Fusion protein containing enzymes for enzyme replacement therapy

Abstract

Provided herein are enzyme replacement therapy enzymes and fusion proteins containing an Fc region, as well as methods for treating lysosomal storage disorders using these proteins. Methods for transporting agents across the blood-brain barrier are also provided herein.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to US Provisional Patent No. 62 / 566,898 filed October 2, 2017; US Provisional Patent No. 62 / 583,276 filed November 8, 2017; US Provisional Patent No. 62 / 626,365 filed February 5, 2018; US Provisional Patent No. 62 / 678,183 filed May 30, 2018; and US Provisional Patent No. 62 / 721,396 filed August 22, 2018, the contents of which are incorporated herein in their entirety for all purposes.

[0003] Sequence list

[0004] The present application comprises a list of sequences submitted electronically in ASCII format, the full text of which is incorporated into the references of this specification. The aforementioned ASCII copy was created on September 28, 2018, is named 102342-000350PC-1103949_SL.txt, and has a size of 580,464 bytes. Background Technology

[0005] background

[0006] Lysosomal storage disorders (LSDs) are relatively rare genetic metabolic disorders resulting from defects in lysosomal function. LSDs are typically caused by a deficiency of a single enzyme involved in the breakdown of metabolites within 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. The majority of LSDs also possess significant neurological components ranging from progressive neurodegeneration and severe cognitive impairment to epilepsy, mobility impairment, and psychiatric disorders. While recombinant forms of the enzyme deficient in LSDs can be used to treat the disorder, such therapies may have little effect on the brain due to difficulties in delivering the recombinant enzyme across the blood-brain barrier (BBB).

[0007] summation

[0008] The present invention provides a method of using fusion proteins containing enzyme replacement therapy (ERT) enzymes to treat lysosomal storage disorders (LSDs).

[0009] In some aspects, the present invention provides a protein comprising the following:

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

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

[0012] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide does not contain an immunoglobulin heavy chain and / or light chain variable region sequence or an antigen-binding portion thereof.

[0013] In some embodiments, the ERT enzyme is iduronate 2-sulfatase (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 respect to any one of SEQ Nos. 91, 92, 114, 230, and 234. In some embodiments, the ERT enzyme comprises any one of SEQ Nos. 91, 92, 114, 230, and 234.

[0014] In some embodiments, the ERT enzyme is N-sulfoglucosamine sulfohydrolase (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 respect to any one of SEQ NOs: 119 and 120. In some embodiments, the ERT enzyme comprises any one of SEQ NOs: 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 respect to any one of SEQ NOs: 121, 122, and 123. In some embodiments, the ERT enzyme comprises any one of SEQ NOs: 121, 122, and 123.

[0016] In some embodiments, the ERT enzyme is β-glucocerebrosidase (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 respect to any one of the amino acid sequences of SEQ NOs: 93 and 94. In some embodiments, the ERT enzyme comprises any one of the amino acid sequences of SEQ NOs: 93 and 94.

[0017] In some embodiments, the first Fc polypeptide is a fusion polypeptide linked to the ERT enzyme, the ERT enzyme variant, or its catalytically active fragment by a peptide bond or a polypeptide linker. In some embodiments, the polypeptide linker is a flexible polypeptide linker. In some embodiments, the flexible polypeptide linker is a glycine-rich linker. In some embodiments, the glycine-rich linker is G4S (SEQ No.: 239) or (G4S)2 (SEQ No.: 240). In some embodiments, the first Fc polypeptide is not linked to the ERT enzyme, the ERT enzyme variant, or its catalytically active fragment by a chemical crosslinking agent, and chamberlain, The above fusion polypeptide does not possess non-peptide bonds or non-polypeptide linkers.

[0018] In certain embodiments, the fusion polypeptide comprises, from the N-terminus to the C-terminus: an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; 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 its catalytically active fragment. In some embodiments, the second Fc polypeptide is a fusion polypeptide linked to the ERT enzyme, the ERT enzyme variant, or its catalytically active fragment by a peptide bond or a polypeptide linker. In some embodiments, the polypeptide linker is a soft polypeptide linker. In some embodiments, the soft polypeptide linker is a glycine-rich linker. In some embodiments, the glycine-rich linker is G4S (SEQ No.: 239) or (G4S)2 (SEQ No.: 240). In some embodiments, the second Fc polypeptide is not linked to the ERT enzyme, the ERT enzyme variant, or its catalytically active fragment by a chemical crosslinking agent, and chamberlain, The above fusion polypeptide does not possess non-peptide bonds or non-polypeptide linkers.

[0020] In some embodiments, the N-terminus of the first Fc polypeptide and / or the N-terminus of the second Fc polypeptide are linked to the 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 are linked to the 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. In some embodiments, the C-terminus of the first Fc polypeptide is linked to one ERT enzyme, and the N-terminus of the second Fc polypeptide is linked to 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 ( chamberlain, It includes exactly two ERT enzymes. In some embodiments, the protein includes exactly one or exactly two ERT enzymes, enzyme variants, or catalytically active fragments thereof.

[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 have a modification that promotes heterodimerization. In some embodiments, the Fc dimer is an Fc heterodimer. In some embodiments, one of the Fc polypeptides has a T366W substitution, and another Fc polypeptide has T366S, L368A, and Y407V substitutions (according to EU numbering). In some embodiments, the first Fc polypeptide has T366S, L368A, and Y407V substitutions, and the second Fc polypeptide has a T366W substitution. In some embodiments, the first Fc polypeptide is linked to the ERT enzyme IDS and comprises any one amino acid sequence of SEQ ID NOs: 117, 232, and 236. In some embodiments, the first Fc polypeptide has a T366W substitution, and the second Fc polypeptide has T366S, L368A, and Y407V substitutions. In some embodiments, the first Fc polypeptide is linked to the ERT enzyme IDS and comprises any one amino acid sequence of SEQ ID NOs: 118, 233, and 237.

[0026] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises an intrinsic FcRn binding site. In some embodiments, the first Fc polypeptide and the second Fc polypeptide do not have an effector function. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises a modification that reduces 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 comprises a substitution of Gly at position 329 (according to EU numbering). In some embodiments, the first Fc polypeptide is linked to the ERT enzyme IDS and comprises any one amino acid sequence of SEQ Nos. 115, 231, and 235. In some embodiments, the first Fc polypeptide is linked to the ERT enzyme SGSH and comprises any one amino acid sequence of SEQ ID NOs: 149, 150, 152, and 153.

[0027] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises amino acid changes that extend the serum half-life compared to the intrinsic Fc sequence. 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 numbering). Alternatively, in other embodiments, the amino acid changes comprise substitutions of Leu at position 428 and Ser at position 434 (according to EU numbering). Alternatively, in additional embodiments, the amino acid changes comprise substitutions of Ser or Ala (according to EU numbering) at position 434.

[0028] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide specifically bind to a 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 positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 (according to EU numbering). In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises at least three, four, five, six, seven, eight, or nine substitutions at these positions.

[0030] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises 1, 2, 3, or 4 substitutions at positions including 380, 391, 392, and 415 (according to EU numbering). In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises 1, 2, or 3 substitutions at positions including 414, 424, and 426 (according to EU numbering).

[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 selected positions at the following locations: 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% identity, at least 90% identity, or at least 95% identity for any one amino acid 111-217 of SEQ NOs: 34-38, 58, and 60-90, 151, and 156-229. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises any one amino acid sequence of SEQ NOs: 156-229. In some embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues at positions corresponding to positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424 and 426 according to any one EU index of sequence numbers: 34-38, 58, and 60-90, 151, and 156-229 are missing or not 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 any one of SEQ ID NOs: 205 and 228 ( chamberlain, It includes the amino acid sequence of SEQ ID NO: 228. In other embodiments, the first Fc polypeptide includes the amino acid sequence of SEQ ID NO: 115, and the second Fc polypeptide includes any one of SEQ ID NO: 169 and 229 ( chamberlain, Includes the amino acid sequence of Sequence 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 any one of SEQ ID NO: 205 and 228 ( chamberlain,It includes the amino acid sequence of SEQ ID NO: 228. In other embodiments, the first Fc polypeptide includes the amino acid sequence of SEQ ID NO: 231, and the second Fc polypeptide is any one of SEQ ID NO: 169 and 229 ( chamberlain, Includes the amino acid sequence of Sequence 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 any one of SEQ ID NO: 205 and 228 ( chamberlain, It includes the amino acid sequence of SEQ ID NO: 228. In other embodiments, the first Fc polypeptide includes the amino acid sequence of SEQ ID NO: 235, and the second Fc polypeptide includes any one of SEQ ID NO: 169 and 229 ( chamberlain, Includes the amino acid sequence of Sequence No. 229.

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

[0041] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide have at least 75%, or at least 80%, 85%, 90%, 92%, or 95% amino acid sequence identity with respect 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, uptake of the ERT enzyme into the brain ( chamberlain,(Using a suitable animal model as described herein) is much larger compared to the intake of the ERT enzyme without the first Fc polypeptide and / or the second Fc polypeptide, or the intake of the ERT enzyme without modification of the first Fc polypeptide and / or the second Fc polypeptide resulting in TfR binding. In some embodiments, the ingestion of the ERT enzyme into the brain is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times greater than the ingestion of the ERT enzyme without the first Fc polypeptide and / or the second Fc polypeptide resulting in TfR binding.

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

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

[0045] In some aspects, polypeptides comprising an ERT enzyme, an ERT enzyme variant, or an Fc polypeptide linked to a catalytically active fragment thereof are provided herein, wherein the Fc polypeptide contains one or more modifications that promote heterodimerization into 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 respect to any one of SEQ Nos. 91, 92, 114, 230, and 234. In some embodiments, the ERT enzyme comprises any one of SEQ Nos. 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 respect to any one of the amino acid sequences of SEQ NOs: 119 and 120. In some embodiments, the ERT enzyme comprises any one of the amino acid sequences of SEQ NOs: 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 respect to any one of SEQ NOs: 121, 122, and 123. In some embodiments, the ERT enzyme comprises any one of SEQ NOs: 121, 122, and 123.

[0049] In some embodiments, the ERT enzyme is 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 respect to any one of the amino acid sequences of SEQ NOs: 93 and 94. In some embodiments, the ERT enzyme comprises any one of the amino acid sequences of SEQ NOs: 93 and 94.

[0050] In some embodiments, the 1 Fc polypeptide is a fusion polypeptide linked to the ERT enzyme, the ERT enzyme variant, or its catalytically active fragment by a peptide bond or a polypeptide linker. In some embodiments, the polypeptide linker is a soft polypeptide linker. In some embodiments, the soft polypeptide linker is a glycine-rich linker. In some embodiments, the glycine-rich linker is G4S (SEQ No.: 239) or (G4S)2 (SEQ No.: 240). In some embodiments, the 2 Fc polypeptide is not linked to the ERT enzyme, the ERT enzyme variant, or its catalytically active fragment by a chemical crosslinking agent, and chamberlain, The above fusion polypeptide does not possess non-peptide bonds or non-polypeptide linkers.

