodiments, patients with Niemann-Pick disease are treated using fusion proteins or agents containing ASM that are engineered to contain SGSH. In some implementations, fusion proteins or agents containing GBA linked to engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies are used to treat patients with Gaucher disease or Parkinson's disease.

[0335] The fusion protein comprising an ERT enzyme (e.g., IDS, SGSH, ASM, or GBA) as described herein is administered to the subject at a therapeutically effective amount or dose. Illustrative doses include a daily dose range of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 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, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 U / mg. In some embodiments, the enzyme activity is at least about 11,000 U / mg, or at least about 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 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 be varied depending on several factors, including the chosen route of administration, the formulation of the composition, patient response, the severity of the condition, the subject's weight, and the prescribing physician's judgment. The dosage may be increased or decreased over time according to the individual patient's needs. In some embodiments, a low dose is initially given to the patient, and then the dose is increased to an effective dose that the patient can tolerate. Determining the effective amount is entirely within the capabilities of those skilled in the art.

[0336] In various embodiments, the fusion protein described herein is administered parenterally. In some embodiments, the protein is administered intravenously. Intravenous administration can be performed by infusion over a period of approximately 10 to 30 minutes or over a period of at least 1 hour, 2 hours, or 3 hours. In some embodiments, the protein is administered as a rapid intravenous injection. A combination of infusion and rapid injection may also be used.

[0337] In some parenteral embodiments, a fusion protein or agent (e.g., a therapeutic agent) linked to an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, a protein or agent linked to an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is administered intradermally or intramuscularly. In some embodiments, the protein or agent is administered intrathecally, such as via epidural or intraventricular administration.

[0338] In other embodiments, the fusion protein or agent (e.g., a therapeutic agent) linked to an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody may be administered orally, via pulmonary administration, intranasal administration, intraocular administration, or via surface administration. Pulmonary administration may also be used, for example, by using an inhaler or nebulizer and in combination with a powder inhaler.

[0339] XIII. Methods of Protein Substitution

[0340] In other aspects, this document provides a method for delivering an agent (e.g., an agent that can be used to treat lysosomal storage diseases (LSD)) across the blood-brain barrier (BBB) ​​in mammals. In some embodiments, the method comprises exposing the BBB to a polypeptide or protein that binds (e.g., specifically binds) to a transferrin receptor (TfR) with an affinity of about 50 nM to about 250 nM. In some embodiments, the polypeptide or protein is linked to the agent and the linked agent is delivered across the BBB. In some embodiments, the maximum concentration (C) of the agent in the mammalian brain is... 最大 ) has improved (e.g., increased).

[0341] In other aspects, this document provides a method for treating LSD. In some embodiments, the method includes administering to a mammal a polypeptide or protein that binds (e.g., specifically binds) to TfR with an affinity of about 50 nM to about 250 nM. In some embodiments, the polypeptide or protein is linked to an agent for treating LSD, thereby exposing the mammalian brain to the agent.

[0342] In some embodiments, the peptide or protein binds to the TfR with an affinity (e.g., specific binding) of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nM. In some embodiments, the peptide or protein binds to the TfR with an affinity of about 100 nM to about 200 nM or about 110 nM to about 150 nM.

[0343] In some embodiments, a peptide or protein (e.g., linked to the agent) causes the agent to be absorbed into the brain by C compared to an agent linked to a reference peptide or protein that binds with a weaker affinity (e.g., specifically to TfR). 最大 To improve (e.g., to increase).

[0344] In some embodiments, the agent is in the brain C 最大 Compared with agents that bind to a reference polypeptide or protein (e.g., which bind to TfR with a weaker affinity), the efficacy was increased (e.g., improved) by at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.4, 2.6, 2.8, 3, 4, 5, or more times.

[0345] In some embodiments, the duration of exposure of the mammalian brain to a therapeutically effective concentration (e.g., a concentration sufficient to treat one or more signs or symptoms of LSD) is shorter than the duration of exposure to an agent bound to a reference polypeptide or protein. In some embodiments, the duration of brain exposure is reduced by at least about 5%, 10%, 25%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, or 98%.

[0346] In some embodiments, brain exposure is quantified by plotting a curve of brain exposure (e.g., the concentration of the agent in the brain) over time and calculating the area under the curve (AUC). A decrease in AUC may indicate a reduction or shortening of brain exposure. In some embodiments, the duration of brain exposure to the agent (e.g., at therapeutically effective concentrations) is shortened.

[0347] In some embodiments, the reference peptide or protein binds to the TfR with an affinity equal to or weaker than about 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, or 600 nM (e.g., specific binding). In some embodiments, the reference peptide or protein binds to the TfR with an affinity equal to or weaker than about 600 nM.

[0348] In some implementations, the mammal is a primate (e.g., a human). In some implementations, the human is a patient requiring treatment for LSD. In some implementations, the patient has one or more signs or symptoms of LSD.

[0349] In some embodiments, the peptide or protein binds (e.g., specifically binds) to the primate TfR. In some embodiments, the primate TfR is the human TfR. In some embodiments, the peptide or protein binds to the apical domain of the TfR.

[0350] In some embodiments, the agent (e.g., a therapeutic agent) is linked to an engineered TfR-binding peptide. In some embodiments, the engineered TfR-binding peptide comprises a CH3 or CH2 domain having modifications that allow the peptide to specifically bind to TfR. Non-limiting examples of suitable engineered TfR-binding peptides are described herein. In some embodiments, the agent is linked to an engineered TfR-binding peptide described in Table 4 or Table 5. In some embodiments, the agent is linked to an engineered TfR-binding peptide 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.

[0351] In some embodiments, the agent (e.g., a therapeutic agent) is linked to a TfR-binding peptide. In some embodiments, the TfR-binding peptide is a short peptide with a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Methods for generating, screening, and identifying suitable peptides (i.e., peptides that bind to TfR with an affinity within the desired range) are known in the art. For example, a phage display strategy using alternating rounds of negative and positive selection can be used to identify suitable peptides. 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.

[0352] In some embodiments, the agent (e.g., a therapeutic agent) is bound to a TfR-binding antibody. Non-limiting examples of suitable TfR-binding antibodies include the OX26 anti-TfR antibody (i.e., having an affinity of about 76 nM, 108 nM, and 174 nM) disclosed in Thom et al., Mol. Pharm., 15(4):1420-1431 (2018). In some embodiments, the agent is bound to a protein comprising an antibody variable region that specifically binds to TfR. In some cases, the protein comprises Fab or scFv.

[0353] In some embodiments, the agent (e.g., a therapeutic agent) is a protein (e.g., an enzyme). In some embodiments, the agent is a protein replacement therapeutic agent. In some embodiments, the agent is a protein or enzyme that is lacking (e.g., poorly expressed or absent) in cells or tissues (e.g., nerve cells or tissues) in mammals. In some embodiments, the agent is a protein or enzyme that is endogenous to or expressed in normal healthy cells or tissues (e.g., nerve cells or tissues) in mammals, but lacking (e.g., in the corresponding cells or tissues) in mammals being treated for LSD.

[0354] In some embodiments, the protein replacement therapy agent is an enzyme. Any number of agents (e.g., protein replacement therapy agents, such as enzymes) can be linked to peptides or proteins (e.g., peptides or proteins bound to TfR) to treat various LSDs. In some embodiments, the agent is an enzyme that, when linked to the peptide or protein, reduces the accumulation of toxic metabolites in the brain of mammals with LSD to a greater extent than when the enzyme is linked to a reference peptide or protein. In some embodiments, the enzyme is iduronate 2-sulfatase (IDS) and LSD is Hunter syndrome. In some cases, the toxic metabolites comprise disaccharides derived from heparin sulfate and / or dermatan sulfate. In some embodiments, the enzyme is N-sulfoglucosamine sulfonylhydrolase (SGSH) and LSD is San Philippe syndrome. In some embodiments, the enzyme is acid sphingomyelinase (ASM) and LSD is Niemann-Pick disease. In some embodiments, the enzyme is β-glucocerebrosidase (GBA) and LSD is Gaucher disease.

[0355] In some embodiments, the agent (e.g., a therapeutic agent) comprises an antibody variable region. In some embodiments, the agent comprises an antibody fragment. In some embodiments, the agent comprises Fab or scFv. In some embodiments, the agent does not contain an antibody variable region. In some cases, the agent does not contain anti-β-secretase 1 (BACE1) Fab.

[0356] Additional implementation schemes and connectors

[0357] A polypeptide (e.g., a modified CH3 or CH2 domain polypeptide as further described herein) may be linked to another domain of the Fc region. In some embodiments, the modified CH3 domain polypeptide is typically linked to a CH2 domain at the C-terminus, which may be a naturally occurring CH2 domain or a variant CH2 domain. In some embodiments, the modified CH2 domain polypeptide is typically linked to a CH3 domain at the N-terminus, which may be a naturally occurring CH3 domain or a variant CH3 domain. In some embodiments, a polypeptide comprising a modified CH2 domain linked to a CH3 domain or a polypeptide comprising a modified CH3 domain linked to a CH2 domain further comprises a portion or all of the hinge region of an antibody, thereby producing a form in which the modified CH3 domain polypeptide or the modified CH2 domain polypeptide is part of an Fc region having a portion or all of the hinge region. The hinge region may be derived from any immunoglobulin subclass or isotype. The illustrative 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).

[0358] In some embodiments, engineered TfR-binding peptides, TfR-binding peptides, or TfR-binding antibodies are fused to peptides or proteins that can be used 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 fused to the engineered TfR-binding peptide, TfR-binding peptide, or TfR-binding antibody may contain protease cleavage sites, such as factor Xa or thrombin cleavage sites.

[0359] In the methods of this disclosure, an agent (e.g., a therapeutic agent) is linked to a polypeptide or protein (e.g., an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody). The linker can be any linker suitable for linking the agent to the polypeptide or protein. In some embodiments, the linking is enzyme-cleavable. In some embodiments, the linking can be cleaved by an enzyme present in the central nervous system.

[0360] In some embodiments, the linker is a peptide linker. The peptide linker can be configured such that it allows the agent (e.g., a therapeutic agent) and the polypeptide or protein to rotate relative to each other; and / or is resistant to digestion by proteases. In some embodiments, the linker can be a flexible linker, which contains, for example, amino acids such as Gly, Asn, Ser, Thr, Ala, etc. Such linkers are designed using known parameters. For example, the linker can have repeats, such as Gly-Ser repeats.

[0361] In various embodiments, the binding of the agent (e.g., a therapeutic agent) to a peptide or protein (e.g., an engineered TfR-binding peptide, TfR-binding peptide, or TfR-binding antibody) can be achieved using well-known chemical crosslinking agents and methods. For example, a large number of chemical crosslinking agents are known to those skilled in the art and can be used to crosslink peptides or proteins with the agent of interest. For example, the crosslinking agent is a heterobifunctional crosslinking agent, which can be used for stepwise binding of molecules. Heterobifunctional crosslinking agents provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrence of unwanted side reactions (such as homologous protein polymers).

[0362] The agent (e.g., a therapeutic agent) may be linked to the N-terminal or C-terminal region of a polypeptide or protein (e.g., an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody), or attached to any region of the polypeptide or protein, provided that the agent does not prevent the polypeptide or protein from binding to the transferrin receptor.

[0363] XIV. Pharmaceutical Compositions and Kits

[0364] In other respects, pharmaceutical compositions and kits comprising the fusion protein described herein are provided.

[0365] Pharmaceutical Composition

[0366] Instructions for preparing formulations for use in this disclosure can be found in a large number of pharmaceutical preparation and formulation manuals known to those skilled in the art.

[0367] In some embodiments, the pharmaceutical composition comprises a fusion protein as described herein and also comprises one or more pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers include any solvent, dispersion medium, or coating agent that is physiologically compatible and does not impair or otherwise inhibit the activity of the active agent.

[0368] In some embodiments, the carrier is suitable for intravenous, intrathecal, ocular, intraventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers may contain one or more physiologically acceptable compounds that, for example, stabilize the composition or increase or decrease peptide absorption. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents, low molecular weight proteins, compositions that reduce the scavenging or hydrolysis of active agents, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are also available in the art.

[0369] The pharmaceutical compositions described herein can be manufactured, for example, by means of conventional mixing, dissolving, granulation, sugar-coated pellet preparation, emulsification, encapsulation, embedding, or lyophilization processes. The following methods and excipients are exemplary.