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

[0052] In some embodiments, the Fc polypeptide contains T366S, L368A, and Y407V substitutions (according to EU numbering). In some embodiments, the polypeptide comprises any one amino acid sequence of SEQ ID NOs: 115, 117, 231, 232, 235, and 236. In some embodiments, the polypeptide comprises any one amino acid sequence of SEQ ID NOs: 149 and 150. In some embodiments, the Fc polypeptide contains T366W substitution. In some embodiments, the polypeptide comprises any one amino acid sequence of SEQ ID NOs: 118, 233, and 237. In some embodiments, the polypeptide comprises any one amino acid sequence of SEQ ID NOs: 152–155. In some embodiments, the polypeptide further comprises another Fc polypeptide. In some embodiments, another Fc polypeptide contains T366W substitution or T366S, L368A, and Y407V substitution and forms an Fc dimer with the ERT enzyme-Fc fusion polypeptide.

[0053] In some embodiments, the Fc polypeptide comprises an intrinsic FcRn binding site. In some embodiments, the Fc polypeptide does not have an operator function. In some embodiments, the Fc polypeptide comprises a modification that reduces the operator function. In some embodiments, the modification that reduces the operator function is the substitution of Ala at position 234 and Ala at position 235 (according to EU numbering). In some embodiments, the modification that reduces the operator function further comprises the substitution of Gly at position 329 (according to EU numbering).

[0054] In some embodiments, the Fc polypeptide comprises amino acid changes that extend the serum half-life compared to the native Fc sequence. 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 numbering).

[0055] In some embodiments, the Fc polypeptide specifically binds to TfR.

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

[0057] In some embodiments, the Fc polypeptide further comprises 1, 2, 3, or 4 substitutions at positions including 380, 391, 392, and 415 (according to EU numbering). In some embodiments, the Fc polypeptide further comprises 1, 2, or 3 substitutions at positions including 414, 424, and 426 (according to EU numbering).

[0058] In some embodiments, the Fc polypeptide comprises Trp at position 388. In some embodiments, the Fc polypeptide comprises 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 selected positions at the following locations: 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% identity, at least 90% identity, or at least 95% identity for any one of the sequence numbers: 34-38, 58, and 60-90, amino acid 111-217. In some embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues of positions corresponding to positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424 and 426 according to any one EU index of sequence numbers: 34-38, 58, and 60-90 are missing or not substituted.

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

[0064] In some embodiments, the Fc polypeptide has at least 75%, or at least 80%, 85%, 90%, 92%, or 95% amino acid sequence identity with respect 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.

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

[0066] In some embodiments, a polynucleotide comprising a nucleic acid encoding a polypeptide comprising an ERT enzyme, an ERT enzyme variant, or an Fc polypeptide linked to a catalytically active fragment thereof is provided herein, wherein the Fc polypeptide contains one or more modifications that promote heterodimerization into another Fc polypeptide. In some embodiments, a vector comprising said polynucleotide is provided herein. In some embodiments, a host cell comprising said polynucleotide or said vector is provided herein. In some embodiments, said host cell further comprises a polynucleotide comprising a nucleic acid sequence encoding another Fc polypeptide. In some embodiments, a method for producing said polypeptide is provided herein, the method comprising culturing a host cell under conditions in which the polypeptide encoded by said polynucleotide is expressed.

[0067] In some aspects, a protein comprising the following is provided herein:

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

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

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

[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 respect to any one of SEQ Nos. 91, 92, 114, 230, and 234. In some embodiments, the ERT enzyme comprises any one of SEQ Nos. 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 respect to any one of the amino acid sequences of SEQ NOs: 119 and 120. In some embodiments, the ERT enzyme comprises any one of the amino acid sequences of SEQ NOs: 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 respect to any one of SEQ NOs: 121, 122, and 123. In some embodiments, the ERT enzyme comprises any one of SEQ NOs: 121, 122, and 123.

[0074] In some embodiments, the ERT enzyme is 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 respect to any one of the amino acid sequences of SEQ NOs: 93 and 94. In some embodiments, the ERT enzyme comprises any one of the amino acid sequences of SEQ NOs: 93 and 94.

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

[0076] In some embodiments, the ERT enzyme is linked to a peptide bond or polypeptide linker to the modified Fc polypeptide of (a) or the Fc polypeptide of (b) ( chamberlain, (Fused) to form a fused polypeptide. In some embodiments, the polypeptide linker is a soft polypeptide linker. In some embodiments, the soft polypeptide linker is a glycine-rich linker. In some embodiments, the glycine-rich linker is G4S (SEQ No.: 239) or (G4S)2 (SEQ No.: 240). In some embodiments, the ERT enzyme is not linked by a chemical crosslinking agent to the modified Fc polypeptide of (a) or the Fc polypeptide of (b), chamberlain, The above fusion polypeptide does not contain non-peptide bonds or non-polypeptide linkers.

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

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

[0079] In some embodiments, the Fc polypeptides of (a) and (b) each contain a modification that promotes heterodimerization. In some embodiments, one of the Fc polypeptides has a T366W substitution, and another Fc polypeptide has T366S, L368A, and Y407V substitutions (according to EU numbering). In some embodiments, the modified Fc polypeptide of (a) contains a T366W substitution, and the Fc polypeptide of (b) contains T366S, L368A, and Y407V substitutions. In some embodiments, the Fc polypeptide of (b) is linked to the ERT enzyme IDS and comprises any one amino acid sequence of SEQ Nos. 117, 232, and 236. In some embodiments, the modified Fc polypeptide of (a) contains T366S, L368A, and Y407V substitutions, and the Fc polypeptide of (b) contains T366W substitutions. In some embodiments, the Fc polypeptide of (b) is linked to the ERT enzyme IDS and comprises any one amino acid sequence of SEQ ID NOs: 118, 233, and 237.

[0080] In some embodiments, the modified Fc polypeptide of (a) and / or the Fc polypeptide of (b) comprises an intrinsic FcRn binding site. In some embodiments, the modified Fc polypeptide of (a) and the Fc polypeptide of (b) do not have an operator function. In some embodiments, the modified Fc polypeptide of (a) and / or the Fc polypeptide of (b) comprises a modification that reduces operator function. In some embodiments, the modification that reduces operator 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 operator function further comprises a substitution of Gly at position 329 (according to EU numbering). In some embodiments, the Fc polypeptide of (b) is linked to the ERT enzyme IDS and comprises any one amino acid sequence of SEQ Nos: 115, 231, and 235. In some embodiments, the Fc polypeptide of (b) is linked to the ERT enzyme SGSH and comprises any one amino acid sequence of SEQ ID NOs: 149, 150, 152, and 153.

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

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

[0083] In some embodiments, the modified Fc polypeptide further comprises 1, 2, 3, or 4 substitutions at positions including 380, 391, 392, and 415 (according to EU numbering). In some embodiments, the modified Fc polypeptide further comprises 1, 2, or 3 substitutions at positions including 414, 424, and 426 (according to EU numbering).

[0084] In some embodiments, the modified Fc polypeptide comprises Trp at position 388. In some embodiments, the modified Fc polypeptide comprises 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 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.

[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 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.

[0088] In some embodiments, the modified Fc polypeptide has a CH3 domain having at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one amino acid 111-217 of SEQ NOs: 34-38, 58, and 60-90, 151, and 156-229. In some embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ NOs: 156-229. In some embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues at positions corresponding to positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424 and 426 according to any one EU index of sequence numbers: 34-38, 58, and 60-90, 151, and 156-229 are missing or not 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 is any one of SEQ ID NOs: 205 and 228 ( chamberlain,The amino acid sequence of SEQ ID NO: 228) is included, and the second polypeptide chain is included in the amino acid sequence of SEQ ID NO: 115. In other embodiments, the first polypeptide chain is any one of SEQ ID NO: 169 and 229 ( chamberlain, It includes the amino acid sequence of sequence number: 229), and the second polypeptide chain includes the amino acid sequence of sequence number: 115.

[0091] In some embodiments, the first polypeptide chain is any one of SEQ ID NOs: 205 and 228 ( chamberlain, The amino acid sequence of SEQ ID NO: 228) is included, and the second polypeptide chain is included in the amino acid sequence of SEQ ID NO: 231. In other embodiments, the first polypeptide chain is any one of SEQ ID NO: 169 and 229 ( chamberlain, It includes the amino acid sequence of SEQ ID NO: 229), and the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 231.

[0092] In some embodiments, the first polypeptide chain is any one of SEQ ID NOs: 205 and 228 ( chamberlain, The amino acid sequence of SEQ ID NO: 228) is included, and the second polypeptide chain is included in the amino acid sequence of SEQ ID NO: 235. In other embodiments, the first polypeptide chain is any one of SEQ ID NO: 169 and 229 ( chamberlain, It includes the amino acid sequence of sequence number: 229), and the second polypeptide chain includes the amino acid sequence of sequence number: 235.

[0093] In some embodiments, the modified Fc polypeptide binds to the apex domain of TfR. In some embodiments, the binding of the protein to TfR does not substantially inhibit the binding of transferrin to 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 with respect 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, intake of the ERT enzyme into the brain ( chamberlain, (Using a suitable animal model as described herein) is much larger compared to the ingestion of the ERT enzyme without the Fc polypeptide, or the ingestion of the ERT enzyme without the modification of the Fc polypeptide resulting in TfR binding. In some embodiments, the ingestion of the ERT enzyme into the brain is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold larger compared to the ingestion of the ERT enzyme without the Fc polypeptide, or the ingestion of the ERT enzyme without the modification of the Fc polypeptide resulting in TfR binding.

[0096] In some aspects, a method for treating LSD is provided herein, said method comprising administering the protein or polypeptide described above to a patient in need thereof. In some embodiments, said method reduces the accumulation of toxic metabolites in the patient, and chamberlain, Toxic metabolites in the patient's brain and / or cerebrospinal fluid (CSF) are reduced.

[0097] In related aspects, a method for reducing the accumulation of toxic metabolites in a patient suffering from LSD is provided herein, said method comprising administering the protein or polypeptide described above to a patient in need thereof. In some embodiments, said method reduces the accumulation of toxic metabolites in the patient's brain and / or CSF.

[0098] In some embodiments, the LSD is Hunter syndrome, and the ERT enzyme is IDS. In some embodiments, the toxic metabolite comprises a heparan sulfate-derived disaccharide and / or a dermatan sulfate-derived disaccharide.

[0099] In some embodiments, the LSD is Sanfilippo syndrome A, and the ERT enzyme is SGSH. In some embodiments, the toxic metabolite is a heparan sulfate-derived oligosaccharide ( chamberlain, It is a hexasaccharide.

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

[0101] In some embodiments, the LSD is Gaucher disease or Parkinson's disease, and the ERT enzyme is GBA. In some embodiments, the toxic metabolite is 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 said toxic metabolites in the absence of said protein or polypeptide. Exemplary tests for measuring ERT enzyme activity and substrate accumulation are described herein.

[0103] In some aspects, a pharmaceutical composition comprising the protein or polypeptide described above and a pharmaceutically acceptable carrier is provided herein.

[0104] In some aspects, a method for monitoring substrate accumulation to measure IDS activity is provided herein, said method comprising:

[0105] (a) a step of destroying cells or tissue samples or cells within microvesicles within a fluid sample of a subject administered a protein or polypeptide as described above, in order to obtain a glycosaminoglycan (GAG) solution to be analyzed and to destroy open microvesicles;

[0106] (b) at least one heparinase ( chamberlain, A GAG solution is cleaved with heparinase I, heparinase II, and heparinase III and chondroitinase B to obtain a GAG-derived disaccharide;

[0107] (c) Mass spectrometry of the above GAG-derived disaccharide ( chamberlain, Analyzed by LC-MS / MS; and

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

[0109] In some embodiments, the step of destroying cells or microvesicles comprises at least one freeze-thaw cycle and / or at least one sonication step. In some embodiments, the cells are from a tissue sample, and the method comprises at least 3, 4, or 5 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 level of heparan sulfate- and / or dermatan 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- and / or dermatan sulfate-derived disaccharide in a control 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 polypeptide. In some embodiments, the control is a cell or tissue sample of the same tissue type known to lack IDS activity. In some embodiments, the protein or polypeptide increases the IDS activity in the sample by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold compared to the IDS activity in the control.

[0111] In other aspects, a method for monitoring substrate accumulation to evaluate SGSH activity is provided herein, said method comprising:

[0112] (a) a step of destroying cells or tissue samples or cells within microvesicles within a fluid sample of a subject administered a protein or polypeptide as described above, in order to obtain a glycosaminoglycan (GAG) solution to be analyzed and to destroy open microvesicles;

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

[0114] (c) Mass spectrometry of the above GAG-derived disaccharide ( chamberlain, Analyzed by LC-MS / MS; and

[0115] (d) Measure the level of heparan sulfate-derived disaccharides and, when compared to a control group lacking SGSH activity, a decrease in the level of heparan sulfate-derived disaccharides indicates that the SGSH activity in the sample has increased compared to the control group.

[0116] In some embodiments, the step of destroying cells or microvesicles comprises at least one freeze-thaw cycle and / or at least one sonication step. In some embodiments, the cells are from a tissue sample, and the method comprises at least 3, 4, or 5 freeze-thaw cycles. In some embodiments, the subject is a mouse lacking SGSH activity. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human patient with Sanfilippo syndrome A.

[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 group lacking SGSH activity. In some embodiments, the control group is a cell or tissue sample of the same tissue type obtained from the subject prior to administration of the protein or polypeptide. In some embodiments, the control group is a cell or tissue sample of the same tissue type known to lack SGSH activity. In some embodiments, the protein or polypeptide increases the SGSH activity in the sample by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold compared to the SGSH activity in the control.

[0118] In other aspects, a method for transporting a formulation across the mammalian BBB is provided herein, the method comprising exposing the BBB to a protein that binds to TfR with an affinity of about 50 nM to about 250 nM, wherein the protein is linked to the formulation and transports the linked formulation across the BBB. In some embodiments, the maximum concentration of the formulation in the mammalian brain (C max ) is improved. In some embodiments, the above preparation is useful for treating LSD.

[0119] Still in other aspects, a method for treating LSD is provided herein, said method comprising administering to a mammal a protein that binds to TfR with an affinity of about 50 nM to about 250 nM, said protein linked to an agent for treating LSD, thereby exposing said mammal to the brain of said mammal. In some embodiments, said protein, compared to an agent linked to a reference protein that binds to TfR with a weaker affinity, C of said agent in the brain max It improves. In some embodiments, the reference protein binds to TfR with an affinity of about 600 nM or weaker.

[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 apex domain of the TfR.

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

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

[0123] In some embodiments, the enzyme reduces the accumulation of toxic metabolites in the brain of mammals suffering from LSD when the enzyme is linked to the protein, compared to when the enzyme is linked to the reference protein. In some embodiments, the enzyme is IDS, and the LSD is Hunter syndrome. In some embodiments, the toxic metabolite is a heparin sulfate-derived disaccharide and / or a dermatan sulfate-derived disaccharide. In some embodiments, the enzyme is SGSH, and the LSD is Sanfilippo syndrome A. In some embodiments, the enzyme is ASM, and the LSD is Niemann-Pick disease. In some embodiments, the enzyme is GBA, and the LSD is Gaucher disease.

[0124] In some embodiments, the formulation comprises an antibody variable region. In some embodiments, the formulation comprises an antibody fragment. In some embodiments, the formulation comprises Fab or scFv.

[0125] In some embodiments, the protein is a modified Fc polypeptide containing a non-inherent binding site capable of binding to 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 embodiments, the protein associated with the above formulation is administered as part of a pharmaceutically acceptable carrier. Brief explanation of the drawing

[0127] Figure 1 shows the purification and analysis of an IDS-Fc fusion protein comprising an Fc polypeptide linked to the iduronate-2-sulfatase (IDS) enzyme and a modified Fc polypeptide bound to the transferrin receptor (TfR). Figure 2 shows the binding affinity test results of the IDS-Fc fusion protein analyzed in Figure 1, which show that the fusion protein binds to TfR. Figure 3 shows the IDS-Fc fusion protein analyzed in Figure 1. examiner Provides data proving IDS activity. Figure 4 shows that when IDS-deficient knockout cells were evaluated using an LC-MS / MS assay, the levels of heparan sulfate-derived disaccharides were increased, and IDS We provide data demonstrating that IDS expression in knockout (KO) cells rescues the corresponding knockout phenotype. Figure 5a shows the same TfR-linked Fc polypeptide ( chamberlain, The IDS-Fc fusion protein as an N-terminal monozyme or C-terminal monozyme containing CH3C.35.21.17) IDS It shows the reversal of heparan sulfate and dermatan sulfate accumulation in KO cells. Figure 5b shows that the N-terminal monozyme ("ETV:IDS 35.21.17") has comparable cellular efficacy against IDS. Figure 5c shows S accumulated in MPS II patient fibroblasts treated with the IDS-Fc fusion protein ("ETV:IDS") or IDS. 35 -Shows a dose-dependent decrease in sulfate-labeled protein; n=8. Fig. 5d shows S in MPS II patient fibroblasts treated with increasing doses of IDS-Fc fusion protein ("ETV:IDS") or IDS in the presence or absence of 5 mM M6P. 35 - Shows the M6PR-dependent elimination evaluation of the labeled protein; n=3. Figs. 5c-5d: "ETV:IDS" = ETV:IDS 35.23.2; the graph shows the mean ± SEM of the experimental replicas. Figure 6 shows wild-type (WT) mice administered the vehicle, or mice administered IDS or IDS-Fc fusion protein ("ETV:IDS"). IDS Provides data describing the levels of heparan and dermatan sulfate in mouse serum over time in KO mice. "ETV:IDS" = ETV:IDS 35.21. Fig. 7 is IDS Aggregation levels of disaccharides D0SO, DOA0, and D0a4 (referred to as "total sGAG levels") in peripheral tissues of KO mice were evaluated on day 7 following a single intravenous injection of 40 mg / kg IDS-Fc fusion protein ("ETV:IDS") or 5.3 mg / kg IDS, and vehicle-treated IDS Provides data showing comparison with that of KO and wild-type mice; IDS For the KO group, n=8, and for the wild-type group, n=3. Data are expressed as mean ± SEM and p-values: one-way ANOVA and Dunnett multiple comparison tests; ** p <0.01 and **** p <0.0001. "ETV:IDS" = ETV:IDS 35.21. Figure 8 shows human TfR knock-in (TfR ms / hu KI) Provides data describing the concentration of IDS-Fc fusion protein in the brain of a mouse. Figure 9a shows TfR after 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 ("IDS:Fc") lacking the mutation conferring TfR binding. ms / hu Provides data describing the concentration of IDS-Fc fusion protein in the brains of KI mice. Figure 9b shows TfR after a single intravenous injection of a 50 mg / kg dose. ms / hu Provides data describing the hepatic concentrations of the IDS-Fc fusion protein ETV:IDS 35.21 or IDS:Fc in KI mice; n=4-5. Graphs represent mean ± SEM. In Figures 10a-10c, ETV:IDS IDS KO x TfR ms / hu It shows that KI reduces GAGs in the brain and peripheral tissues of mice. IDS KO x TfR ms / hu KI mice were administered a single intravenous injection or a 4-week course of 40 mg / kg ETV:IDS or 14.2 mg / kg IDS as described in Example 2. IDS concentrations in serum (Fig. 10a) and tissue (Fig. 10b) were after a single administration IDS KO x TfR ms / hu Measured in KI mice. Tissue PK represents 2 h after administration; n=4. Graphs show mean ± SEM and p-values: unpaired t-test analysis. Fig. 10c is IDS KO x TfR ms / hu Levels of disaccharides D0SO, DOA0, and D0a4 ("total sGAG levels") were measured in the brain, CSF, and peripheral tissues of KI mice after single or multiple administrations of ETV:IDS or IDS, and compared to vehicle-treated and wild-type mice; IDS KO x TfR ms / hu For the KI group, n=8, and for the wild-type group, n=5. Graphs show mean ± SEM and p-values: one-way ANOVA and Dunnett multiple comparison test; ** p <0.01, *** p ≤0.001, and **** p ≤0.0001. Figure 11 shows the purification and analysis of an ASM-Fc fusion protein containing an Fc region linked to two acid sphingomyelinase (ASM) enzymes. Figure 12 shows the ASM-Fc fusion protein analyzed in Figure 11. examiner Provides data describing ASM activity. Figure 13 shows that the ASM-Fc fusion protein, as analyzed in Figure 11, is ASM Provides data showing that sphingomyelin accumulation is reduced using imaging-based assays in KO cells. Figure 14 shows that the ASM-Fc fusion protein, as analyzed in Figure 11, is ASM We provide data showing that LC-MS / MS-based assays reduce sphingomyelin accumulation in KO cells. Figure 15 shows the SGSH-Fc fusion protein as described in the examples. examiner Provides data explaining N-sulfoglucosamine sulfohydrolase (SGSH) activity. Figure 16 provides data showing that SGSH-deficient knockout (KO) cells have increased levels of heparan sulfate-derived disaccharides when evaluated using the LC-MS / MS assay. n=3–4 distinct cell lines; data are mean ± ± sem. Figure 17 shows the SGSH-Fc fusion protein analyzed in Figure 15 SGSH Provides data showing the reversal of heparan sulfate accumulation in KO cells. Figure 18 shows TfR ms / hu This shows the correlation between the affinity of hTfR and engineered TfR-binding polypeptides in KI mice and brain exposure over time. The dots represent TfR ms / hu This shows the cumulative brain exposure (AUC) over time of differently engineered TfR-binding polypeptide affinity variants following a single administration of 50 mg / kg in KI mice. Brain concentrations of the polypeptide (as measured by huIgG1) were calculated at various 24 hours post-administration (ranging from 1 to 10 days). Data represent summaries of three independent studies; in each study, n=4–5 mice per group. In Fig. 19, TfR ms / hu This shows the correlation between hTfR affinity and maximum brain concentration of engineered TfR-binding polypeptides in KI mice. The dots represent the maximum brain concentrations of different polypeptide affinity variants measured 1-day after administration of a single 50 mg / kg dose. The data represent a summary of three independent studies; in each study, one group consisted of n=4-5 mice. In Fig. 20, TfR ms / hu This shows the correlation between the affinity of the engineered TfR-binding polypeptide hTfR and the ratio of plasma concentration to the brain in KI mice. The dots represent the maximum plasma concentrations to the brain of different polypeptide affinity variants measured 1-day after administration of a single 50 mg / kg dose. Data represent a summary of three independent studies; in each study, one population consisted of n=4-5 mice. Figures 21a and 21b show TfR after systemic injection of a single 50 mg / kg anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusion (mean ± SEM, n=5 per population). ms / hu huIgG1 concentrations in plasma (Fig. 21a) and brain lysate (Fig. 21b) of krypton-phosphorus (KI) mice are shown. Figure 21c shows TfR after a single 50 mg / kg systemic administration of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusion (mean ± SEM, n=5 per population). ms / hu This represents the concentration of endogenous mouse Aβ in brain lysates of KI mice. Figure 21d shows TfR after systemic administration of a single 50 mg / kg anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusion (mean ± SEM, n=5 per population). ms / hu This shows the Western blot quantification of actin-standardized brain TfR protein in brain lysates of KI mice. Specific details for implementing the invention