[0370] For oral administration, the fusion protein described herein can be formulated by combining it with a pharmaceutically acceptable carrier well known in the art. Such carriers enable the fusion protein to be formulated as tablets, pills, sugar-coated pills, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be treated. Pharmaceutical formulations for oral use can be obtained by mixing the fusion protein with a solid excipient, optionally grinding the resulting mixture, and processing the particulate mixture after adding suitable adjuvants (if desired) to obtain a tablet or sugar-coated pill core. Suitable excipients include, for example, fillers such as sugars, including 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 necessary, a disintegrant such as croscarmellose, agar, or alginate or their salts, such as sodium alginate, may be added.

[0371] As disclosed above, fusion proteins as described herein can be formulated for parenteral administration by injection (e.g., by rapid injection or continuous infusion). For injection, fusion proteins can be formulated by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent (such as vegetable oil or other similar oils, synthetic aliphatic acid glycerides, esters of high-carbon fatty acids, or propylene glycol); and, if desired, using conventional additives such as solubilizers, isotonics, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the fusion proteins can be formulated in an aqueous solution, such as a physiologically compatible buffer, non-limiting examples of which include Hanks's solution, Ringer's solution, and physiological saline buffer. The formulation for injection can be in a single dosage form along with added preservatives in, for example, ampoules or multi-dose containers. The composition may be in the form of a suspension, solution or emulsion, such as in an oily or aqueous medium, and may contain formulations such as suspending agents, stabilizers and / or dispersants.

[0372] In some embodiments, the fusion protein as described herein is prepared, for example, in a semi-permeable matrix of a solid hydrophobic polymer containing an active agent, for delivery in the form of a sustained-release, controlled-release, extended-release, time-limited-release, or delayed-release formulation. Various types of sustained-release materials have been identified and are well known to those skilled in the art. Extended-release formulations include film-coated tablets, multiparticle or spheroidal systems, matrix techniques using hydrophilic or lipophilic materials, and wax-based tablets using pore-forming excipients. Typically, sustained-release formulations can be prepared using naturally occurring or synthetic polymers, such as polymeric vinylpyrrolidones, such as polyvinylpyrrolidone; carboxyvinyl hydrophilic polymers; hydrophobic and / or hydrophilic aqueous colloids, such as methylcellulose, ethylcellulose, hydroxypropylcellulose, and hydroxypropyl methylcellulose; and carboxypolymethylene.

[0373] Typically, pharmaceutical compositions intended for internal administration are sterile. Sterilization can be achieved using methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or radiation.

[0374] The dosage and desired drug concentration of the pharmaceutical compositions described herein may vary depending on the intended specific use. Suitable dosages are also described in Section XII above.

[0375] medicine box

[0376] In some embodiments, a kit is provided for treating LSD (e.g., Hunter syndrome, St. Philippian syndrome A, Niemann-Pick disease, Gaucher's disease, or Parkinson's disease), the kit containing a fusion protein as described herein.

[0377] In some embodiments, the kit also contains one or more additional therapeutic agents. For example, in some embodiments, the kit contains a fusion protein as described herein and also contains one or more additional therapeutic agents for treating neurological symptoms of LSD. In some embodiments, the kit also contains instructional material containing guidance (i.e., protocols) on practicing the methods described herein (e.g., instructions on using the kit to cross the blood-brain barrier to administer a fusion protein containing an ERT enzyme). While instructional material typically includes written or printed material, it is not limited to written or printed material. This disclosure covers any medium capable of storing such instructional material and enabling it to communicate with the end user. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, cassette tapes, chips), optical media (e.g., CD-ROMs), etc. Such media may include website URLs providing such instructional material.

[0378] XV. Example

[0379] This disclosure will be described in more detail by way of specific embodiments. The following embodiments are provided for illustrative purposes only and are not intended to limit this disclosure in any way. Those skilled in the art will readily identify several non-critical parameters that can be changed or modified to obtain substantially the same results. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and biases may exist. Unless otherwise indicated, the practice of this disclosure will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of the art. Such techniques are well explained in the literature. Furthermore, it will be apparent to those skilled in the art that methods used for engineering certain libraries can also be applied to other libraries described herein.

[0380] Example 1. Construction of a fusion protein containing iduronate 2-sulfatase (IDS).

[0381] Design and cloning

[0382] The IDS-Fc fusion protein is designed to contain (i) a fusion polypeptide in which a mature human IDS enzyme is fused to a human IgG1 fragment including an Fc region (“IDS-Fc fusion polypeptide”), and (ii) a modified human IgG1 fragment containing a mutation in the Fc region conferring transferrin receptor (TfR) binding (“modified Fc polypeptide”). Specifically, the IDS-Fc fusion polypeptide is formed in which the IDS fragment is fused to the N-terminus or C-terminus of the human IgG1 Fc region. In some cases, a linker is placed between the IDS and the IgG1 fragment to alleviate any steric hindrance between the two fragments. In all constructs, a signal peptide from κ chain V-III, amino acids 1-20 (UniProtKB ID-P01661), is inserted upstream of the fusion to promote secretion, and the IDS is truncated to consist of amino acids S26-P550 (UniProtKB ID-P22304). The fragment of the human IgG1 Fc region used corresponds to amino acid D104-K330 (positions 221-447, EU number, which includes 10 amino acids of the hinge (positions 221-230)) in UniProtKB ID P01857. In some embodiments, a second Fc polypeptide derived from human IgG1 residues D104-K330 but lacking IDS fusion is co-transfected with an IDS-Fc fusion polypeptide to generate a heterodimeric fusion protein (“single enzyme”) containing one IDS enzyme. In some constructs, the IgG1 fragment contains an additional mutation to promote heterodimerization of both Fc regions. Similarly, control IDS-Fc fusion proteins lacking mutations conferring TfR binding were designed and constructed, the difference being that these proteins lacked the mutations conferring TfR binding. As another control, we generated an IDS (amino acids S26-P550) containing a C-terminal hexahistine tag (SEQ ID NO: 241) to facilitate detection and purification.

[0383] The TfR-binding IDS-Fc fusion protein used in the examples is a dimer formed by the IDS-Fc fusion polypeptide and the modified Fc polypeptide bound to TfR. For the IDS enzyme-linked dimer to the N-terminus of the Fc region, the IDS-Fc fusion polypeptide may have the sequence of any one of SEQ ID NO: 115, 231, and 235. In these sequences, the IDS sequence is underlined, and the cysteine ​​residue at position 59 (double underlined) is modified to formylglycine. The IDS is linked to the Fc polypeptide via a GGGGS linker (SEQ ID NO: 239). The N-terminus of the Fc polypeptide includes a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 113). The CH2 domain sequence begins at position 541 in SEQ ID NO: 115, 231, and 235.

[0384] The IDS-Fc fusion protein ETV:IDS 35.21 used in the examples is a dimer formed by an IDS-Fc fusion polypeptide having the sequence of any one of SEQ ID NO:115, 231, and 235 and a modified Fc polypeptide bound to TfR and having the sequence of SEQ ID NO:116. The first 10 amino acids are part of the IgG1 hinge region. The CH2 domain sequence begins at position 11 of SEQ ID NO:116.

[0385] The IDS-Fc fusion protein ETV:IDS 35.21.17.2 used in the examples is a dimer formed by an IDS-Fc fusion polypeptide having the sequence of any one of SEQ ID NO:115, 231, and 235 and a modified Fc polypeptide bound to TfR and having the sequence of SEQ ID NO:228. The first 10 amino acids are part of the IgG1 hinge region. The CH2 domain sequence begins at position 11 of SEQ ID NO:228.

[0386] The IDS-Fc fusion protein ETV:IDS 35.23.2 used in the examples is a dimer formed by an IDS-Fc fusion polypeptide having the sequence of any one of SEQ ID NO:115, 231, and 235 and a modified Fc polypeptide bound to TfR and having the sequence of SEQ ID NO:229. The first 10 amino acids are part of the IgG1 hinge region. The CH2 domain sequence begins at position 11 of SEQ ID NO:229.

[0387] The IDS-Fc fusion protein ETV:IDS 35.21.17 used in the examples is a dimer formed by an IDS-Fc fusion polypeptide having the sequence of any one of SEQ ID NO:115, 231, and 235 and a modified Fc polypeptide bound to TfR and 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).

[0388] Recombinant protein expression and purification

[0389] To express the 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 according to the manufacturer's instructions (Thermo Fisher Scientific). Cells were grown in ExpiCHO™ expression medium at 37°C, 6% CO2, and 120 rpm in an Infortrex HT Multitron. In short, at 6 x 10⁻⁶ cells / mL… 6 Log-growing ExpiCHO™ cells were transfected at a density of 0.8 μg of DNA plasmid per mL of culture volume. Following transfection, cells were returned to 37°C and the transfected cultures were fed as directed 18–22 hours post-transfection. The supernatant of the transfected cell culture was harvested at 120 hours post-transfection by centrifugation at 3,500 rpm for 20 min. The clarified supernatant was filtered (using a 0.22 μM membrane) and stored at 4°C. With minor modifications, the expression of an epitope-tagged IDS enzyme (used as a control) was performed as described above. In short, the IDS enzyme with a C-terminal hexahistine tag (SEQ ID NO: 241) was expressed in ExpiCHO cells.

[0390] IDS-Fc fusion proteins with (or without) engineered Fc regions conferring TfR binding were purified from cell culture supernatants using protein A affinity chromatography. The supernatant was loaded onto a HiTrap MabSelect SuRe protein A affinity column (GE Healthcare Life Sciences, using the Akta Pure system). The column was then washed with >20 column volumes (CV) of PBS. The bound protein was 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 protein in the eluted fraction was assessed by reducing and non-reducing SDS-PAGE.

[0391] To purify the hexahistidine-labeled (SEQ ID NO: 241) IDS enzyme, the transfection supernatant was thoroughly dialyzed overnight with 15 μL of 20 mM HEPES pH 7.4 containing 100 mM NaCl. The dialyzed supernatant was then bound to a HisTrap column (GE Healthcare Life Sciences, using the Akta Pure system). After binding, the column was washed with 20 CV of PBS. The bound protein was eluted with PBS containing 500 mM imidazole. The homogeneity of the IDS enzyme in the elution fraction was assessed by reducing and non-reducing SDS-PAGE. The pooled fraction containing the IDS enzyme was diluted 1:10 in 50 mM Tris pH 7.5 and further purified using a high-performance Q agarose gel (GE Healthcare). After binding, the column was washed with 10 CV of 50 mM Tris pH 7.5. The bound proteins were eluted using a linear gradient of 50 mM Tris at pH 7.5 and 0.5 M NaCl and collected in 1 CV fractions. Fraction purity was assessed by non-reducing SDS-PAGE. Figure 1 As shown, the purification yields homogeneous IDS-Fc fusion protein and IDS enzyme labeled with six histidine (SEQ ID NO:241).

[0392] Example 2. Characterization of IDS fusion protein.

[0393] The IDS-Fc fusion protein with an engineered TfR binding site binds to human TfR.

[0394] To determine whether the engineered TfR-binding IDS-Fc fusion protein affects the ability of the modified Fc domain to interact with human TfR, Biacore was used. ™ Surface plasmon resonance analysis was used to assess the affinity of this protein for human TfR. Biacore was used... ™ The SCM5 sensor chip was immobilized using anti-human Fab (from GE Healthcare's Human Fab Capture Kit). 5 μg / mL of the IDS-Fc fusion protein was captured in each flow cell for 1 minute, followed by injection of serially 3-fold dilutions of human apical domain TfR at a flow rate of 30 μL / min. Each sample was analyzed under 3-minute association and 3-minute dissociation conditions. After each injection, the chip was regenerated using 10 mM glycine-HCl (pH 2.1). The binding reaction was corrected by subtracting RU from flow cells that captured irrelevant IgG at similar densities. Biacore was used. ™The T200 evaluation software v3.1 obtains steady-state affinity by fitting the concentration-equilibrium response. For example... Figure 2 As shown, Biacore ™ Analysis confirmed that the IDS-Fc fusion protein, with a TfR binding site engineered to the Fc region, binds to human TfR. (Biacore) ™ The analysis also confirmed that the IDS-Fc fusion protein ETV:IDS 35.21 binds to human TfR with an affinity of approximately 200 nM.

[0395] The IDS-Fc fusion protein with engineered TfR binding sites is active in vitro, in cells, and in vivo.