[0128] details

[0129] I. Overview

[0130] We have developed fusion proteins containing enzyme replacement therapy (ERT) enzymes linked to Fc polypeptides. These proteins can be used to treat lysosomal storage disorders (LSDs). In some cases, the proteins contain dimeric Fc polypeptides, wherein one of the Fc polypeptide monomers is linked to the ERT enzyme. The Fc polypeptides can increase the enzyme half-life and, in some cases, can be modified to confer additional functional properties to the proteins. Fusion proteins that enable the transport of ERT enzymes across the blood-brain barrier (BBB) ​​are also described herein. These proteins comprise a dimeric Fc polypeptide, a modified Fc polypeptide, and an ERT enzyme linked to said Fc region and / or said 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 iduronate 2-sulfatase (IDS), or a catalytically active variant or fragment of wild-type IDS, chamberlain, It is a wild-type human IDS. In other embodiments, the ERT enzyme is N-sulfoglucosamine sulfohydrolase (SGSH), acid sphingomyelinase (ASM), β-glucocerbrosidase (GBA), or wild-type SGSH, ASM, or GBA, chamberlain, It is a catalytically active variant or fragment of wild-type human SGSH, ASM, or GBA.

[0131] We also developed a method to transport therapeutic agents linked to TfR-binding polypeptides and proteins across the BBB for the treatment of diseases. We discovered that the TfR binding affinity desirable for therapeutic agent transport across the BBB depends on the therapeutic agent's target as well as the mechanism of action that induces efficacy in treating the disease. In particular, we found that using polypeptides and proteins with stronger TfR affinity C max It was found that although it becomes larger, it leads to faster removal.

[0132] In the case of some treatments, for example, protein replacement therapy used to treat LSDs involves ERT enzymes, such as IDS ( chamberlain, (for the treatment of Hunter syndrome), as well as the use of other things, including the high brain C of the said therapeutic agent max Achieving [this] across the dosing window is desirable because higher extracellular concentrations will, in turn, lead to an increase in intracellular protein concentration. Once delivered into the cell, the intracellular half-life of the delivered protein persists for a longer period compared to its plasma residence time. Additionally, since high enzyme concentrations can induce increased substrate turnover by the enzyme, high C max Having [it] may be advantageous for enzyme replacement. Brain C max To improve this, polypeptides and proteins with a TfR affinity range of 50-250 nM are particularly useful.

[0133] II. Definition

[0134] As used herein, the singular forms (“a,” “an,” and “the”) include plural concepts unless otherwise explicitly stated. Thus, for example, “polypeptide” may contain two or more of these molecules and similar ones.

[0135] As used herein, when the terms “about” and “approximately” are used to modify a quantity specified by a numerical value or range, values ​​and numerical values ​​known to those skilled in the art and reasonable deviations, e.g., ± 20%, ± 10%, or ± 5%, are within the intended meaning of the quoted value.

[0136] "Enzyme replacement therapy enzyme" or "ERT enzyme" refers to an enzyme deficient in lysosomal storage disorder. "ERT enzyme variant" refers to a functional variant including splice variants of the wild-type ERT enzyme or its fragments, wherein the ERT enzyme variant has, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type ERT enzyme or its fragment when tested under the same conditions. The "catalytically active fragment" of the ERT enzyme refers to a portion of the full length of the ERT enzyme or its variant, wherein the catalytically active fragment has, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full length of the ERT enzyme or its variant when tested under the same conditions.

[0137] 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. A deficiency of IDS is associated with Mucopolysaccharidosis II, also known as Hunter syndrome. As a component of proteins containing the Fc polypeptide, the term "IDS" as used herein is catalytically active and encompasses functional variants, including alleles and splice variants of wild-type IDS or fragments thereof. The sequence of human IDS isoform I is a human sequence designated as canonical, available as UniProt entry P22304, and encoded by the human IDS gene at Xq28. The full-length sequence is provided at SEQ No. 91. As used herein, "mature" IDS sequence refers to a polypeptide chain form lacking the signal and propeptide sequences of the naturally occurring full-length polypeptide chain. The amino acid sequence of the mature human IDS polypeptide is provided at SEQ No. 92, which corresponds to amino acids 34–550 of the full-length human sequence. As used herein, "truncated" IDS sequence refers to a catalytically active fragment of the naturally occurring full-length polypeptide chain. An exemplary amino acid sequence of the truncated human IDS polypeptide is provided at SEQ No. 114, which corresponds to amino acids 26–550 of the full-length human sequence. The structure of human IDS is well-characterized. Exemplary structures are available under PDB access code 5FQL. The structures also Nat. Comm.It is described in 8:15786 doi: 10.1038 / ncomms15786, 2017. Non-human primate IDS sequences, including chimpanzee (UniProt entry K7BKV4) and rhesus monkey (UniProt entry H9FTX2), have also been described. Mouse IDS sequences are available in Uniprot entry Q08890. IDS variants are chamberlain, For example, when tested under the same conditions, it 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 IDS or its fragment. For example, when tested under the same conditions, the catalytically active IDS 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 IDS or its variant.

[0138] As used herein, "sulfoglucosamine sulfohydroase," "N-sulfoglucosamine sulfohydroase," or "SGSH" is used to refer to N-sulfoglucosamine sulfohydroase (EC 3.10.1.1), an enzyme involved in the lysosomal degradation of heparan sulfate. Mutations in this gene are associated with Sanfilippo syndrome A, a type of lysosomal storage disorder mucopolysaccharidosis III, which results from impaired degradation of heparan sulfate. As a component of proteins containing the Fc polypeptide, the term "SGSH" as used herein is catalytically active and encompasses functional variants, including alleles and splice variants of wild-type SGSH or fragments thereof. The sequence of human SGSH is available in UniProt entry P51688 and is encoded by human SGSH at 17q25.3. The full-length sequence is provided at SEQ No. 119. As used herein, the term "mature" SGSH sequence refers to a polypeptide chain form lacking the signal sequence of the naturally occurring full-length polypeptide chain. The amino acid sequence of the mature human SGSH polypeptide is provided at SEQ No. 120, which corresponds to amino acids 21–502 of the full-length human sequence. As used herein, the term "truncated" SGSH sequence refers to the catalytically active fragment of the naturally occurring full-length polypeptide chain. The structure of the human SGSH is well-characterized. An exemplary structure is available under PDB access code 4MHX. Non-human primate SGSH sequences, including chimpanzee (UniProt entry K7C218), have also been described. Mouse SGSH sequences are available at UniProt entry Q9EQ08. SGSH variants chamberlain,For example, when tested under the same conditions, it 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 SGSH or its fragment. For example, when tested under the same conditions, the catalytically active SGSH 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 SGSH or its variant.

[0139] As used herein, "acid sphingomyelinase," "sphingomyelin phosphodiesterase," or "ASM" refers to sphingomyelin phosphodiesterase 1 (EC 3.1.4.12), a lysosomal enzyme that converts sphingomyelin into ceramide. A ring associated with ASM deficiency is Niemann-Pick disease ( chamberlain,Includes type A or type B). As a component of a protein containing an Fc polypeptide, the term “ASM” as used herein encompasses catalytically active functional variants, including alleles and splice variants of wild-type ASM or fragments thereof. The sequence of human ASM isoform I is a human sequence designated as a standard sequence, available as UniProt entry P17405, and encoded by the human SMPD1 gene at 11p15.4. The full-length sequence is provided at SEQ ID NO: 121. As used herein, the “mature” ASM sequence refers to a polypeptide chain form lacking the signal sequence of the naturally occurring full-length polypeptide chain. The amino acid sequence of the mature human ASM polypeptide is provided at SEQ ID NO: 122, which corresponds to amino acids 47–629 of the full-length human sequence. As used herein, the “truncated” ASM sequence refers to the catalytically active fragment of the naturally occurring full-length polypeptide chain. An exemplary truncated human ASM polypeptide amino acid sequence is provided as Sequence No. 123, which corresponds to amino acids 47–620 of the full human sequence. The structure of the human ASM is well-characterized. An exemplary structure is available under PDB access code 5I81. Non-human primate ASM sequences, including chimpanzee (UniProt entry H2Q319), have also been described. The mouse ASM sequence is available at Uniprot entry Q04519. ASM variants chamberlain,For example, when tested under the same conditions, it 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 ASM or its fragment. The catalytically active ASM fragment has, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length ASM or its variant when tested under the same conditions.

[0140] "β-glucocerebrosidase" or "GBA" is also known as glucosylceramidase (EC 3.2.1.45). As used herein, the term refers to a lysosomal enzyme possessing glucosylceramidase activity that catalyzes the degradation of glucosylceramide into ceramide and glucose. GBA deficiency is associated with Gaucher disease and Parkinson's disease. As a component of proteins containing the Fc polypeptide, the term "GBA" as used herein is catalytically active and encompasses functional variants, including alleles and splice variants of wild-type GBA or fragments thereof. The sequence of human GBA, the long isoform, is designated as the standard sequence, is available as UniProt entry P04062-1, and is encoded by the human GBA gene at 1q22. The full sequence is provided as Sequence No. 93. As used herein, the term "mature" GBA sequence refers to a polypeptide chain form 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 at SEQ No. 94, which corresponds to amino acids 40–536 of the full-length human sequence. As used herein, the term "truncated" GBA sequence refers to the catalytically active fragment of the naturally occurring full-length polypeptide chain. The structure of human GBA is 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 at UniProt entry P17439. GBA variants chamberlain,For example, when tested under the same conditions, it 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 GBA or its fragment. The catalytically active GBA fragment has, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length GBA or its variant when tested under the same conditions.

[0141] As used herein, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is presented as SEQ ID No. 96. Sequences of transferrin receptor protein 1 of other species are also known ( chamberlain, Chimpanzee, accession number XP_003310238.1; rhesus, NP_001244232.1; dog, NP_001003111.1; cattle, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term "transferrin receptor" also refers to exemplary reference sequences, chamberlain, This encompasses allelic variants of the human sequence encoded by the gene at the chromosomal locus 1 of the transferrin receptor protein. The full-length transferrin receptor protein contains 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 presented as sequence number 238.

[0142] As used herein, "fusion protein" or "[ERT enzyme]-Fc fusion protein" refers to a first Fc polypeptide linked (e.g., fused) to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof ( chamberlain, "[ERT]-Fc fusion polypeptide"); and refers to a dimeric protein comprising a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide. The second Fc polypeptide may also be linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof ( chamberlain, (Fused). The first Fc polypeptide and / or the second Fc polypeptide may be linked to the ERT enzyme, an ERT enzyme variant, or its catalytically active fragment by a peptide bond or a polypeptide linker. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that promote the dimerization of another Fc polypeptide. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that confer binding to a transferrin receptor. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that reduce operator function. The first Fc polypeptide and / or the second Fc polypeptide may be modified Fc polypeptides containing one or more modifications that extend the serum half-life.

[0143] As used herein, "fusion polypeptide" or "[ERT enzyme]-Fc fusion polypeptide" refers to an Fc polypeptide linked (e.g., fused) to an ERT enzyme, an ERT enzyme variant, or its catalytically active fragment. The Fc polypeptide may be linked to the ERT enzyme, the ERT enzyme variant, or its catalytically active fragment by a peptide bond or a polypeptide linker. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that promote the dimerization of another Fc polypeptide. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that confer binding to a transferrin receptor. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that reduce operator function. The Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that prolong serum half-life.

[0144] As used herein, the term “Fc polypeptide” refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. The Fc polypeptide comprises a constant region sequence containing at least a CH2 domain and / or a CH3 domain, and may contain at least a portion of a hinge region. Generally, the Fc polypeptide does not contain a variable region.

[0145] "Modified Fc polypeptide" refers to at least one mutation when compared to the wild-type immunoglobulin heavy chain Fc polypeptide sequence, chamberlain, It refers to an Fc polypeptide that has substitutions, deletions, or insertions, but retains the structure of the entire Ig fold or the intrinsic Fc polypeptide.

[0146] The term "FcRn" refers to a nascent Fc receptor. Binding of Fc polypeptides to FcRn reduces clearance and increases the serum half-life of said Fc polypeptides. The human FcRn protein is a dimer composed of a protein approximately 50 kDa in size similar to major histocompatibility (MHC) class I proteins and a β2-microglobulin approximately 15 kDa in size.

[0147] As used herein, "FcRn binding site" refers to a region of the Fc polypeptide that binds to FcRn. In human IgG, the FcRn binding sites include T250, L251, M252, I253, S254, R255, T256, T307, E380, M428, H433, N434, H435, and Y436 when numbered using the EU index. These positions correspond to positions 20-26, 77, 150, 198, and 203-206 of SEQ ID NO: 1.

[0148] As used herein, "inherent FcRn binding site" refers to an Fc polypeptide region that binds to FcRn and has the same amino acid sequence as the region of the naturally occurring Fc polypeptide that binds to FcRn.

[0149] The terms “CH3 domain” and “CH2 domain” refer to immunoglobulin constant region domain polypeptides as used herein. For the purposes of this application, the CH3 domain polypeptide refers to the amino acid segment at position approx. 341 to position approx. 447 when numbered according to the EU, and the CH2 domain polypeptide refers to the amino acid segment at position approx. 231 to position approx. 340 when numbered according to the EU numbering system, and does not contain a hinge region sequence. The CH2 and CH3 domain polypeptides may also be numbered according to the IMGT (ImMunoGeneTics) numbering system, where the CH2 domain numbering is 1-110 and the CH3 domain numbering is 1-107 according to the IMGT Scientific chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc region of the immunoglobulin. The Fc region refers to a segment of amino acids from approximate position 231 to approximate position 447 when numbered according to the EU numbering scheme, but may include at least a portion of the hinge region of the antibody as used herein. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (Sequence No.: 95).

[0150] With respect to CH3 or CH2 domains, the terms "wild type," "inherent," and "naturally occurring" refer to domains having sequences that occur in nature.

[0151] As used herein, in relation to mutant polypeptides or mutant polynucleotides, the term "mutant" is used interchangeably with "variant." Variants for a given wild-type CH3 or CH2 domain reference sequence include naturally occurring allelic variants. "Non-naturally occurring" CH3 or CH2 domains do not exist in naturally occurring cells, and genetic modification, chamberlain,It refers to a variant or mutant domain of a native CH3 domain or CH2 domain polynucleotide or polypeptide produced by genetic engineering or mutagenesis technology. "Variant" includes any domain containing at least one amino acid mutation relative to the wild type. Mutations may include substitutions, insertions, and deletions.

[0152] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids.

[0153] Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are subsequently modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphorine. "Amino acid analogs" are compounds having the same basic chemical structure as naturally occurring amino acids, e.g., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. These analogs possess modified R groups (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refer to chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0154] 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. Stereoisomers of naturally occurring α-amino acids include, but are not limited to: D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and their Combinations.

[0155] Acids and amino acids may be referred to in this specification by commonly known three-letter or one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0156] The terms “polypeptide” and “peptide” are interchangeable herein as comprising a polymer of amino acid residues in a single chain. These terms apply to amino acid polymers, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, wherein one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid. The amino acid polymer may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L-amino acids and D-amino acids.

[0157] The term "protein" as used herein refers to a polypeptide or a dimer of single-stranded polypeptides ( in other words, 2) or a multimer ( chamberlain, It refers to three or more). Single-stranded polypeptides of proteins are covalently bonded, chamberlain, They can be connected by disulfide bonds or non-covalent interactions.

[0158] terminology "Conservative substitution," "conservative mutation," or "conservatively modified variant" refers to a change in which an amino acid is substituted for another amino acid that can be classified as having similar characteristics. Examples of categories of conservative amino acid groups defined in this way include the following: "Charged / polar group" includes Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); "aromatic group" includes Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); And the “aliphatic group” includes Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Within each group, subgroups may also be specified. For example, the group of charged or polar amino acids may be subdivided into subgroups including a “positively charged subgroup” including Lys, Arg, and His; a “negatively charged subgroup” including Glu and Asp; and a “polar subgroup” including Asn and Gln. In another embodiment, the aromatic or cyclic group may be subdivided into subgroups including a “nitrogenous cyclic subgroup” including Pro, His, and Trp; and a “phenyl subgroup” including Phe and Tyr. In another additional embodiment, the aliphatic group may be partially subdivided into subgroups including, for example, an "aliphatic non-polar subgroup" including Val, Leu, Gly, and Ala; and an "aliphatic micro-polar subgroup" including Met, Ser, Thr, and Cys.Examples of the category of conservative mutations include, but are not limited to, amino acid substitutions of amino acids within the above subgroups: Lys for Arg or its reverse, thereby retaining a positive charge; Glu for Asp or its reverse, thereby retaining a negative charge; Ser for Thr or its reverse, thereby retaining a free -OH; and Gln for Asn or its reverse, thereby retaining a free -NH2. In some embodiments, the hydrophobic amino acid is chamberlain, It is substituted for a naturally occurring hydrophobic amino acid to preserve hydrophobicity at the active site.

[0159] With respect to the content of two or more polypeptide sequences, the term "identical" or "identical" percentage refers to being identical for a specified area, or a specified percentage, when measured by a sequence comparison algorithm, or by manual alignment and visual inspection, or when compared and aligned for maximum correspondence across a comparison window or designated area. chamberlain, It refers to two or more sequences or subsequences having residues having at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more percentages.

[0160] For the sequence comparison of polypeptides, typically a single amino acid sequence serves as a reference sequence to be compared with candidate sequences. Alignment may be performed using various methods available to those skilled in the art, such as visual alignment, or using publicly available software employing algorithms known to achieve maximum alignment. Such programs include BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment may be determined by those skilled in the art. For the sequence comparison of polypeptide sequences for the purposes of this application, the standard protein BLAST algorithm is used to align two protein sequences, along with default parameters.

[0161] When used in the context of identifying a given amino acid residue in a polypeptide sequence, the terms “corresponding to,” “determined by reference,” or “numbered by reference” refer to the position of a specific residue when the given amino acid sequence is maximally aligned and compared with a reference sequence. Thus, for example, when the residue is optimally aligned to sequence number: 1, the amino acid residue of the modified Fc polypeptide “corresponds” to the amino acid of sequence number: 1. A polypeptide aligned with a reference sequence does not need to be the same length as the reference sequence.

[0162] "Bonding affinity," as used herein, refers to two molecules, chamberlain, Single binding site and target on polypeptide, chamberlain,It refers to the strength of non-covalent interactions between the transferrin receptors that bind to it. Thus, for example, the above term may refer to a 1:1 interaction between a polypeptide and its target unless otherwise noted or explicitly stated in the content. Binding affinity is the dissociation rate constant (k d , hour -1 ) is the combined rate constant (k a , hour -1 M -1 Equilibrium dissociation constant (K) meaning divided by ) D It can be quantified by measuring ). K D For example, using the Surface Plasmon Resonance (SPR) method, e.g., the Biacore™ system; KinExA ® Motion exclusion analysis such as; and BioLayer interferometers (e.g., ForteBio ® Octet ® It can be determined by measuring the kinetics of complex formation and dissociation using a platform. As used herein, "binding affinity" includes not only the formal binding affinity reflecting the 1:1 interaction between the polypeptide and its target, but also the calculated K which can reflect avid binding. D It includes apparent affinity for.

[0163] As used herein, when referring to the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody described herein, the target, chamberlain,The terms “specifically binding” or “selectively binding” to TfR refer to a binding reaction that causes a engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody to bind to said target with greater affinity, greater avidity, and / or for a longer period than binding to a structurally different target. In typical embodiments, said engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody, when tested under the same affinity assay conditions, compared to an irrelevant target, a specific target, chamberlain, It has at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for TfR. The terms "specific binding," "binds specifically to," or specific target ( chamberlain, "Specific" for TfR) as used herein refers, for example, to the parallel dissociation constant K with respect to the target to which the molecules bind. D , chamberlain, 10 -4 M or smaller, chamberlain, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 It may be represented by a molecule having M. In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is conserved between species ( chamberlain, Structurally preserved across species), chamberlain, Conserved between non-human primates and human species ( chamberlain, It specifically binds to epitopes on TfR (structurally conserved between non-human primates and human species). In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody can exclusively bind to human TfR.

[0164] The term “variable region” or “variable domain” refers to a domain of the antibody heavy or light chain derived from germline variable (V) genes, diversity (D) genes, or linkage (J) genes (and not derived from invariant (Cμ and Cδ) gene segments) that confers its specificity to the antibody for binding to an antigen. Typically, the antibody variable region comprises four conserved “framework” regions interspersed with three hypervariable “complementarity determining regions.”

[0165] The terms “antigen-binding portion” and “antigen-binding fragment” are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen through the variable region of the antibody. Examples of antigen-binding fragments include, but are not limited to: the Fab fragment (a monovalent fragment consisting of VL, VH, CL, and CH1 domains), the F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at 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).

[0166] The terms "treatment," "treating," and similar terms are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. "Treating" or "treatment" refers to lysosomal storage disorders, chamberlain,In the treatment or improvement of Hunter syndrome, Sanfilippo syndrome A, Niemann-Pick disease, Gaucher disease, or Parkinson's disease, any index of success may be referred to, including objective or subjective parameters such as remission, remission, improvement in patient survival, increase in survival time or survival rate, reduction in symptoms, or making the patient more tolerant of disability, slowing the rate of regression or decline, or improvement in the patient's physical or mental well-being. Treatment or improvement of symptoms may be based on subjective or objective parameters. Treatment effects may be compared to an untreated individual or pool of individuals, or to the same patient at different time points—before or during treatment.

[0167] The terms "subject," "entity," and "patient" are interchangeable herein, and refer to humans, non-human primates, rodents ( chamberlain, It refers to mammals including, but not limited to, rats, mice, and guinea pigs), rabbits, cattle, pigs, horses, and other mammalian species. In one embodiment, the patient is a human.