[0396] The in vitro and cellular activities of the engineered TfR-binding IDS-Fc fusion protein were evaluated to confirm that IDS maintains its enzymatic activity upon fusion into a human IgG fragment. In vitro activity was measured using a two-step fluorescent enzyme assay with an artificial substrate. Specifically, 20 μL of 1 mM 4-methylumbelliferyl α-L-pyranouronic acid 2-sulfate disodium salt substrate (Carbosynth Limited, #EM03201) was diluted in analytical 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 h and the reaction was terminated with 60 μL of 0.2 M phosphate-citrate buffer, pH 5.0. A second reaction was then performed in the presence of 15 μg of cell lysate from HEK 293T cells transiently transfected with human α-iduronidase (IDUA), incubated at 37°C for 16 h, and stopped by adding 100 μL of 0.5 M sodium carbonate buffer, pH 10.5. Fluorescence of the reaction solution was then measured (excitation at 365 nm and emission at 450 nm). The amount of product was calculated by fitting a 4-methylumbelliferone standard curve using linear regression, and was verified to be less than 10% of total substrate lysis. Specific activity (nanomoles of product per nanomoles of IDS per minute) was calculated by dividing the amount of product by the reaction time and the molar amount of IDS.

[0397] In vitro enzyme activity analysis confirmed that the IDS-Fc fusion protein was active and indicated that the fusion of the Fc region into IDS did not impair enzyme activity. Figure 3 ).

[0398] IDS knockout (KO) cells were generated using CRISPR / CAS9 to provide a cellular system for testing the cellular viability of engineered IDS-Fc fusion proteins. HEK 293T cells (ATCC) were transfected with a CRISPR / CAS9 pCas-Guide-EF1a-GFP vector (Origene) containing a guide sequence targeting the latter half of exon 1 in human IDS. Individual cell clones were analyzed for the presence of insertions and deletions within the IDS genome sequence after using the Guide-it Mutation Detection Kit (Clontech) according to the manufacturer's instructions. To identify IDS KO cells, the lysate of insertion-deletion-positive clones was analyzed using the in vitro IDS enzyme assay described above. In summary, in vitro activity assays were performed using 12.5, 25, 50, and 100 μg of cell lysate as previously described in lead acetate assay buffer at pH 5.0 (100 mM sodium acetate, 10 mM lead acetate, 0.02% sodium azide) (Vozyni et al., J. Inherit. Metab. Dis., 24:675-80 (2001)). The reaction was initiated by combining 10 μL of normalized cell lysate (in water) with 1 mM substrate in 20 μL of lead acetate buffer. The first reaction mixture was incubated at 37°C for 4 h and terminated using 60 μL of 0.2 M phosphate-citrate buffer at pH 5.0. A second reaction was then performed by adding 10 μg / 10 μL of cell lysate from HEK 293T cells transiently transfected with human α-iduronidase (IDUA) and incubating at 37°C for 24 hours. The reaction was stopped by adding 100 μL of 0.5 M sodium carbonate buffer, pH 10.3. Fluorescence of the reaction solution was then measured (excited at 365 nm and emitted at 450 nm). IDS activity in the HEK 293T CRISPR clone was compared with recombinant IDS, HEK wild-type (WT) lysate, and HEK cell lysate overexpressing IDS as analytical standards. Following the mini-Topo (ThermoFisher) clone, clones with enzyme activity levels similar to the background signal were sequence validated and identified as KO clones. Subsequent cell analyses used three unique and validated IDS KO clones and three independent batches of WT HEK 293T cells.

[0399] To test the cellular activity of naked IDS enzymes or IDS-Fc fusion proteins, LC-MS / MS-based glycosomnography was performed, allowing for the monitoring of substrate accumulation (heparan sulfate and dermatan sulfate) as an indicator of IDS activity. Substrate accumulation in IDS KO cells was measured before and after the addition of IDS or IDS-Fc fusion proteins to the cell culture medium. In short, cells were washed three times with PBS, aggregated into pellets, and frozen. Cell pellets were sonicated in disaccharide digestion buffer (111 mM NH4OAc, 11 mM CaOAc, pH 7.0). Protein concentration was measured using BCA analysis (Pierce). Total protein (100 μg) was added to 100 μL of digestion buffer along with 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 digestion with heparan sulfate and dermatan sulfate for three hours at 30°C, 20 ng of the internal standard (4UA-2S-GlcNCOEt-6S HD009 [Galen]) was added to each sample. The enzymes were deactivated by adding 6 μL of 250 mM EDTA, and the samples were boiled at 95°C for 10 minutes. The samples were then centrifuged at 16,000 x G for 5 minutes at room temperature. The supernatant was transferred to an Amicon Ultra 30KD centrifuge filter (Millipore) and centrifuged at 14,000 x G for 15 minutes. The disaccharide was concentrated in the flow-through and then resuspended in a mixture of [1:1, v / v] analytical buffer and acetonitrile, and then transferred to a mass spectrometry vial for further analysis.

[0400] Disaccharides produced by enzymatic digestion of heparan sulfate and dermatan sulfate were analyzed by liquid chromatography coupled with electrospray mass spectrometry (Sciex 6500+ QTRAP, Sciex, Framingham, MA, USA) using a Shimadzu Nexera X2 system (Shimadzu Scientific Instrument, Columbia, MD, USA). For each analysis, 10 μL of sample was injected onto 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 with 10 mM ammonium formate and 0.1% formic acid. Mobile phase B consisted of acetonitrile with 0.1% formic acid. The gradient was programmed as follows: 0.0–1.0 min at 85% B, 1.0–5.0 min from 85% B to 50% B, 5.0–6.0 min from 50% B to 85% B, and held at 85% B for 6–8.0 min. Electrospray ionization was performed in anion mode using the following settings: curtain gas, 30; collision gas set to medium; ion spray voltage, -4500; temperature, 450; ion source gas 1, 50; ion source gas 2, 60. Data acquisition was performed using Analyst 1.6.3 (Sciex) in multiple reaction monitoring (MRM) mode with a residence time of 25 milliseconds. Collision energy, -30; declustering potential, -80; inlet potential, -10; collision chamber outlet potential, -10. GAG was detected using the following MRM transitions in the form of [M--H]-: D0A0 > 87.0 at m / z 378.1; D0a0 > 175.0 at m / z 378.1; D0S0 > 138.0 at m / z 416.1; D0a4 > 300.0 at m / z 458.1; D0A6, D2A0, D0a6, D2a0 > 97.0 at m / z 458.1; D0S6, D2S0 > 416.1 at m / z 496.0; D2a4, D2a6, D0a10, D2A6 > 458.0 at m / z 538.0; D0S6 > 97.0 at m / z 575.95; at m / z 472.0 The fragment ion 4UA-2S-GlcNCOEt-6S> 97.0 was used as an internal standard (IS). GAGs were identified based on residence time and MRM transients matched with a commercially available reference standard (Iduron Ltd, Manchester, UK).Quantification was performed using MultiQuant 3.0.2 (Sciex) by area ratio to IS. GAG was normalized to total protein content. Protein concentration was measured using BCA analysis (Pierce).

[0401] Compared to control cell lines, significant substrate accumulation, as reflected by the amount of disaccharides observed after digestion with heparan sulfate and dermatan sulfate, was observed in IDS KO cells. This effect could be rescued by adding recombinant IDS to the cells. Figure 4 This confirms that LC-MS / MS-based analysis can be used to assess the cellular viability of IDS and IDS-Fc fusion proteins.

[0402] Using this analysis, it was confirmed that treating cells with either an N-terminal single enzyme (i.e., ETV:IDS 35.21.17) or a C-terminal single enzyme form of the IDS-Fc fusion protein containing the same TfR-binding Fc polypeptide (i.e., CH3C.35.21.17) reduced the levels of heparan sulfate and dermatan sulfate-derived disaccharides back to the levels observed in wild-type cells. Figure 5A Furthermore, the activity of the N-terminal single enzyme is similar to that of IDS (…). Figure 5B In summary, these data confirm that the IDS-Fc fusion protein maintains enzyme activity and reduces substrate accumulation in IDS KO cells.

[0403] As previously described 35 S-pulse tracking analysis was used to examine the cellular activity of the IDS-Fc fusion protein in fibroblasts from MPS II patients and healthy controls. 35 S is integrated into the newly synthesized GAG (Lu et al., Bioconjugate Chemistry, 21:151-156 (2010)). MPS II patient fibroblasts lack detectable IDS activity, resulting in approximately 10-fold substrate accumulation and 2.5-fold [unclear - possibly referring to a specific type of protein or component]. 35 S-signal accumulation ( 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, regarding the reduction of S... 35 Accumulation of labeled proteins indicates low skin molar EC cells 50 ( Figure 5C Furthermore, the cellular activity of IDS-Fc fusion proteins such as ETV:IDS 35.23.2 was confirmed to be M6PR-dependent, as excessive M6P inhibition was observed in fibroblasts from MPS II patients treated with the protein. 35 Clearance of S-labeled proteins ( Figure 5DIn summary, these data confirm that M6PR-dependent transport and cellular activity of IDS can be maintained in the IDS-Fc fusion protein mode.

[0404] To measure heparan sulfate and dermatan sulfate-derived disaccharides in vivo, LC-MS / MS-based glycosylchemistry analysis was adapted for tissue and fluid analysis. In short, all tissues and fluids were collected, immediately frozen, and stored at -80°C. Samples were subjected to five freeze-thaw cycles and processed for cellular analysis as described above. Significant accumulation of heparan sulfate and dermatan sulfate-derived disaccharides was observed in all tissues and fluids analyzed from male IDS KO mice compared to male wild-type littermates (Table 1). This analysis was used for in vivo efficacy studies of the fusion protein. IDS KO mice were obtained from Jackson Laboratories (JAX strain 024744).

[0405] Table 1. Glycosomal analysis of tissues and fluids from IDS KO mice.

[0406]

[0407] Using this method, the levels of heparan sulfate and dermatan sulfate-derived disaccharides in the serum of wild-type (WT) mice administered the vector and IDS KO mice administered IDS or the IDS-Fc fusion protein (i.e., ETV: IDS 35.21) were assessed. Baseline measurements prior to administration showed a significant accumulation of heparan sulfate and dermatan sulfate-derived disaccharides in the serum of IDS KO mice compared to WT mice. Following administration of the IDS-Fc fusion protein, the levels of heparan sulfate and dermatan sulfate-derived disaccharides in the serum of IDS KO mice were significantly reduced, showing a reduction similar to that observed in the serum of IDS KO mice administered IDS. Figure 6 These data confirm that the IDS-Fc fusion protein is active in vivo and reduces substrate accumulation in IDS KO mice. Based on these data, the distribution and pharmacodynamic (PD) response in tissues of IDS KO mice were assessed seven days after a single dose of the IDS-Fc fusion protein. IDS KO mice were administered 40 mg / kg of the IDS-Fc fusion protein or 5.3 mg / kg of IDS (25% molar equivalent) intravenously as a positive control, and GAG levels were assessed. The distribution of both molecules in peripheral tissues was confirmed two hours after administration. A significant reduction in substrate was observed in the liver, spleen, and lungs of IDS KO mice seven days after administration of the IDS-Fc fusion protein. Figure 7 ).

[0408] To determine whether the TfR-binding IDS-Fc fusion protein showed improved brain delivery compared to the control IDS-Fc fusion protein, human TfR knock-in (TfR) was administered. ms / hu TfR-binding IDS-Fc fusion protein ETV:IDS35.21 or a control IDS-Fc fusion protein lacking a mutation conferring TfR binding (“IDS:Fc”) was administered to mice at 50 mg / kg, and the concentration of IDS-Fc fusion protein in the brain was measured using a sandwich ELISA-based assay as described in Example 3 below at 4 hours post-administration. TfR was generated by expressing the human Tfrc apical domain within the mouse Tfrc gene using CRISPR / Cas9 technology, as described in International Patent Publication No. WO 2018 / 152285. ms / hu KI mice; the obtained 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 (…). Figure 8 Using TfR ms / hu KI mice were used to assess brain uptake of two additional TfR-binding IDS-Fc fusion proteins, ETV:IDS 35.21.17.2 and ETV:IDS 35.23.2. TfR was administered... ms / hu KI mice were administered 50 mg / kg of ETV:IDS 35.21.17.2, ETV:IDS 35.23.2, or the control IDS-Fc fusion protein (“IDS:Fc”), and the concentration of IDS-Fc fusion protein in the brain was measured using a sandwich ELISA-based assay at 2 and 8 hours post-administration. At 2 hours post-administration, administration of the TfR-binding IDS-Fc fusion protein resulted in a 5-fold increase in brain uptake compared to the control IDS-Fc fusion protein, and at 8 hours, it resulted in a 10-20-fold increase in brain concentration. Figure 9A The serum PK and complete fusion protein accumulation in the liver of ETV:IDS 35.21 and IDS:Fc were equivalent. Figure 9B The data indicate that the IDS portion primarily determines the distribution phase of plasma clearance. Brain levels of the TfR-binding IDS-Fc fusion protein remained elevated for eight hours, decreasing slightly with peripheral clearance. In summary, these data confirm that the interaction between the TfR-binding IDS-Fc fusion protein and TfR generally maintains peripheral distribution while significantly improving brain exposure.