[0168] The term "pharmaceuticalally acceptable excipients" refers to non-active pharmaceutical components that are biologically or pharmacologically compatible for use in humans or animals, including but not limited to buffers, carriers, or preservatives.

[0169] As used herein, the "therapeutic amount," "therapeutically effective amount," or "therapeutically effective concentration" of a preparation refers to the subject ( chamberlain, It is the amount or concentration of a substance used to treat the signs or symptoms of a disease (e.g., LSD) in mammals.

[0170] The term "administer" refers to a method of delivering a preparation, compound, or composition to a desired site of biological action. These methods include, but are not limited to, local delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the polypeptides described herein are administered intravenously.

[0171] III. Enzyme Replacement Therapy (ERT) Enzymes

[0172] Lysosomal storage disorders (LSDs) are genetic metabolic disorders characterized by the accumulation of undigested or partially digested macromolecules, which ultimately lead to cellular dysfunction and clinical abnormalities. Generally, LSDs have been defined as deficiencies in lysosomal function, typically classified by accumulated substrates, and include sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses, lipoprotein storage disorders, neurogenic ceroid lipofuscinoses, and similar conditions. The classification of these disorders has recently been expanded to include other defects or deficiencies in proteins that result in the accumulation of macromolecules, such as proteins required for the normal post-detoxification modification of lysosomal enzymes or proteins critical for proper lysosomal trafficking.

[0173] In some aspects, the fusion protein described herein includes: (i) Fc polypeptide, which is a modified ( chamberlain, It may contain one or more modifications that promote heterodimerization, or it may be a wild-type Fc polypeptide; and ERT enzyme; and (ii) Fc polypeptide, which is a modification ( chamberlain,It may contain 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 of the Fc polypeptides are blood-brain barrier (BBB) ​​receptors, chamberlain, It may contain modifications that cause binding to the transferrin receptor (TfR). The ERT enzyme may be any enzyme deficient in LSD. The ERT enzyme incorporated into the fusion protein is catalytically active, and chamberlain, This enzyme maintains enzymatic activity bound to LSD. In some embodiments, the ERT enzyme is iduronate 2-sulfatase (IDS), which is deficient in Hunter syndrome. In some embodiments, the ERT enzyme is N-sulfoglucosamine sulfohydrolase (SGSH), which is deficient in Sanfilippo 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 disease and Parkinson's disease.

[0174] In some embodiments, an ERT enzyme and an optionally modified Fc polypeptide that binds to a BBB receptor, chamberlain,The fusion protein comprising the TfR-binding Fc polypeptide 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 the IDS protein comprising any one amino acid sequence of SEQ Nos. 91, 92, 114, 230, and 234. In some embodiments, the catalytically active variant or fragment of the IDS enzyme has an activity of 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 than the activity of the wild-type IDS enzyme.

[0175] In some embodiments, an ERT enzyme and an optionally modified Fc polypeptide that binds to a BBB receptor, chamberlain, The fusion protein comprising the TfR-binding Fc polypeptide comprises a catalytically active fragment or variant of wild-type SGSH. In some embodiments, the SGSH enzyme is a variant or catalytically active fragment of the SGSH protein comprising any one amino acid sequence of SEQ Nos. 119 and 120. In some embodiments, the catalytically active variant or fragment of the SGSH enzyme has an activity of 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 than the activity of the wild-type SGSH enzyme.

[0176] In some embodiments, an ERT enzyme and an optionally modified Fc polypeptide that binds to a BBB receptor, chamberlain,The fusion protein comprising the TfR-binding Fc polypeptide comprises a catalytically active fragment or variant of wild-type ASM. In some embodiments, the ASM enzyme is a variant or catalytically active fragment of the ASM protein comprising any one amino acid sequence of SEQ Nos. 121, 122, and 123. In some embodiments, the catalytically active variant or fragment of the ASM enzyme has an activity of 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 than the activity of the wild-type ASM enzyme.

[0177] In some embodiments, an ERT enzyme and an optionally modified Fc polypeptide that binds to a BBB receptor, chamberlain, The fusion protein comprising the TfR-binding Fc polypeptide comprises a catalytically active fragment or variant of wild-type GBA. In some embodiments, the GBA enzyme is a variant or catalytically active fragment of a GBA protein comprising any one amino acid sequence of SEQ Nos. 93 and 94. In some embodiments, the catalytically active variant or fragment of the GBA enzyme has an activity of 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 than the activity of the wild-type GBA enzyme.

[0178] In some embodiments, the ERT enzyme present in the fusion protein described herein, chamberlain,IDS, SGSH, ASM, or GBA, or a catalytically active variant or fragment thereof, retains at least 25% of its activity when not linked to the Fc polypeptide or TfR-linked Fc polypeptide. In some embodiments, the ERT enzyme, or a catalytically active variant or fragment thereof, retains at least 10% of its activity when not linked to the Fc polypeptide or TfR-linked Fc polypeptide, or at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of its activity when not linked to the Fc polypeptide or TfR-linked Fc polypeptide. In some embodiments, the ERT enzyme, or a catalytically active variant or a fragment thereof, retains at least 80%, 85%, 90%, or 95% of its activity when not connected to the Fc polypeptide or the TfR-linked Fc polypeptide. In some embodiments, due to fusion to the Fc polypeptide, the ERT enzyme, chamberlain, The activity of IDS, SGSH, ASM, or GBA, or a catalytically active variant or fragment thereof, is not reduced. In some embodiments, the activity of the ERT enzyme is not reduced by fusion to the TfR-binding Fc polypeptide.

[0179] IV. Fc polypeptide modification for blood-brain barrier (BBB) ​​receptor binding

[0180] In some aspects, fusion proteins capable of being transported across the blood-brain barrier (BBB) ​​are provided herein. These proteins comprise a modified Fc polypeptide that binds to a BBB receptor. BBB receptors are expressed in the BBB endothelium, as well as in other cell and tissue types. In some embodiments, the BBB receptor is a transferrin receptor (TfR).

[0181] BBB receptor, chamberlain,Amino acid residues specified in various Fc modifications, including those introduced into modified Fc polypeptides that bind to TfR, are numbered herein according to EU index numbering. Any Fc polypeptide, chamberlain, IgG1, IgG2, IgG3, or IgG4 Fc polypeptides are modified at one or more of the positions described herein, chamberlain, It can have amino acid substitutions.

[0182] Modified ( chamberlain, The Fc polypeptide (heterodimerization and / or enhancement of BBB receptor-binding) for the intrinsic Fc region sequence or a fragment thereof, chamberlain, For a fragment of at least 50 amino acids or at least 100 amino acids, or a longer length, it may have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity. In some embodiments, the intrinsic Fc amino acid sequence is the Fc region sequence of SEQ ID NO: 1. In some embodiments, the modified Fc polypeptide is amino acids 1-110 of SEQ ID NO: 1, or amino acids 111-217 of SEQ ID NO: 1, or a fragment thereof, chamberlain, It has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity for a fragment of at least 50 amino acids or at least 100 amino acids, or a longer length.

[0183] In some specific examples, modified ( chamberlain,The Fc polypeptide (heterodimerization and / or enhancement of BBB receptor-binding) comprises at least 50 amino acids corresponding to the intrinsic Fc region amino acid sequence, or at least 60, 65, 70, 75, 80, 85, 90, or 95 or more, or at least 100 amino acids, or more. In some embodiments, the modified Fc polypeptide has at least 25 contiguous amino acids corresponding to the intrinsic Fc region amino acid sequence, e.g., SEQ ID NO: 1, or at least 30, 35, 40, or 45 contiguous amino acids, or 50 contiguous amino acids, or at least 60, 65, 70, 75, 80, 85, 90, or 95 or more contiguous amino acids, or 100 or more contiguous amino acids.

[0184] In some embodiments, the domain modified for BBB receptor-binding activity is a human Ig CH3 domain, e.g., an IgG1 CH3 domain. The CH3 domain is any IgG subtype, chamberlain, It may be IgG1, IgG2, IgG3, or IgG4. In the context of IgG1 antibodies, the CH3 domain refers to the amino acid segment at position 341 to position 447 when numbered according to the EU numbering system.

[0185] In some embodiments, the domain modified for BBB receptor-binding activity is a human Ig CH2 domain, e.g., an IgG CH2 domain. The CH2 domain is any IgG subtype, chamberlain, It may be IgG1, IgG2, IgG3, or IgG4. In the context of IgG1 antibodies, the CH2 domain refers to the amino acid segment at position 231 to position 340 when numbered according to the EU numbering system.

[0186] In some specific embodiments, the modified (present in the fusion protein described herein) chamberlain, The BBB receptor-binding) Fc polypeptide comprises at least 1, 2, or 3 substitutions; and in some embodiments, comprises at least 4, 5, 6, 7, 8, 9, or 10 substitutions at amino acid positions including positions 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 according to the EU numbering scheme.

[0187] In some specific embodiments, the modified (present in the fusion protein described herein) chamberlain, The BBB receptor-binding) Fc polypeptide comprises at least 1, 2, or 3 substitutions; and in some embodiments, comprises at least 4, 5, 6, 7, 8, or 9 substitutions at amino acid positions including positions 274, 276, 283, 285, 286, 287, 288, 289, and 290 according to the EU numbering scheme.

[0188] In some specific embodiments, the modified (present in the fusion protein described herein) chamberlain, The BBB receptor-binding) Fc polypeptide comprises at least 1, 2, or 3 substitutions; and in some embodiments, comprises at least 4, 5, 6, 7, 8, 9, or 10 substitutions at amino acid positions including positions 268, 269, 270, 271, 272, 292, 293, 294, 296, and 300 according to the EU numbering scheme.

[0189] In some specific embodiments, the modified (present in the fusion protein described herein) chamberlain,The BBB receptor-binding) Fc polypeptide comprises at least 1, 2, or 3 substitutions; and in some embodiments, comprises at least 4, 5, 6, 7, 8, or 9 substitutions at amino acid positions including positions 272, 274, 276, 322, 324, 326, 329, 330, and 331 according to the EU numbering scheme.

[0190] In some specific embodiments, the modified (present in the fusion protein described herein) chamberlain, The BBB receptor-binding) Fc polypeptide comprises at least 1, 2, or 3 substitutions; and in some embodiments, comprises at least 4, 5, 6, or 7 substitutions at amino acid positions including positions 345, 346, 347, 349, 437, 438, 439, and 440 according to the EU numbering scheme.

[0191] In some specific embodiments, the modified (present in the fusion protein described herein) chamberlain, The BBB receptor-binding) Fc polypeptide comprises at least 1, 2, or 3 substitutions; and in some embodiments, comprises at least 4, 5, 6, 7, 8, or 9 substitutions at amino acid positions including positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering scheme.

[0192] FcRn binding site

[0193] In certain aspects, the modified (present in the fusion protein described herein) chamberlain, BBB receptor-binding) Fc polypeptides, or Fc polypeptides that do not specifically bind to BBB receptors, may also include an FcRn binding site. In some embodiments, the FcRn binding site is located within the Fc polypeptide or a fragment thereof.

[0194] In some embodiments, the FcRn binding site comprises an intrinsic FcRn binding site. In some embodiments, the FcRn binding site does not include an amino acid change compared to the amino acid sequence of the intrinsic FcRn binding site. In some embodiments, the intrinsic FcRn binding site is an IgG binding site, chamberlain, It is a human IgG binding site. In some embodiments, the FcRn binding site includes a modification that alters the FcRn binding.

[0195] In some specific examples, the FcRn binding site is mutated, chamberlain, It has one or more substituted amino acid residues, wherein the mutation(s) increase the serum half-life or substantially do not decrease the serum half-life ( chamberlain, When tested under identical conditions, the serum half-life is reduced to less than 25% compared to a counterpart modified Fc polypeptide having wild-type residues at the mutated positions. 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 scheme.

[0196] In some embodiments, the serum half-life of the modified polypeptide is extended by mutating one or more residues at or near the FcRn binding site relative to the native human IgG sequence. In some embodiments, mutations are introduced at one, two, or three of positions 252, 254, and 256. In some embodiments, the mutations are M252Y, S254T, and T256E. In some embodiments, the modified Fc polypeptide further comprises the mutations M252Y, S254T, and T256E. In some embodiments, the modified Fc polypeptide comprises substitutions at one, two, or all three of positions T307, E380, and N434 according to the EU numbering scheme. In some embodiments, the mutations are T307Q and N434A. In some embodiments, the modified Fc polypeptide comprises mutations T307A, E380A, and N434A. In some embodiments, the modified Fc polypeptide comprises substitutions at positions T250 and M428 according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide comprises mutations T250Q and / or M428L. In some embodiments, the modified Fc polypeptide comprises substitutions at positions M428 and N434 according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide comprises mutations M428L and N434S. In some embodiments, the modified Fc polypeptide comprises the N434S or N434A mutation.

[0197] V. Transferrin receptor-binding FC polypeptide

[0198] This section describes the generation of the modified Fc polypeptide described herein that binds to the transferrin receptor (TfR) and can be transported across the blood-brain barrier (BBB).

[0199] TfR-binding Fc polypeptides containing mutations in the CH3 domain

[0200] In some embodiments, the modified Fc polypeptide that specifically binds to TfR includes a substitution in the CH3 domain. In some embodiments, the modified Fc polypeptide includes a human Ig CH3 domain modified for TfR-binding activity, such as an IgG CH3 domain. The CH3 domain is any IgG subtype, chamberlain, It may be IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH3 domain refers to the amino acid segment at approximate position 341 to approximate position 447 when numbered according to the EU numbering system.

[0201] In some embodiments, a modified Fc polypeptide that specifically binds to TfR may bind to the apex domain of said TfR and bind to TfR without blocking or otherwise inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is not substantially inhibited. In some embodiments, the binding of transferrin to TfR is less than about 50% ( chamberlain, It is inhibited to about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 5%). In some embodiments, the binding of transferrin to TfR is inhibited to about 20% or less ( chamberlain, It is suppressed to approximately 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1%.

[0202] In some embodiments, the modified Fc polypeptide specifically binding to TfR comprises at least 2, 3, 4, 5, 6, 7, 8, or 9 substitutions at positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering scheme. Exemplary substitutions that may be introduced at these positions are presented in Tables 4 and 5. In some embodiments, the amino acid at position 388 and / or 421 is an aromatic amino acid, chamberlain, It is 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.

[0203] In some embodiments, at least one position is substituted as follows: 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 at position 388, chamberlain, Trp; 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 is a conservative substitution, chamberlain, Identical charge clusters, hydrophobic clusters, and side chain loop structure clusters of the specified amino acids at one or more of the positions within the set ( chamberlain,It may include amino acids within aromatic amino acids), size clusters, and / or polar or non-polar clusters. Thus, for example, Ile may be present at positions 384, 386, and / or position 413. In some embodiments, one or two of positions 387, 413, and 416, or at each of these positions, the acidic amino acid is Glu. In other embodiments, one or two of positions 387, 413, and 416, or at each of these positions, the acidic amino acid is Asp. In some embodiments, two, three, four, five, six, seven, or eight of positions 384, 386, 387, 388, 389, 413, 416, and 421 all have the amino acid substitutions specified in this paragraph.

[0204] In some embodiments, the modified Fc polypeptide as described in the preceding two paragraphs comprises an intrinsic Asn at position 390. In some embodiments, the modified Fc polypeptide comprises Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 390. In some embodiments, the modified Fc polypeptide comprises one, two, three, or four additional substitutions at positions including 380, 391, 392, and 415 according to the EU numbering scheme. In some embodiments, Trp, Tyr, Leu, or Gln may be present at position 380. In some embodiments, Ser, Thr, Gln, or Phe may be present at position 391. In some embodiments, Gln, Phe, or His may be present at position 392. In some embodiments, Glu may be present at position 415.

[0205] In certain embodiments, the modified Fc polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from the following: 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 11 positions as follows: 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.

[0206] In certain embodiments, the modified Fc polypeptide comprises Leu or Met at position 384; Leu, His, or Pro at position 386; Val at position 387; Trp at position 388; Val or Ala at position 389; Pro at position 413; Thr at position 416; and / or Trp at position 421. In some embodiments, the modified Fc polypeptide further comprises Ser, Thr, Gln, or Phe at position 391. In some embodiments, the modified Fc polypeptide further comprises Trp, Tyr, Leu, or Gln at position 380 and / or Gln, Phe, or His at position 392. In some embodiments, Trp is at position 380, and / or Gln is at position 392. In some embodiments, the modified Fc polypeptide does not have Trp at position 380.

[0207] 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 an intrinsic Asn at position 390. In certain embodiments, the modified Fc polypeptide further comprises Trp, Tyr, Leu, or Gln at position 380; and / or Glu at position 415. In some embodiments, the modified Fc polypeptide further comprises Trp at position 380 and / or Glu at position 415.

[0208] In additional embodiments, the modified Fc polypeptide further comprises one, two, or three substitutions at positions including positions 414, 424, and 426 according to the EU numbering scheme. In some embodiments, position 414 is Lys, Arg, Gly, or Pro; position 424 is Ser, Thr, Glu, or Lys; and / or position 426 is Ser, Trp, or Gly.

[0209] In some embodiments, the modified Fc polypeptide comprises one or more of the following substitutions: according to the EU numbering scheme, Trp at position 380; Thr at position 386; Trp at position 388; Val at position 389; Thr or Ser at position 413; Glu at position 415; and / or Phe at position 421.

[0210] In some embodiments, the modified Fc polypeptide is any one of sequence numbers: 4-90, 97-100, and 105-108 ( chamberlain, (SEQ NOs: 34-38, 58, and 60-90) has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 111-217. In some embodiments, the modified Fc polypeptide is any one of SEQ NOs: 4-90, 97-100, and 105-108 ( chamberlain, It has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ NOs: 34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide is any one of SEQ NOs: 4-90, 97-100, and 105-108 ( chamberlain,SEQ ID NOs: 34-38, 58, and 60-90) comprises the said amino acid at EU index positions 384-390 and / or 413-421. In some embodiments, the modified Fc polypeptide is any one of SEQ ID NOs: 4-90, 97-100, and 105-108 ( chamberlain, SEQ ID NOs: 34-38, 58, and 60-90) comprises the said amino acid at EU index positions 380-390 and / or 413-421. In some embodiments, the modified Fc polypeptide comprises any one of SEQ ID NOs: 4-90, 97-100, and 105-108 ( chamberlain, Sequence numbers: 34-38, 58, and 60-90) The above amino acids are included at EU index positions 380-392 and / or 413-426.

[0211] In some embodiments, the modified Fc polypeptide is at least one of sequence numbers: 4-90, 97-100, and 105-108 ( chamberlain, Having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ numbers: 34-38, 58, and 60-90), and further comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of these positions when numbered according to the EU index as follows: 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; an aromatic amino acid at position 388, chamberlain,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 acid, 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 acid 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.

[0212] In some embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ NOs: 34-38, 58, and 60-90. In other embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ NOs: 34-38, 58, and 60-90, provided that 1, 2, or 3 amino acids are substituted.

[0213] In some embodiments, the modified Fc polypeptide comprises additional mutations, including but not limited to the following, such as the mutations described in Section VI below: knob mutations ( chamberlain, T366W when numbered according to EU numbering), hole mutation ( chamberlain, When numbered according to EU numbering, T366S, L368A, and Y407V), mutations that regulate actuator function ( chamberlain, When numbered according to EU numbering, L234A, L235A, and / or P329G ( chamberlain, L234A and L235A), and / or mutations that increase serum stability or serum half-life ( chamberlain,(i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to EU numbering). For illustrative purposes, SEQ ID NOs: 156-229 provide non-limiting examples of modified Fc polypeptides having mutations in the CH3 domain, comprising one or more of these additional mutations ( chamberlain, 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).

[0214] In some embodiments, the modified Fc polypeptide is a Knob mutation ( chamberlain, It includes T366W when numbered according to EU numbering, and has at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one of sequence numbers: 156, 168, 180, 192, 204, and 216. In some embodiments, the modified Fc polypeptide comprises any one of sequence numbers: 156, 168, 180, 192, 204, and 216.

[0215] In some embodiments, the modified Fc polypeptide is a Knob mutation ( chamberlain, When numbered according to EU numbering, T366W) and mutations that adjust actuator functions ( chamberlain, When numbered according to EU numbering, L234A, L235A, and / or P329G ( chamberlain,It includes L234A and L235A)) and has at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one of sequence numbers: 157, 158, 169, 170, 181, 182, 193, 194, 205, 206, 217, 218, 228, and 229. In some embodiments, the modified Fc polypeptide comprises any one of sequence numbers: 157, 158, 169, 170, 181, 182, 193, 194, 205, 206, 217, and 218.

[0216] In some embodiments, the modified Fc polypeptide is a Knob mutation ( chamberlain, T366W when numbered according to EU numbering) and mutations that increase serum stability or serum half-life ( chamberlain, (i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to EU numbering, having at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one of sequence numbers: 159, 171, 183, 195, 207, and 219. In some embodiments, the modified Fc polypeptide comprises any one of sequence numbers: 159, 171, 183, 195, 207, and 219.

[0217] In some embodiments, the modified Fc polypeptide is a Knob mutation ( chamberlain, T366W when numbered according to EU numbering), mutation that adjusts actuator function ( chamberlain, When numbered according to EU numbering, L234A, L235A, and / or P329G ( chamberlain, L234A and L235A), and mutations that increase serum stability or serum half-life ( chamberlain,(i) when numbered according to EU numbering, M252Y, S254T, and T256E, or (ii) when numbered according to EU numbering, with or without M428L, N434S) having at least 85% identity, at least 90% identity, or at least 95% identity for any one of sequence numbers: 160, 161, 172, 173, 184, 185, 196, 197, 208, 209, 220, and 221. In some embodiments, the modified Fc polypeptide comprises any one of sequence numbers: 160, 161, 172, 173, 184, 185, 196, 197, 208, 209, 220, and 221.

[0218] In some embodiments, the modified Fc polypeptide is a hole mutant ( chamberlain, It includes T366S, L368A, and Y407V when numbered according to EU numbering, and has at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one of sequence numbers: 162, 174, 186, 198, 210, and 222. In some embodiments, the modified Fc polypeptide comprises any one of sequence numbers: 162, 174, 186, 198, 210, and 222.