[0409] ETV: Intravenous administration of IDS reduces GAGs in the brain.

[0410] To examine whether the improved brain exposure observed in the TfR-binding IDS-Fc fusion protein (referred to herein as ETV:IDS) prepared according to Example 1 as described above resulted in a corresponding reduction in the accumulated substrate in the brain, an IDS-deficient mouse model with the human TfR apical domain of knock-in mouse TfR (referred herein as IDS KO xTfR) was generated. ms / hu KI mice). Simply put, it makes TfR ms / hu Male KI mice were bred with female IDS heterozygous mice to achieve TfR ms / hu IDS KO mice were generated from a KI homozygous background. All mice used in this study were male and housed under a 12-hour light-dark cycle with free access to food (LabDiet JL irradiation 6F) and water.

[0411] For IDS KO x TfR ms / hu KI mice were administered intravenously with a single or four-weekly active equivalent dose of ETV:IDS or IDS (747 μmol product / min / kg or 40 mg / kg and 14.2 mg / kg, respectively), and pharmacokinetic and pharmacodynamic responses were evaluated. Specifically, 2-month-old IDS KO x TfR mice were administered intravenously (iv) once (n=8) or weekly for 4 weeks (n=8) with saline, IDS (14.2 mg / kg body weight), or ETV:IDS (40 mg / kg body weight). ms / hu KI mice assay of the effect of peripheral ETV:IDS on IDS KO x TfR ms / hu Effects of intravenous saline injection on brain and tissue GAG ​​in KI mice. Two-month-old littermate TfR mice were injected intravenously with saline once (n=5) or weekly for 4 weeks (n=5). ms / hu KI mice were used as controls. For animals administered IDS or ETV:IDS, serum samples were collected at various time points via submandibular exsanguination. All animals were euthanized 7 days after a single dose or 7 days after the final 4-week dose. Urine, serum, CSF, liver, kidneys, spleen, lungs, heart, and right hemisphere of the brain were dissected and rapidly frozen on dry ice.

[0412] Following a single dose, as assessed using the ELISA-based assay for detecting IDS concentration described in Example 3 below, ETV:IDS exhibited a similar serum clearance pattern to IDS, thus providing additional support for the enzyme's primary effect on peripheral clearance. Figure 10A Two hours after administration, ETV:IDS showed a significant increase in brain levels compared to IDS, with mean concentrations of 8.4 and 1.6 nM, respectively, and significantly increased liver and spleen levels compared to IDS. Figure 10B ).

[0413] To determine whether ETV:IDS reduces substrate levels in the brain, the levels of the enzyme after a single dose or four weeks of treatment with IDS KO x TfR were evaluated as described in Example 2. ms / hu GAG levels in KI mice. At early time points, IDS slightly reduced brain GAG levels, but after four weeks of treatment, it was ineffective in significantly reducing GAG levels. Figure 10C However, ETV:IDS reduced brain GAG levels by approximately 58% after a single dose and by 71% after four weeks of treatment. Figure 10C This resulted in a reduction of approximately 75% in CSF GAG after a single dose, which was maintained four weeks after administration. Figure 10C Both molecules effectively reduced GAG levels in the liver and spleen after one week, and the response was maintained with repeated administration. Figure 10C This confirms that TfR binding does not negatively affect pharmacokinetic responses in these tissues. In summary, these data demonstrate that ETV:IDS significantly increases brain exposure to the enzyme and greatly reduces substrate accumulation in the peripheral and CNS.

[0414] Example 3. Pharmacokinetic characterization of the IDS fusion protein.

[0415] This example describes the pharmacokinetic (PK) characterization of an engineered IDS-Fc fusion protein in mouse plasma.

[0416] To determine the plasma half-life and clearance of TfR-binding IDS-Fc fusion proteins, 7-8 week old male C57BL / 6 mice were administered 10 mg / kg of two IDS-Fc fusion protein molecules (N-terminal and C-terminal monoenzymes) via tail vein injection. The concentration of IDS-Fc fusion proteins retained in plasma over 24 hours was measured using an ELISA-based assay. In short, the concentration of IDS-Fc fusion proteins in mouse plasma was quantified using a sandwich ELISA. Anti-Fc capture antibody (Abcam #ab124055) was coated at 3 μg / mL onto 384-well MaxiSorp™ plates (Thermo Scientific #464718). The plates were blocked with 5% BSA and then incubated with plasma diluted 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 developed using TMB substrate, the reaction was stopped with sulfuric acid, and the absorbance was measured at 450 nm using a BioTek plate reader. Standard curves were prepared for individual constructs in a 4-fold dilution series ranging from 200 to 0.1 ng / mL and fitted using four-parameter Rogers regression.

[0417] Using this analysis, the terminal plasma half-life of the IDS-Fc fusion protein was confirmed to be 7.7–10 hours (Table 2). No unexpected PK tendency was observed in vivo with the IDS-Fc fusion protein.

[0418] Table 2. Pharmacokinetics of the IDS-Fc fusion protein in mice over 24 hours.

[0419]

[0420] Example 4. Construction of a fusion protein containing acid sphingomyelinase (ASM).

[0421] Design and cloning

[0422] The ASM-Fc fusion protein is designed as a dimer of a fusion polypeptide (“ASM-Fc fusion polypeptide”) fused to a mature human ASM enzyme and including a human IgG1 fragment containing the Fc region. In some embodiments, the ASM-Fc fusion polypeptide comprises a modified Fc region containing a mutation conferring transferrin receptor (TfR) binding. Specifically, the ASM-Fc fusion polypeptide is formed by fusing an ASM fragment to the N-terminus of the human IgG1 Fc region. In some cases, a linker is placed between the ASM and IgG1 fragments to alleviate any steric hindrance between the two fragments. In all constructs, the native ASM signal sequence amino acids 1-46 (UniProtKB ID- P17405) was removed and secreted with amino acids 1-20 from the κ chain V-III (UniProtKB ID- P01661 To improve ASM secretion, a replacement was performed. Additionally, in the fusion protein, ASM was truncated at its C-terminus, ending at amino acid Q620, to prevent any unwanted cleavage between ASM and the human IgG1 Fc region. Then, a fragment of the human IgG1 Fc region (UniProtKB ID-) was... P01857 The C-terminus of ASM is placed within the frame starting at amino acid E99, with a cysteine ​​residue at position 103 mutated to a serine residue. In some embodiments, the IgG1 fragment contains additional mutations to promote heterodimerization of the two Fc regions. Additionally, an ASM-Fc fusion protein containing one or two ASM molecules is generated. As a control, an ASM-hexahistidine (SEQ ID NO:241) fusion protein is designed to consist of ASM amino acids 1-628 truncated to remove the C-terminal cysteine ​​and promote enzyme activation, along with a C-terminal fused hexahistidine tag (SEQ ID NO:241).

[0423] Recombinant protein expression and purification

[0424] To express the recombinant ASM enzyme fused to the Fc region, ExpiCHO-S cells (Thermo Fisher) were thawed at 6 x 10⁻⁶ cells / mL. 6 Transfected cells at a density of 1,000 cells were transfected using the Expifectamine CHO / plasmid DNA complex according to the manufacturer's instructions (ThermoFisher Scientific). Following transfection, cells were incubated in a rotary shaker (Infors HT Multitron) at 32°C under a humidified atmosphere of 6-8% CO2. On day 1 post-transfection, the Expifectamine enhancer and Expifectamine feed were added to the culture. After 48-72 hours of expression time, the culture supernatant was harvested by centrifugation. The clarified supernatant was supplemented with an EDTA-free protease inhibitor (Roche) and stored at -80°C.

[0425] For the purification of the ASM-Fc fusion protein, 200 μM zinc acetate (Sigma-Aldrich) was added to the clarified culture supernatant. The supernatant was loaded onto a HiTrap MabSelect SuRe protein A affinity column (GE Healthcare Life Sciences) and washed with 200 mM arginine and 137 mM succinate buffer at pH 5.0 (arginine-succinate buffer). The fusion protein was eluted in 100 mM QB citrate buffer at pH 3.0 supplemented with 200 μM zinc acetate. Immediately after elution, arginine-succinate buffer was added to adjust the pH. The protein aggregates were separated from the ASM-Fc fusion protein by size exclusion chromatography (SEC) on a Superdex 200increase 10 / 300 GL column (GE Healthcare Life Sciences). The SEC mobile phase was maintained in arginine-succinate buffer at pH 5.0 supplemented with 200 μM zinc acetate. All chromatographic steps were performed using the Akta Pure or Akta Avant system (GE Healthcare LifeSciences). Fraction purity was assessed by non-reducing SDS-PAGE. Figure 11 As shown in the figure, purification yields a homogeneous ASM-Fc fusion protein.

[0426] Example 5. Characterization of ASM fusion protein.

[0427] The ASM-Fc fusion protein is active in vitro and in cells.

[0428] To confirm that ASM maintains its enzymatic activity upon fusion into the human IgG heavy chain, the in vitro and cellular activities of the ASM-Fc fusion protein were evaluated. The in vitro activities of the recombinant ASM enzyme or the recombinant ASM-Fc fusion protein were measured using a synthetic chromogenic analog of sphingomyelin. Specifically, 2.5 mM 2-(N-hexadecylamino)-4-nitrophenylphosphocholine (EMD Millipore) was mixed with 0.75 nM ASM in 100 mM sodium acetate buffer (pH 5.3; final concentration in a 100 μL reaction volume). The reaction mixture was incubated at 37 °C for 16 h and stopped by adding an equal volume of 0.2 M NaOH. The absorbance of the reaction solution was then measured at 410 nm. The amount of product was calculated by fitting a p-nitrophenol standard curve using linear regression, and was verified to be less than 10% total substrate cleavage. Specific activities (nanomoles of product per nanomoles of ASM per minute) were calculated by dividing the amount of product by the reaction time and the molar amount of ASM. In vitro enzyme activity analysis confirmed that the ASM-Fc fusion protein was active and indicated that the fusion of the Fc region into ASM did not impair its enzymatic activity. Figure 12 ).

[0429] ASM KO cells were generated using CRISPR / CAS9 to provide a cellular system for testing the cellular activity of the ASM-Fc fusion protein. HEK 293T cells (ATCC) were transfected with the CRISPR / CAS9 pCas-Guide-EF1a-GFP vector (Origene) containing a guide sequence targeting the latter half of exon 2 in human SMPD1. Single cell clones were analyzed for the presence of insertions and deletions within the ASM genome sequence after using the Guide-it Mutation Detection Kit (Clontech) according to the manufacturer's instructions. In vitro ASM enzyme analysis was performed on the lysate of insertion / deletion positive clones using the ASM chromogenetic substrate 2-N-hexadecylamino-4-nitrophenylphosphocholine (EMDMillipore). In short, in vitro activity analysis was performed using 12.5, 25, 50, and 100 μg of cell lysate in 100 mM sodium acetate buffer (pH 5.3). The reaction was initiated by adding 2.5 mM of substrate and stopped by adding 0.2 M NaOH after 20 hours. ASM activity in the HEK293T CRISPR clone was compared with recombinant ASM, HEK wild-type (WT) lysate, and HEK cell lysate overexpressing ASM, which were used as analytical standards. Following the mini-Topo (Thermo Fisher Scientific) cloning, clones with enzyme activity levels similar to the background signal were sequence validated and identified as KO clones. Subsequent cell analyses used three unique and validated ASM KO clones and three independent batches of WT HEK293T cells.

[0430] To test the cellular activity of naked ASM enzyme or ASM-Fc fusion protein, two cellular analytes were developed for colorimetric analysis, allowing for monitoring of substrate accumulation (sphingomyelin) in ASM KO cells at baseline and after treatment with ASM or ASM-Fc fusion. First, imaging-based analysis was performed to monitor the accumulation of BODIPY-conjugated C5-sphingomyelin in ASM KO cells. Specifically, HEK293T WT and ASM KO cells were low-density plated in DMEM (Gibco) supplemented with 10% FBS onto PDL-coated 96-well plates (Perkin Elmer). Four hours after plating, 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 then removed and replaced with fresh medium containing 1 μM BODIPY-C5-sphingomyelin (Thermo Fisher Scientific) and incubated at 37°C for 16 hours. Cells were then washed with PBS, fixed with 4% paraformaldehyde, and stained with nuclear (DAPI, Thermo Fisher) and cytoplasmic (FAR) cell mask, Thermo Fisher Scientific. Images were acquired using an Opera Phenix confocal microscope (Perkin Elmer) with 63X objectives, multiple fields of view per well, and three replicates per condition. Image analysis was performed using Harmony software (Perkin Elmer), which detected and analyzed the mean total intensity, number of spots, and spot intensity of BODIPY-C5-sphingomyelin in each cell. These values ​​for each cell were then averaged for the values ​​of each well, which were used to analyze the effect of genotype and / or treatment on BODIPY-C5-sphingomyelin accumulation. Significant BODIPY-C5-sphingomyelin accumulation was observed in ASM KO cells compared to the control cell line, an effect that could be rescued by the addition of recombinant ASM enzyme and ASM-Fc fusion protein. Figure 13 ).

[0431] To further confirm the maintenance of the ASM-Fc fusion protein's activity in cells, LC-MS / MS analysis was performed to monitor endogenous sphingomyelin accumulation in ASM KO cells. HEK293T WT and ASM KO cells were cultured and treated with the enzymes described above. At 68 hours post-spreading, cells were thoroughly washed with PBS with or without ASM or ASM-Fc fusion protein treatment, and lipids were extracted with a water:methanol [1:1, v / v] mixture containing appropriate internal standards. Lipids were extracted using methyl tert-butyl ether (MTBE), vortexed, and centrifuged at 10,000 × g and 4 °C for 10 min. The upper MTBE fraction containing lipids was then evaporated to dryness under a gentle nitrogen stream. The lipids were resuspended in a mixture of isopropanol:acetonitrile:water [2:1:1, v / v / v] and transferred to mass spectrometry vials for further analysis.

[0432] Lipid analysis was performed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled with electrospray ionization mass spectrometry (Sciex 6500+ QTRAP, Sciex, Framingham, MA, USA). For each analysis, 5 μL of sample was injected at 0.25 mL / min onto a BEH C18 1.7 μm, 2.1 × 100 mm column (Waters Corporation, Milford, Massachusetts, USA) at 55 °C. Mobile phase A consisted of 60:40 acetonitrile / water (v / v) plus 10 mM ammonium formate + 0.1% formic acid. Mobile phase B consisted of 90:10 isopropanol / acetonitrile (v / v) plus 10 mM ammonium formate + 0.1% formic acid. The gradient was programmed as follows: 0.0–8.0 min from 45% B to 99% B, 8.0–10.0 min at 99% B, 10.0–10.1 min at 45% B, and 10.1–12.0 min at 45% B. Electrospray ionization was performed in cation mode using the following settings: curtain gas, 20; collision gas set to medium; ion spray voltage, 5200; temperature, 250; ion source gas 1, 50; ion source gas 2, 60. Data acquisition was performed using Analyst 1.6 (Sciex) in multiple reaction monitoring (MRM) mode. Collision energy, 40; declustering potential, 80; inlet potential, 10; collision chamber outlet potential, 12.5. The following MRM transients were used for [M-H₂O+H]... +Ceramide (Cer) was detected in the following forms: Cer d18:1 / 16:0>264.3 at m / z 538.5; Cer d18:1 / 18:0>264.3 at m / z 566.6; Cer d18:1 / 20:0>264.3 at m / z 594.6; Cer d18:1 / 22:0>264.3 at m / z 622.6; Cer d18:1 / 24:0>264.3 at m / z 650.6; Cer d18:1 / 24:1>264.3 at m / z 648.6; and Cer d18:1 / 17:0>264.3 at m / z 552.4. Cer d18:1 / 17:0>264.3 was used as an internal standard. The following MRM transients were used in [M+H]... + The following parameters were used for the formal determination of sphingomyelin (SM): SM d18:1 / 16:0 > 184.1 for m / z 703.7; SM d18:1 / 18:0 > 184.1 for m / z 731.7; SM d18:1 / 20:0 > 184.1 for m / z 759.7; SM d18:1 / 22:0 > 184.1 for m / z 787.7; SM d18:1 / 24:0 > 184.1 for m / z 815.7; SM d18:1 / 24:1 > 184.1 for m / z 813.7; and SM d18:1 / 18:1 (d9) > 184.1 for m / z 738.7. These were used as internal standards. Lipids were identified based on residence time and MRM characteristics of a commercially available reference standard (Avanti Polar Lipids, Birmingham, AL, USA). Quantification was performed using MultiQuant 3.02 (Sciex). Lipids were normalized against total protein content. Protein concentration was measured using BCA analysis (Pierce). LC-MS / MS analysis confirmed that the ASM-Fc fusion protein reduced endogenous sphingomyelin levels in ASM KO cells back to levels observed in wild-type cells. Figure 14 In addition, in both analyses, the ASM-Fc fusion protein was as effective as the naked ASM enzyme in reducing sphingomyelin (Table 3).

[0433] Table 3. The ASM-Fc fusion protein showed similar efficacy to ASM in cellular analysis.

[0434]

[0435] In summary, these data confirm that ASM-Fc fusion proteins retain their activity and can rescue substrate accumulation in ASM-deficient cells.

[0436] Example 6. Construction of a fusion protein containing N-sulfoglucosamine sulfonylhydrolase (SGSH).

[0437] Design and cloning

[0438] The SGSH-Fc fusion protein was designed to contain (i) a fusion polypeptide in which a mature human SGSH enzyme is fused to a human IgG1 fragment including an Fc region (“SGSH-Fc fusion polypeptide”), and (ii) a modified human IgG1 fragment containing a mutation in the Fc region conferring transferrin receptor (TfR) binding (“modified Fc polypeptide”). Specifically, the SGSH-Fc fusion polypeptide was formed in which an SGSH fragment was fused to the N-terminus or C-terminus of the human IgG1 Fc region. In some cases, a linker was placed between the SGSH and IgG1 fragments to alleviate any steric hindrance between the two fragments. In all constructs, a signal peptide from κ chain V-III, amino acids 1-20 (UniProtKB ID-P01661), was inserted upstream of the fusion to promote secretion, and the SGSH was truncated to consist of amino acids R21-L502 (UniProtKB ID-P51688). The fragment of the human IgG1 Fc region used corresponds to amino acid D104-K330 (positions 221-447, EU number, including 10 amino acids of the hinge (positions 221-230)) in UniProtKB ID P01857. In some embodiments, a second Fc polypeptide derived from human IgG1 residue D104-K330 and containing a mutation conferring TfR binding in the Fc region but lacking SGSH fusion is co-transfected with an SGSH-Fc fusion polypeptide to produce a heterodimeric fusion protein with one SGSH enzyme (“single enzyme”). In other embodiments, a second Fc polypeptide derived from human IgG1 residue D104-K330 and containing a mutation conferring TfR binding in the Fc region and fused to SGSH is co-transfected with an SGSH-Fc fusion polypeptide to produce a heterodimeric fusion protein with two SGSH enzymes (“double enzyme”). In some constructs, the IgG1 fragment contains additional mutations to promote heterodimerization of both Fc regions. Similarly, a control SGSH-Fc fusion protein lacking the mutation conferring TfR binding was designed and constructed. As another control, SGSH (amino acid R21-L502) with a C-terminal six-histidine tag (SEQ ID NO:241) was generated to facilitate detection and purification.

[0439] The SGSH-Fc fusion protein containing TfR binding used in the examples is a dimer formed by an SGSH-Fc fusion polypeptide and a modified Fc polypeptide bound to TfR, wherein the modified Fc polypeptide lacks SGSH fusion (“single enzyme”) or is fused to a second SGSH molecule (“double enzyme”).

[0440] An SGSH-Fc fusion polypeptide comprising a mature human SGSH sequence fused to the N-terminus of an IgG1 Fc polypeptide sequence having galvanic and LALA mutations has the sequence SEQ ID NO:149. The SGSH enzyme is linked to the Fc polypeptide via a GGGGS linker (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).

[0441] The SGSH-Fc fusion polypeptide comprising a mature human SGSH sequence fused to the C-terminus of an IgG1 Fc polypeptide sequence having galvanic and LALA mutations has the sequence of SEQ ID NO:150. The SGSH enzyme is linked to the Fc polypeptide via a GGGGS adapter (SEQ ID NO:239), and the N-terminus of the Fc polypeptide may include a portion of the IgG1 hinge region (e.g., SEQ ID NO:113).

[0442] A TfR-modified Fc polypeptide containing the sequence of clone CH3C.35.21.17 (SEQ ID NO:58) with the cleavage 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).

[0443] An "N-terminal monoenzyme" containing a single SGSH molecule is formed at the N-terminus of the Fc polypeptide between SEQ ID NO: 149 and 151. A "C-terminal monoenzyme" containing a single SGSH molecule is formed at the C-terminus of the Fc polypeptide between SEQ ID NO: 150 and 151.

[0444] A modified Fc polypeptide containing a mature human SGSH sequence fused to the N-terminus of clone CH3C.35.21.17 (SEQ ID NO:58) with the sigmata and LALA mutations, and having the sequence SEQ ID NO:154, is a TfR-bound Fc polypeptide. The SGSH enzyme is linked to the modified Fc polypeptide via a GGGGS adapter (SEQ ID NO:239), and the N-terminus of the modified Fc polypeptide may include a portion of the IgG1 hinge region (e.g., SEQ ID NO:113).

[0445] An “N-terminal double enzyme” 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 is formed between SEQ ID NO:149 and 154.

[0446] Recombinant protein expression and purification

[0447] To express the recombinant SGSH enzyme fused to the Fc region, ExpiCHO cells (ThermoFisher Scientific) were transfected using the Expifectamine™ CHO transfection kit according to the manufacturer's instructions (ThermoFisher Scientific) with the relevant DNA construct. Cells were grown in ExpiCHO™ expression medium at 37°C, 6% CO2, and 120 rpm in an Infortrex HT Multitron. Specifically, 0.8 μg of DNA plasmid per mL of culture volume was used in 6 x 10⁻⁶ cells / mL of medium. 6 Log-growing ExpiCHO™ cells were transfected at a density of 1 / 3 cells. Following transfection, cells were returned to 37°C and the transfected cultures were fed as directed 18–22 hours post-transfection. The supernatant of the transfected cell cultures was harvested at 120 hours post-transfection by centrifugation at 3,500 rpm for 20 min. The clarified supernatant was filtered (using a 0.22 μM membrane) and stored at 4°C. Expression of an epitope-tagged SGSH enzyme (used as a control) was performed as described above with minor modifications. In short, an SGSH enzyme with a C-terminal hexahistine tag (SEQ ID NO: 241) was expressed in ExpiCHO cells.

[0448] Affinity chromatography was used to purify SGSH-Fc fusion proteins, with or without engineered Fc regions conferring TfR binding, from cell culture supernatants. The supernatant was loaded onto a HiTrap MabSelect SuRe protein A affinity column (GE Healthcare Life Sciences, using the Akta Pure system). The column was then washed with >20 column volumes (CV) of PBS. The bound protein was 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 protein in the eluted fraction was assessed by reducing and non-reducing SDS-PAGE.

[0449] To purify hexahistidine-labeled (SEQ ID NO:241) SGSH, the transfection supernatant was thoroughly dialyzed overnight with 15 μL of 20 mM HEPES pH 7.4 containing 100 mM NaCl, and 20 mM imidazole was added to the dialysate supernatant before purification. The dialysate supernatant was then bound to a HisTrap column (GE Healthcare Life Sciences, using an AktaPure system). After binding, the column was washed with 20 CV of PBS. The bound protein was eluted with PBS containing 500 mM imidazole. The homogeneity of the SGSH enzyme in the elution fraction was assessed by reducing and non-reducing SDS-PAGE. The pooled fraction containing SGSH could be diluted 1:10 in 50 mM Tris pH 7.5 and further purified using a high-performance Q agarose gel (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 of 50 mM Tris at pH 7.5 and 0.5 M NaCl and collected in 1 CV fractions. Fraction purity was assessed by non-reducing SDS-PAGE. Purification yielded homogeneous SGSH-Fc fusion protein and hexahistine-labeled (SEQ ID NO:241) SGSH.

[0450] Example 7. Characterization of SGSH fusion protein.

[0451] The SGSH-Fc fusion protein with an engineered TfR binding site binds to human TfR.

[0452] To determine whether an engineered TfR-binding SGSH-Fc fusion protein affects the ability of the modified Fc domain to interact with human TfR, Biacore can be used. ™ Surface plasmon resonance analysis was used to assess the affinity of this protein for human TfR. Biacore was used... ™ The SCM5 sensor chip was immobilized using anti-human Fab (Human Fab Capture Kit from GE Healthcare). 5 μg / mL of IDS-Fc fusion protein was captured onto each cell for 1 minute, followed by injection of serially 3-fold dilutions of human apical domain TfR at a flow rate of 30 μL / min. Each sample was analyzed under 3-minute association and 3-minute dissociation conditions. After each injection, the chip was regenerated using 10 mM glycine-HCl (pH 2.1). The binding reaction was corrected by subtracting RU from cells containing similar densities of unrelated IgG. (Biacore was used for further analysis.) ™The T200 evaluation software v3.1 uses a concentration-equilibrium-state response fitting method to obtain steady-state affinity. (Biacore) ™ Analysis confirmed that the SGSH-Fc fusion protein, which has a TfR binding site engineered into the Fc region, binds to human TfR.

[0453] The SGSH-Fc fusion protein with engineered TfR binding sites is active in vitro and in cells.

[0454] The in vitro and cellular activities of engineered TfR-binding SGSH-Fc fusion protein were evaluated to confirm that SGSH maintains its enzymatic activity upon fusion into a human IgG fragment. The in vitro activity of recombinant SGSH was measured using a two-step fluorescent enzyme assay with artificial substrates. Specifically, 20 μL of 1 mM 4-methylumbelliferyl ketone 2-deoxy-2-sulfonamido-aD-glucopyranoside sodium salt substrate (Carbosynth Limited, #EM06602), diluted in analytical buffer (0.03 M sodium acetate, 0.12 M NaCl, pH 6.5), was mixed with 10 μL of 40 nM SGSH. The first reaction mixture was incubated at 37 °C for 17 h, and then the reaction was terminated with 10 μL of 0.2 M phosphate-citrate buffer, pH 6.7. Next, the second reaction was initiated by adding 10 μL (0.5 U) of yeast α-glucosidase (Sigma, #G0660-750UN), incubated at 37°C for 24 h, and stopped by adding 100 μL of 0.5 M sodium carbonate buffer, pH 10.3. The fluorescence of the reaction solution was then measured (excited at 365 nm and emitted at 450 nm). The amount of product was calculated by fitting a standard curve of 4-methylumbelliferone using linear regression, and was verified to be less than 10% of the total substrate cleavage. Specific activity (moles of product per picomolar SGSH per minute) was calculated by dividing the amount of product by the reaction time and the molar amount of SGSH.

[0455] In vitro enzyme activity analysis confirmed that the SGSH-Fc fusion protein was active and indicated that the fusion of the Fc region into SGSH did not impair enzyme activity. Figure 15 ).

[0456] SGSH knockout (KO) cells were generated using CRISPR / CAS9 to provide a cellular system for testing the cellular activity of engineered SGSH-Fc peptides. HEK 293T cells (ATCC) were transfected with a CRISPR / CAS9 pCas-Guide-EF1a-GFP vector (Origene) containing a guide sequence targeting exon 2 upstream of the reactive cysteine ​​site in human SGSH that produces formylglycine. To identify SGSH KO cells, single-cell clones were grown and the cell lysates were subjected to the in vitro SGSH enzyme assays described above. Briefly, in vitro activity assays were performed using 12.5, 25, 50, and 100 μg of cell lysates in lead acetate assay buffer pH 5.0 (100 mM sodium acetate, 10 mM lead acetate). The reaction was initiated by combining 20 μL of normalized cell lysates (in water) with 1 mM substrate in 10 μL of lead acetate buffer (3X) and incubated at 37°C for seventeen hours. The first reaction was stopped by adding 70 μL of 4x citrate phosphate buffer to pH 6.7 and 0.5 U NAGLU (Sigma). The reaction was carried out at 37°C for 24 hours and then stopped by adding 100 μL of 0.5 M sodium carbonate to pH 10.3. SGSH activity in the HEK293T CRISPR clone was compared with recombinant SGSH (R&D), HEK wild-type (WT) lysate, and SGSH-overexpressing HEK cell lysate as analytical standards. Following the mini-Topo (ThermoFisher) clone, clones with enzyme activity levels similar to the background signal were sequence validated and identified as KO clones. Subsequent cell analyses used three unique and validated SGSH KO clones and three independent batches of WT HEK293T cells.

[0457] To test the cellular activity of naked SGSH enzymes or SGSH-Fc fusion proteins, LC-MS / MS-based glycosomnography was performed, allowing for the monitoring of substrate accumulation (heparan sulfate) as an indicator of SGSH activity. Substrate accumulation was measured in SGSH KO cells and WT HEK293T cells. SGSH KO cells and WT HEK293T cells were cultured for 24 hours, washed three times with PBS, aggregated into granules, and frozen. Cell granules were sonicated in disaccharide digestion buffer (111 mM NH4OAc, 11 mM CaOAc, pH 7.0). Protein concentration was measured using BCA analysis (Pierce). Total protein (100 μg) was added to 100 μL of digestion buffer along with 2 mM DTT, 1.25 mIU heparinase I (Galen), 1.25 mIU heparinase II (Galen), and 1.25 mIU heparinase III (Galen). After three hours of digestion with heparan sulfate at 30°C, 20 ng of internal standard (4UA-2S-GlcNCOEt-6S HD009 [Galen]) was added to each sample. The enzyme was deactivated by adding 6 μL of 250 mM EDTA, and the samples were boiled at 95°C for 10 min. The samples were then centrifuged at 16,000 x G for 5 min at room temperature. The supernatant was transferred to an Amicon Ultra 30KD centrifuge filter (Millipore) and centrifuged at 14,000 x G for 15 min. The disaccharide was concentrated in the eluent and resuspended in a [1:1, v / v] mixture of analysis buffer and acetonitrile, and then transferred to a mass spectrometry vial for further analysis.

[0458] GAG analysis was performed by liquid chromatography coupled with electrospray mass spectrometry (Sciex 6500+ QTRAP, Sciex, Framingham, MA, USA) using a Shimadzu Nexera X2 system (Shimadzu Scientific Instrument, Columbia, MD, USA). For each analysis, 10 μL of sample was injected onto 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 with 10 mM ammonium formate and 0.1% formic acid. Mobile phase B consisted of acetonitrile with 0.1% formic acid. The gradient was programmed as follows: 0.0–1.0 min at 85% B, 1.0–5.0 min from 85% B to 50% B, 5.0–6.0 min from 50% B to 85% B, and held at 85% B for 6–8.0 min. Electrospray ionization was performed in anion mode using the following settings: curtain gas, 30; collision gas set to medium; ion spray voltage, -4500; temperature, 450; ion source gas 1, 50; ion source gas 2, 60. Data acquisition was performed using Analyst 1.6.3 (Sciex) in multiple reaction monitoring (MRM) mode with a residence time of 25 milliseconds. Collision energy, -30; declustering potential, -80; inlet potential, -10; collision chamber outlet potential, -10. GAG was detected using the following MRM transients in the form of [M--H]-: D0A0 > 87.0 at m / z 378.1; D0a0 > 175.0 at m / z 378.1; D0S0 > 138.0 at m / z 416.1; D0a4 > 300.0 at m / z 458.1; D0A6, D2A0, D0a6, D2a0 > 97.0 at m / z 458.1; D0S6, D2S0 > 416.1 at m / z 496.0; D2a4, D2a6, D0a10, D2A6 > 458.0 at m / z 538.0; D0S6 > 97.0 at m / z 575.95; and D0S6 > 97.0 at m / z 472.0. The 4UA-2S-GlcNCOEt-6S (fragment ions) value > 97.0 was used as an internal standard (IS). GAGs were identified based on residence time and MRM transients matched with 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 against total protein content.Protein concentration was measured using BCA analysis (Pierce).

[0459] Compared to the control cell line, significant substrate accumulation was observed in SGSH KO cells, as reflected by the amount of disaccharide observed after heparan sulfate digestion. Figure 16 Glycosomal analysis based on LC-MS / MS confirmed that treatment of cells with the TfR-binding SGSH-Fc fusion protein reduced the level of heparan sulfate-derived disaccharides back to the levels observed in wild-type cells. Figure 17 In summary, these data confirm that the SGSH-Fc fusion protein maintains enzyme activity and reduces substrate accumulation in SGSHKO cells.

[0460] Example 8. In vitro analysis of SGSH activity.

[0461] This embodiment provides an alternative in vitro activity assay for the SGSH-Fc fusion protein. The assay is adapted from Karpova et al., J. Inherit. Metab. Dis., 19:278-285 (1996).

[0462] The standard reaction mixture consisted of 10–15 μg protein and 20 μL of Michaelis' barbital sodium acetate buffer (5 or 10 mmol / L, respectively), pH 6.5 (29 mmol / L sodium barbital, 29 mmol / L sodium acetate, 0.68% (w / v) NaCl, 0.02% (w / v) sodium azide; adjusted to pH 6.5 with HCl), and the reaction mixture was incubated at 37 °C for 17 h. MU-α-GlcNS was available from Moscerdam Substrates. After the first incubation, a second incubation was performed at 37°C for 24 h, adding 6 μl of twice-concentrated McIlvain's phosphate / citrate buffer (pH 6.7) containing 0.02% sodium azide and water containing 10 μl (0.1 U) yeast α-glucosidase (Sigma). Longer incubation (17–24 h) was then performed in 96-well plates sealed with wide tape to limit evaporation to <15%. Next, 200 μL of 0.5 mol / L Na₂CO₃ / NaHCO₃ was added at pH 10.7, and the fluorescence of released 4-methylumbelliferone (MU) was measured on a Fluoroskan (Titertek) fluorometer. Protein assays were performed as previously described (van Diggelen et al., Clin. Chim. Acta., 187:131–139 (1990)).

[0463] Example 9. Modified Fc peptide bound to TfR.

[0464] This embodiment describes the modification of the Fc peptide to enable it to bind to the transferrin receptor (TfR) and be transported across the blood-brain barrier (BBB).

[0465] Unless otherwise indicated, the positions of amino acid residues in this section are numbered based on the EU index number of the wild-type Fc region of human IgG1.

[0466] Positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 contain modified Fc polypeptides (CH3C). The generation and characterization of cysts

[0467] A yeast library containing an Fc region was generated as described below, the Fc region having modifications introduced at positions including amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and 421. Illustrative clones bound to TfR are shown in Tables 4 and 5.

[0468] After two more rounds of sorting, individual clones were sequenced and four unique sequences were identified. These sequences all possess a conserved Trp at position 388 and an aromatic residue (i.e., Trp, Tyr, or His) at position 421. Significant diversity was observed at other positions.

[0469] Four clones selected from the library were expressed as Fc fusions with the Fab fragment in CHO or 293 cells and purified by protein A and size exclusion chromatography. They were then screened for binding to human TfR by ELISA in the presence or absence of holo-Tf. All clones bound to human TfR, and the binding was not affected by the addition of excess (5 μM) holo-Tf. The clones were also tested for binding to 293F cells endogenously expressing human TfR. The clones bound to 293F cells, although the overall binding was generally weaker than that of the high-affinity positive control.

[0470] Next, clone CH3C.3 was used as a test clone to test whether the clone could be internalized in TfR-expressing cells. Attached HEK 293 cells were grown in 96-well plates to approximately 80% confluence, the medium was removed, and samples were added at a concentration of 1 μM: clone CH3C.3, anti-TfR baseline positive control antibody (Ab204), anti-BACE1 baseline negative control antibody (Ab107), and human IgG isotype control (obtained from Jackson Immunoresearch). Cells were incubated at 37°C and 8% CO2 for 30 min, then washed with 0.1% Triton X-rays. ™ X-100 permeabilization, and with anti-human-IgG-Alexa Fluor ® 488 secondary antibody staining. After rewashing, the cells were stained with a high-throughput fluorescence microscope (i.e., Opera Phenix). ™ Imaging was performed using a system that quantified the number of spots per cell. At 1 μM, clone CH3C.3 showed a similar tendency to internalize as the positive anti-TfR control, while the negative control showed no internalization.

[0471] Further engineering of cloning

[0472] Additional libraries were generated using a soft randomization approach to improve the affinity of the initial hits for human TfR, where DNA oligonucleotides were generated based on each of the original four hits to introduce soft mutagenesis. Additional clones binding to TfR were identified and selected. The selected clones belonged to two general sequence groups. Group 1 clones (i.e., clones CH3C.18, CH3C.21, CH3C.25, and CH3C.34) possess a semi-conserved Leu motif at position 384, a Leu or His motif at position 386, conserved and semi-conserved Val motifs at positions 387 and 389 respectively, and semi-conserved PTW motifs at positions 413, 416, and 421 respectively. Group 2 clones possess a conserved Tyr motif at position 384, a TXWSX motif at positions 386-390, and a conserved S / TEF motif at positions 413, 416, and 421 respectively. The clones CH3C.18 and CH3C.35 were used for additional studies as representative members of their respective sequence groups.

[0473] Epitope localization

[0474] To determine whether the engineered Fc region binds to the apical domain of the TfR, enabling the TfR apical domain to be expressed on the phage surface, one of the loops must be truncated and the sequence needs to be circularized for proper folding and visualization of the apical domain. Clones CH3C.18 and CH3C.35 were coated onto ELISA plates and subjected to a phage ELISA protocol. In short, after washing and blocking with 1% PBSA, a dilution of the phage display was added and incubated at room temperature for 1 hour. The plate was then washed and anti-M13-HRP was added, and after a second wash, the plate was developed with TMB substrate and the reaction was stopped with 2N H2SO4. In this analysis, both clones CH3C.18 and CH3C.35 bound to the apical domain.

[0475] Complementary bit positioning

[0476] To understand which residues in the Fc domain are most important for TfR binding, a series of mutant clones, CH3C.18 and CH3C.35, were formed, with each mutant having a single position mutated back to wild-type in a TfR binding registry. The resulting variants were recombinated and expressed as Fc-Fab fusions and tested for binding to human or canine TfR. For clone CH3C.35, positions 388 and 421 were important for binding; reverting to wild-type at either of these positions completely eliminated binding to human TfR.

[0477] Binding characterization of mature clones

[0478] As described above, binding ELISA was performed using purified Fc-Fab fusion variants in the presence of human or canine TfR coated on the plate. Variant clones CH3C.3.2-1, CH3C.3.2-5, and CH3C.3.2-19 from the mature CH3C.18 clone library were used with approximately equal EC50 values. 50 The binding values ​​of human and canine TfR were positive, while the parental clones CH3C.18 and CH3C.35 showed a binding factor greater than 10 times better than canine TfR to human TfR.

[0479] Next, the internalization of the modified Fc peptide was tested in human and monkey cells. Using the protocol described above, internalization was tested in human HEK 293 cells and rhesus monkey LLC-MK2 cells. Similarly, the variant clones CH3C.3.2-5 and CH3C.3.2-19, which bind to human and canine TfR, showed significantly improved internalization in LLC-MK2 cells compared to clone CH3C.35.

[0480] Additional engineering for cloning

[0481] Further engineering of other affinity-mature clones CH3C.18 and CH3C.35 involved adding additional mutations to sites where binding is enhanced through direct interactions, second-shell interactions, or structural stabilization. This was achieved by generating and selecting from “NNK walk” or “NNK patch” libraries. NNK walk libraries involve forming individual NNK mutations on residues close to their complementary sites. By examining the structure of Fc binding to FcγRI (PDB ID: 4W4O), 44 residues close to the original modification sites were identified as query candidates. Specifically, NNK mutagenesis was performed targeting the following residues: K248, R255, Q342, R344, E345, Q347, T359, K360, N361, Q362, S364, K370, E380, E382, S383, G385, Y391, K392, T393, D399, S400, D401, S403, K409, L410, T411, V412, K414, S415, Q418, Q419, G420, V422, F423, S424, S426, Q438, S440, S442, L443, S444, P4458, G446, and K447. A 44-spot NNK library was generated using Kunkel mutagenesis, and the product was collected and introduced into yeast via electroporation as described above for other yeast libraries.

[0482] Combinations of these mini-libraries (each containing a single mutation site, resulting in 20 variants) produce small libraries, which are then selected using yeast surface display for any sites that induce high-affinity binding. Selection is performed using TfR apical domain proteins as described above. After three rounds of sorting, clones from the enriched yeast libraries are sequenced, and several “hotspot” sites are identified, where certain point mutations significantly improve binding to apical domain proteins. For clone CH3C.35, these mutations include E380 (mutated to Trp, Tyr, Leu, or Gln) and S415 (mutated to Glu). Sequences of single and combined mutant clones of CH3C.35 are illustrated in SEQ ID NO:27-38. For clone CH3C.18, these mutations include E380 (mutated to Trp, Tyr, or Leu) and K392 (mutated to Gln, Phe, or His). The sequences of the cloned CH3C.18 single mutant are described in SEQ ID NO:21-26.

[0483] Additional mature libraries for improving the affinity of clone CH3C.35

[0484] As described in the previous yeast library, another library was generated to identify mutant combinations from the NNK walk library, with additional positions added around these sites. In this library, the YxTEWSS (SEQ ID NO:242) and TxxExxxxF motifs remained constant, and six positions were completely randomized: E380, K392, K414, S415, S424, and S426. Positions E380 and S415 were included because they are “hotspots” in the NNK walk library. Positions K392, S424, and S426 were included because they form part of the core of the localizable binding region, while K414 was chosen because of its proximity to position 415.

[0485] The library was sorted using only the canine TfR apical domain as previously described. The enriched pool was sequenced after five rounds, and the sequences of the modified regions of the identified unique clones are illustrated in SEQ ID NO:42-59.

[0486] Subsequent libraries were designed to further explore acceptable diversity at key binding complement sites. Each of the original positions (384, 386, 387, 388, 389, 390, 413, 416, and 421) was individually randomized with two hotspots (380 and 415) using the NNK codon to generate a series of yeast-based single-site saturation mutagenesis libraries. Furthermore, each position was individually restored to wild-type residues, and these individual clones were displayed on yeast. It should be noted that positions 380, 389, 390, and 415 are the only positions that retain substantial binding to TfR after restoration to wild-type residues (for 413, restoration to wild-type showed some residual but greatly weakened binding).

[0487] Single-position NNK libraries were sorted three times against the top domain of human TfR to collect the first approximately 5% of binders, and then at least 16 clones from each library were sequenced. The results indicate what amino acids are tolerable at each position without significantly reducing binding to human TfR in the context of clone CH3C.35. A summary is as follows:

[0488] Position 380: Trp, Leu, or Glu;

[0489] Position 384: Tyr or Phe;

[0490] Position 386: Thr only;

[0491] Position 387: Glu only;

[0492] Location 388: TRP only;

[0493] Position 389: Ser, Ala, or Val (although wild-type Asn residues appear to retain some binding, it does not appear after library sorting);

[0494] Position 390: Ser or Asn;

[0495] Position 413: Thr or Ser;

[0496] Position 415: Glu or Ser;

[0497] Position 416: Glu only; and

[0498] Position 421: Phe only.

[0499] When these residues are substituted as a single change or in combination into clone CH3C.35, they exhibit complementary site diversity in binding to the TfR apical domain. Clones with mutations at these positions include those shown in Table 5, and the sequences of the CH3 domain of these clones are described in SEQ ID NO:34-38, 58, and 60-90.

[0500] Example 10. It can be modified to give additional Fc sites for TfR binding.

[0501] Additional modified Fc polypeptides binding to the transferrin receptor (TfR) are generated in the Fc region at substitution sites, such as at the following locations:

[0502] Positions 274, 276, 283, 285, 286, 287, 288, and 290 (CH2A2 clone);

[0503] Positions 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 (CH2C clones);

[0504] Positions 268, 269, 270, 271, 272, 292, 293, 294 and 300 (CH2D clone);

[0505] Positions 272, 274, 276, 322, 324, 326, 329, 330, and 331 (CH2E3 clone); or

[0506] Positions 345, 346, 347, 349, 437, 438, 439 and 440 (CH3B clone).

[0507] Illustrative CH3B clones bound to TfR are described in SEQ ID NO:124-128. Illustrative CH2A2 clones bound to TfR are described in SEQ ID NO:129-133. Illustrative CH2C clones bound to TfR are described in SEQ ID NO:134-138. Illustrative CH2D clones bound to TfR are described in SEQ ID NO:139-143. Illustrative CH2E3 clones bound to TfR are described in SEQ ID NO:144-148.

[0508] Example 11. Method.

[0509] Generation of phage display libraries

[0510] A DNA template encoding the wild-type human Fc sequence was synthesized and incorporated into a phage vector. The phage vector contained an ompA or pelB leader sequence, an Fc insert fused to the c-Myc and 6xHis (SEQ ID NO:241) epitope tags, and an amber stop codon following the M13 capsid protein pIII.

[0511] Primers containing the "NNK" triple codon at the desired position for modification are generated, where N is any DNA base (i.e., A, C, G, or T) and K is G or T. Alternatively, primers for "soft" randomization are used, where each randomization position uses a base mixture corresponding to 70% wild-type bases and 10% of each of the other three bases. A library is generated by PCR amplification of the Fc region corresponding to the randomized region, then assembled using end primers containing SfiI restriction sites, digested with SfiI, and ligated into a phage vector. Alternatively, primers are used for Konkel mutagenesis. The ligation product or Konkel product is transformed into strain TG1 (obtained from Lucigen). ® The library was electrotransformed into competent *E. coli* cells. After recovery and overnight growth, the *E. coli* cells were infected with M13K07 helper phage. The phage library was then precipitated with 5% PEG / NaCl, resuspended in PBS containing 15% glycerol, and frozen until use. Typical library sizes are between approximately 10 μL. 9 To about 10 11 Within the range of transformants, the pairing between Fc fused via pIII and soluble Fc not attached to pIII (the latter being generated by the amber stop codon preceding pIII) causes the Fc-dimer to be displayed on the phage.

[0512] Generation of yeast display libraries

[0513] DNA templates encoding the wild-type human Fc sequence were synthesized and incorporated into yeast display vectors. For CH2 and CH3 libraries, the Fc peptides were displayed on Aga2p cell wall proteins. Both vectors contained a prepro leader peptide with a Kex2 cleavage sequence and a c-Myc epitope tag fused to the Fc terminus.

[0514] Yeast display libraries were assembled using methods similar to those described for phage libraries, except that primers containing ends homologous to the vector were used for fragment amplification. Freshly prepared electroporated competent yeast cells (i.e., strain EBY100) were electroporated with the linearized vector and the assembled library inserts. Electroporation methods are known to those skilled in the art. After recovery in selective SD-CAA medium, the yeast was allowed to confluence and divide twice, and then protein expression was induced by transfer to SG-CAA medium. Typical library sizes range from approximately 10 μL. 7 To about 10 9 Within the range of transformants, Fc-dimers are formed through pairing of adjacent Fc monomers.

[0515] General methods for phage selection

[0516] The phage method is adapted from Phage Display: A Laboratory Manual (Barbas, 2001). Additional protocol details can be found in this reference.

[0517] Plate sorting method

[0518] The antigen was coated onto MaxiSorp at 4°C. ® Incubate overnight on a microtiter plate (typically 1–10 μg / mL). Add the phage library to each well and incubate overnight for binding. Tweeze the microtiter wells with a solution containing 0.05% Tween. ® The bound phages were thoroughly washed with 20 μL (PBST) of PBS and eluted by incubation for 30 min in acidic conditions (typically 50 mM HCl plus 500 mM KCl, or 100 mM glycine, pH 2.7). The eluted phages were neutralized with 1 M Tris (pH 8) and amplified using TG1 cells and M13 / KO7 helper phages, and grown overnight at 37°C in 2YT medium containing 50 μg / mL carbenacillin and 50 μg / mL kanamycin. Enrichment was assessed by comparing the titers of phages eluted from wells containing the target with those recovered from wells without the target. Selectivity was increased by subsequently reducing the incubation time during binding and increasing the washing time and number of washes.

[0519] Bead sorting method

[0520] NHS-PEG4-Biotin (obtained from Pierce) ™The antigen was biotinylated via free amines. For the biotinylation reaction, a 3 to 5 molar excess of biotin reagent was used in PBS. The reaction was stopped with Tris, followed by thorough dialysis in PBS. The biotinylated antigen was immobilized on streptavidin-coated magnetic beads (i.e., M280-streptavidin beads obtained from Thermo Fisher). The phage display library was incubated with the antigen-coated beads at room temperature for 1 hour. Unbound phages were then removed and the beads were washed with PBST. Bound phages were eluted by incubation for 30 minutes in 50 mM HCl (or 0.1 M glycine, pH 2.7) containing 500 mM KCl, followed by neutralization and proliferation as described above regarding plate sorting.

[0521] After three to five rounds of panning, individual clones are screened by expressing Fc on phages or by soluble expression in the periplasm of *E. coli*. Such expression methods will be known to those skilled in the art. Individual phage supernatants or periplasmic extracts are exposed to blocked ELISA plates coated with antigens or negative controls, and subsequently detected using HRP-conjugated goat anti-Fc (from Jackson Immunoresearch) for periplasmic extracts or anti-M13 (GE Healthcare) for phages, followed by color development with TMB reagent (from Thermo Fisher). 450 Wells with values ​​greater than approximately 5 times the background were considered positive clones and sequenced. Subsequently, some clones were expressed as soluble Fc fragments or as fused to Fab fragments.

[0522] General methods for yeast selection

[0523] Bead sorting (Magnetic Assisted Cell Sorting (MACS))

[0524] Similar to the method described in Ackerman et al., Biotechnol. Prog., 25(3):774 (2009), MACS and FACS selection were performed. Streptavidin magnetic beads (e.g., M-280 streptavidin beads from ThermoFisher) were labeled with biotinylated antigens and incubated with yeast (typically 5-10x library diversity). Unbound yeast was removed, the beads were washed, and bound yeast was grown in selective media to induce subsequent rounds of selection.

[0525] Fluorescence-activated cell sorting (FACS)

[0526] Yeast was labeled with anti-c-Myc antibody to monitor the expression and phacotinized antigen (concentration varied depending on each round of sorting). In some experiments, the antigen was mixed with streptavidin-Alexa Fluor. ® 647 was premixed to enhance the affinity of the interaction. In other experiments, it was used in combination with streptavidin-Alexa Fluor. ® Biotinylated antigens were detected after washing with 647. Bound singlet yeast cells were sorted using a FACS Aria III cell sorter. The sorted yeast cells were then grown in a selective medium, followed by subsequent rounds of selection.

[0527] After enriching the yeast population, the yeast cells were plated on SD-CAA agar plates and allowed to grow individual colonies and be induced to express. They were then labeled as described above to determine their tendency to bind to the target. Subsequently, single positive clones binding to the antigen were sequenced, and some clones were expressed as soluble Fc fragments or as fused to Fab fragments.

[0528] General methods of screening

[0529] Screening via ELISA

[0530] Clones were selected from the panning output and grown individually in the wells of a 96-well deep-well plate. Periplasmic expression of the clones was induced using automated induction medium (from EMD Millipore) or by infecting them with helper phages to induce phage display of individual Fc variants on the phages. Typically, ELISA plates were coated overnight with 0.5 mg / mL antigen, blocked with 1% BSA, and then phage or periplasmic extract was added. After 1 hour of incubation and washing away unbound proteins, an HRP-conjugated secondary antibody (i.e., anti-Fc or anti-M13 for soluble Fc or phage-displayed Fc, respectively) was added and incubated for 30 minutes. The plates were washed again, developed with TMB reagent, and the reaction was stopped with 2N sulfuric acid. The plate was read using a BioTek plate reader. ® Quantify the absorbance at 450 nm and plot the binding curve using Prism software as appropriate. In some analyses, soluble transferrin or other competitors are typically added in a significant molar excess during the binding step.

[0531] Screening by flow cytometry

[0532] Fc variant peptides (expressed on phages, in periplasmic extracts, or soluble as fusions with Fab fragments) were added to the wells of 96-well V-plates (approximately 100,000 cells per well in PBS + 1% BSA (PBSA)) and incubated at 4°C for 1 hour. The plates were then centrifuged and the culture medium removed, followed by a single wash with PBSA. The cells were then resuspended in a secondary antibody (typically goat anti-human IgG-Alexa Fluor). ® Cells were placed in PBSA (647 cells, obtained from Thermo Fisher). After 30 minutes, the plate was centrifuged and the culture medium was removed. Cells were washed 1-2 times with PBSA, and then the plate was read on a flow cytometer (i.e., FACSCanto™ II flow cytometer). Median fluorescence values ​​for each condition were calculated using FlowJo software, and binding curves were plotted using Prism software.

[0533] Example 12. Selection of TfR-binding peptide affinity.

[0534] This example illustrates the relationship between the affinity of the TfR-binding peptide for the transferrin receptor (TfR) and the resulting brain exposure to therapeutic agents linked to the TfR-binding peptide.

[0535] Figure 18 This indicates that when the therapeutic agent is bound to a peptide with a relatively stronger affinity for TfR, brain exposure to the therapeutic agent (as assessed by measuring the area under the curve (AUC) of brain concentration versus time) is shortened. In particular, brain exposure is substantially shortened when the therapeutic agent is bound to a peptide with a stronger affinity for TfR than about 250 nM.

[0536] like Figure 19 As shown, when the therapeutic agent is bound to a peptide with a relatively stronger affinity for TfR, a higher maximum concentration (C0) is observed in the brain. 最大 Specifically, when the TfR-binding peptide has a stronger affinity than approximately 250 nM, brain C 最大 The value is significantly higher.

[0537] Figure 20 This demonstrates the effects of binding to peptides with a series of affinities for TfR in brain C. 最大 The ratio of plasma concentration to therapeutic agent.

[0538] method

[0539] TfR ms / hu The generation of KI

[0540] Methods for generating knock-in / knock-out mice have been published in the literature and are well known to those skilled in the art. In summary, TfR mice are generated using CRISPR / Cas9 technology. ms / hu KI mice express the human Tfrc apical domain within the mouse Tfrc gene; the resulting chimeric TfR is expressed in vivo under the control of an endogenous promoter. As described in International Patent Publication No. WO 2018 / 152285, which is incorporated herein by reference in its entirety, knock-in of a human apical TfR mouse strain is achieved by microinjecting C57Bl6 mice into single-cell embryos via pronuclear injection, followed by embryo transfer to pseudopregnant females. Specifically, Cas9, a single guide RNA, and donor DNA are introduced into the embryos. The donor DNA contains a human apical domain coding sequence, which has been codon-optimized for expression in mice. The apical domain coding sequence is flanked by the left and right homologous arms. The donor sequence is designed such that the apical domain is inserted after the fourth mouse exon and tightly flanked at the 3' end by the ninth mouse exon. The founder males of the offspring from the receiving females can be bred with wild-type females to produce F1 heterozygous mice. Then, homozygous mice are generated by breeding F1 generation heterozygous mice.

[0541] Mouse PK / PD

[0542] For PK / PD assessment, TfR was determined by intravenous injection at 50 mg / kg via the tail vein. ms / hu KI mice were administered a single systemic dose. Prior to perfusion, blood was collected in EDTA plasma tubes via cardiac puncture and centrifuged at 14,000 rpm for 5 minutes. The plasma was then separated for subsequent PK / PD analysis. After perfusion, the brain was extracted and the hemispheres were separated for homogenization in 10 times the tissue weight of PBS containing 1% NP-40 (for PK) or 5 M GuHCl (for PD).

[0543] The concentrations of engineered TfR-binding peptides in mouse plasma and brain lysates were quantified using the Universal Human IgG Assay (MSD Human IgG Kit #K150JLD) following the manufacturer's instructions. In short, pre-coated plates were blocked with MSD blocking agent A for 30 minutes. Plasma samples were diluted 1:10,000 using a Hamilton Nimbus liquid processor and added in duplicate to the blocked plates. Brain samples were homogenized in 1% NP-40 lysis buffer, and the lysates were diluted 1:10 for PK analysis. Dosing solutions were also analyzed on the same plates to confirm appropriate dosage. A standard curve (0.78–200 ng / mL IgG) was fitted using a four-parameter Rogers regression.

[0544] Example 13. Using Biacore™ Binding characterization of CH3C variants.

[0545] Use Biacore ™ The T200 instrument uses surface plasmon resonance to determine the affinity of clonal variants for the recombinant TfR apical domain. (Biacore) ™ The SCM5 series sensor chip was immobilized using anti-human Fab (from GE Healthcare's Human Fab Capture Kit). 5 μg / mL of the IDS-Fc fusion protein was captured in each cell for 1 minute, and serially 3-fold dilutions of the human or canine apical domain were injected at a flow rate of 30 μL / min. Each sample was analyzed under 45-second association and 3-minute dissociation conditions. After each injection, the chip was regenerated using 10 mM glycine-HCl (pH 2.1). The binding reaction was corrected by subtracting RU from the cells containing similar densities of unrelated IgG. Biacore was used. ™ The T200 evaluation software v3.1 obtains steady-state affinity by fitting the concentration to the response under equilibrium conditions.

[0546] To determine the affinity of clonal variants for the extracellular domain (ECD) of recombinant TfR, Biacore was used. ™ The SCM5 series sensor chip was immobilized with streptavidin. Biotinylated human or canine TfR ECDs were captured on each liquid cell for 1 minute, and serially 3-fold dilutions of the clonal variants were injected at a flow rate of 30 μL / min at room temperature. Each sample was analyzed under conditions of 45 seconds of association and 3 minutes of dissociation. The binding reaction was corrected by subtracting RU from the liquid cell containing TfR ECDs of similar density. Biacore was used. ™ The T200 evaluation software v3.1 obtains steady-state affinity by fitting the concentration to the response under equilibrium conditions.

[0547] Table 6 summarizes the binding affinity. Affinity was obtained through steady-state fitting.

[0548] Table 6. Binding affinity of exemplary CH3C variants.

[0549]

[0550] Example 14. In TfR ms / hu Brain and plasma PKPD of the peptide-Fab fusion in mice: CH3C.35.21, CH3C.35.20, CH3C.35, CH3C.35.23, CH3C.35.23.3.

[0551] To assess the effect of TfR binding affinity on PK and brain uptake, anti-BACE1 Ab153 and TfR-binding peptide fusions (CH3C.35.21:Ab153, CH3C.35.20:Ab153, CH3C.35:Ab153 fusions) were generated, said fusions being analyzed by Biacore. ™ The measured binding affinities for apical human TfR differed. The binding affinities of CH3C.35.21:Ab153, CH3C.35.20:Ab153, and CH3C.35:Ab153 fusions to human TfR were 100 nM, 170 nM, and 620 nM, respectively. Using 50 mg / kg as the TfR... ms / hu Knock-in...

Claims

1. A protein, said protein comprising: (a) a first Fc polypeptide, the first Fc polypeptide being linked to an enzyme replacement therapy (ERT) enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and (b) A second Fc polypeptide, wherein the first Fc polypeptide forms an Fc dimer with the first Fc polypeptide. in, The first Fc polypeptide and / or the second Fc polypeptide do not include the variable region sequence of the immunoglobulin heavy chain and / or light chain or its antigen-binding portion.

2. The protein of claim 1, wherein the ERT enzyme is iduronate 2-sulfatase (IDS), an IDS variant, or a catalytically active fragment thereof.

3. The protein of claim 2, wherein the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 91, 92, 114, 230, and 234.

4. The protein of claim 3, wherein the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NO: 91, 92, 114, 230, and 234.

5. The protein of claim 1, wherein the ERT enzyme is N-sulfoglucosamine sulfonylhydrolase (SGSH), an SGSH variant, or a fragment thereof with catalytic activity.

6. The protein of claim 5, wherein the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 119 and 120.

7. The protein of claim 6, wherein the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NO: 119 and 120.

8. The protein of claim 1, wherein the ERT enzyme is acid sphingomyelinase (ASM), an ASM variant, or a catalytically active fragment thereof.

9. The protein of claim 8, wherein the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 121, 122, and 123.

10. The protein of claim 9, wherein the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NO: 121, 122 and 123.

Images