[0219] In some embodiments, the modified Fc polypeptide is a hole mutant ( chamberlain, When numbered according to EU numbering, T366S, L368A, and Y407V) and mutations that adjust actuator functions ( chamberlain, When numbered according to EU numbering, L234A, L235A, and / or P329G ( chamberlain,It includes L234A and L235A)) and has at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one of sequence numbers: 163, 164, 175, 176, 187, 188, 199, 200, 211, 212, 223, and 224. In some embodiments, the modified Fc polypeptide comprises any one of sequence numbers: 163, 164, 175, 176, 187, 188, 199, 200, 211, 212, 223, and 224.

[0220] In some embodiments, the modified Fc polypeptide is a hole mutant ( chamberlain, (when numbered according to EU numbering, T366S, L368A, and Y407V) and mutations that increase serum stability or serum half-life ( chamberlain, (i) M252Y, S254T, and T256E when numbered according to EU numbering, or (ii) N434S with or without M428L when numbered according to EU numbering, having at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one of sequence numbers: 165, 177, 189, 201, 213, and 225. In some embodiments, the modified Fc polypeptide comprises any one of sequence numbers: 165, 177, 189, 201, 213, and 225.

[0221] In some embodiments, the modified Fc polypeptide is a hole mutant ( chamberlain, When numbered according to EU numbering, T366S, L368A, and Y407V), mutations that adjust actuator functions ( chamberlain, When numbered according to EU numbering, L234A, L235A, and / or P329G ( chamberlain, L234A and L235A), and mutations that increase serum stability or serum half-life ( chamberlain,(i) when numbered according to EU numbering, M252Y, S254T, and T256E, or (ii) when numbered according to EU numbering, with or without M428L, N434S), and having at least 85% identity, at least 90% identity, or at least 95% identity for any one of sequence numbers: 166, 167, 178, 179, 190, 191, 202, 203, 214, 215, 226, and 227. In some embodiments, the modified Fc polypeptide comprises any one of sequence numbers: 166, 167, 178, 179, 190, 191, 202, 203, 214, 215, 226, and 227.

[0222] In some embodiments, the modified Fc polypeptide specifically binding to TfR comprises at least 2, 3, 4, 5, 6, 7, or 8 substitutions at positions 345, 346, 347, 349, 437, 438, 439, and 440 according to the EU numbering scheme. Modified Fc polypeptides for description are presented in SEQ ID NOs: 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 certain embodiments, the modified Fc polypeptide comprises 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, all of 2, 3, 4, 5, 6, 7, or 8 positions of 345, 346, 347, 349, 437, 438, 439, and 440 have the substitutions specified in this paragraph. In some embodiments, the modified Fc polypeptide has conservative substitutions, chamberlain, Identical charge clusters, hydrophobic clusters, and side chain loop structure clusters of the specified amino acids at one or more of the positions within the set ( chamberlain, It may include aromatic amino acids), or amino acids within size clusters, and / or polar or non-polar clusters.

[0223] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one amino acid 111-217 of SEQ NO: 124-128. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ NO: 124-128. In some embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ NO: 124-128. In other embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ ID NOs: 124-128, provided that 1, 2, or 3 amino acids are substituted.

[0224] TfR-binding Fc polypeptides containing mutations in the CH2 domain

[0225] In some embodiments, the modified Fc polypeptide that specifically binds to TfR includes a substitution in the CH2 domain. In some embodiments, the modified Fc polypeptide includes a human Ig CH2 domain modified for TfR-binding activity, such as an IgG CH2 domain. The CH2 domain is any IgG subtype, chamberlain, It may be IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH2 domain refers to the amino acid segment at position 231 to position 340 when numbered according to the EU numbering system.

[0226] In some embodiments, a modified Fc polypeptide that specifically binds to TfR may bind to the apex domain of said TfR and bind to TfR without blocking or otherwise inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is not substantially inhibited. In some embodiments, the binding of transferrin to TfR is less than about 50% ( chamberlain, It is inhibited to about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 5%). In some embodiments, the binding of transferrin to TfR is inhibited to about 20% or less ( chamberlain, It is suppressed to approximately 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1%.

[0227] In some embodiments, the modified Fc polypeptide specifically binding to TfR comprises at least 2, 3, 4, 5, 6, 7, 8, or 9 substitutions at positions 274, 276, 283, 285, 286, 287, 288, and 290 according to the EU numbering scheme. Modified Fc polypeptides for illustrative purposes are presented in SEQ ID NOs: 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 comprises at least one substitution at the following positions: Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp at position 274; At position 276, Ile, Val, Asp, Glu, Thr, Ala, or Tyr; at position 283, Asp, Pro, Met, Leu, Ala, Asn, or Phe; at position 285, Arg, Ser, Ala, or Gly; at position 286, Tyr, Trp, Arg, or Val; at position 287, Glu; at position 288, Trp or Tyr; at position 289, Gln, Tyr, His, Ile, Phe, Val, or Asp; or at position 290, Leu, Trp, Arg, Asn, Tyr, or Val. In some embodiments, all 2, 3, 4, 5, 6, 7, 8, or 9 positions of 274, 276, 283, 285, 286, 287, 288, and 290 have the substitutions specified in this paragraph. In some embodiments, the modified Fc polypeptide is a conservative substitution, chamberlain, Identical charge clusters, hydrophobic clusters, and side chain loop structure clusters of the specified amino acids at one or more of the positions within the set ( chamberlain, It may include aromatic amino acids), or amino acids within size clusters, and / or polar or non-polar clusters.

[0228] In some embodiments, the modified Fc polypeptide is 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 is Arg at position 285; Tyr or Trp at position 286; Glu at position 287; Trp at position 288; and / or include Arg or Trp at position 290.

[0229] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one amino acid 1-110 of SEQ NOs: 129-133. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ NOs: 129-133. In some embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ NOs: 129-133. In other embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ ID NOs: 129-133, provided that 1, 2, or 3 amino acids are substituted.

[0230] In some embodiments, the modified Fc polypeptide specifically binding to TfR comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions at positions 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 according to the EU numbering scheme. Modified Fc polypeptides for illustrative purposes are presented in SEQ ID NOs: 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 comprises 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, all of 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 have the substitutions specified in this paragraph. In some embodiments, the modified Fc polypeptide has conservative substitutions, chamberlain, Identical charge clusters, hydrophobic clusters, and side chain loop structure clusters of the specified amino acids at one or more of the positions within the set ( chamberlain, It may include aromatic amino acids), or amino acids within size clusters, and / or polar or non-polar clusters.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one amino acid 1-110 of SEQ NO: 134-138. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ NO: 134-138. In some embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ NO: 134-138. In other embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ ID NOs: 134-138, provided that 1, 2, or 3 amino acids are substituted.

[0235] In some embodiments, the modified Fc polypeptide specifically binding to TfR comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions at positions 268, 269, 270, 271, 272, 292, 293, 294, and 300 according to the EU numbering scheme. Modified Fc polypeptides for illustrative purposes are presented in SEQ ID NOs: 139-143. In some embodiments, the modified Fc polypeptide comprises 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, all 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of 268, 269, 270, 271, 272, 292, 293, 294, and 300 have the substitutions specified in this paragraph. In some embodiments, the modified Fc polypeptide has conservative substitutions, chamberlain, Identical charge clusters, hydrophobic clusters, and side chain loop structure clusters of the specified amino acids at one or more of the positions within the set ( chamberlain, It may include aromatic amino acids), or amino acids within size clusters, and / or polar or non-polar clusters.

[0236] In some embodiments, the modified Fc polypeptide 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.

[0237] 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.

[0238] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one amino acid 1-110 of SEQ NOs: 139-143. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ NOs: 139-143. In some embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ NOs: 139-143. In other embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ ID NOs: 139-143, provided that 1, 2, or 3 amino acids are substituted.

[0239] In some embodiments, the modified Fc polypeptide specifically binding to TfR has at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions at positions 272, 274, 276, 322, 324, 326, 329, 330, and 331 according to the EU numbering scheme. Modified Fc polypeptides for illustrative purposes are presented in SEQ ID NOs: 144-148. In some embodiments, the modified Fc polypeptide comprises Trp at position 330. In some embodiments, the modified Fc polypeptide comprises 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, all of 2, 3, 4, 5, 6, 7, 8, or 9 positions of 272, 274, 276, 322, 324, 326, 329, 330, and 331 have the substitutions specified in this paragraph. In some embodiments, the modified Fc polypeptide has conservative substitutions, chamberlain, Identical charge clusters, hydrophobic clusters, and side chain loop structure clusters of the specified amino acids at one or more of the positions within the set ( chamberlain, It may include aromatic amino acids), or amino acids within size clusters, and / or polar or non-polar clusters.

[0240] In some embodiments, the modified Fc polypeptide comprises 2, 3, 4, 5, 6, 7, 8, or 9 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 comprises Val or Ile at position 272; Gly at position 274; Arg at position 276; Includes 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.

[0241] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one amino acid 1-110 of SEQ NO: 144-148. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ NO: 144-148. In some embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ NO: 144-148. In other embodiments, the modified Fc polypeptide comprises any one amino acid sequence of SEQ ID NOs: 144-148, provided that 1, 2, or 3 amino acids are substituted.

[0242] VI. Additional FC polypeptide mutations

[0243] In some aspects, the fusion protein described herein comprises two Fc polypeptides, each of which may comprise an independently selected modification, or a wild-type Fc polypeptide, chamberlain, It may be a human IgG1 Fc polypeptide. In some embodiments, one or both of the Fc polypeptides are blood-brain barrier (BBB) ​​receptors, chamberlain, It may contain one or more modifications that cause transferrin receptor (TfR) binding. Non-limiting examples of other mutations that may be introduced into one or both of the Fc polypeptides are chamberlain, Includes mutations that increase serum stability or serum half-life, mutations that modulate effector function, mutations that affect glycosylation, mutations that reduce immunogenicity in humans, and / or mutations that provide Knob and Hall dimerization for the said Fc polypeptides.

[0244] In some embodiments, the Fc polypeptides present in the fusion protein are the corresponding wild-type Fc polypeptides ( chamberlain, It 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 independently with respect to human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide.

[0245] In some embodiments, the Fc polypeptide present in the fusion protein includes knob and hole mutations that promote heterodimer formation and inhibit homodimer formation. Generally, the modification introduces a protuberance ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") at the interface of the second polypeptide so that the protuberance may be positioned within the cavity to promote heterodimer formation and thus inhibit homodimer formation. The protuberance is constructed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protuberance is created at the interface of the second polypeptide by replacing a large amino acid side chain with a smaller one (e.g., alanine or threonine). In some embodiments, these additional mutations are located within the Fc polypeptide at positions that do not negatively affect the polypeptide's binding to BBB receptors, e.g., TfR.

[0246] In one exemplary embodiment of the Knob and Hall approach for dimerization, position 366 (numbered according to the EU numbering system) of one of the Fc polypeptides present in the fusion protein contains tryptophan at the native threonine site. In the dimer, another Fc polypeptide has valine instead of the native tyrosine at position 407 (numbered according to the EU numbering system). The other Fc polypeptide may contain further substitutions, wherein the native threonine at position 366 (numbered according to the EU numbering system) is substituted with serine, and the native leucine at position 368 (numbered according to the EU numbering system) is substituted with alanine. Accordingly, one of the Fc polypeptides of the fusion proteins described herein has a T366W knob mutation, and another Fc polypeptide has a Y407V mutation, which are typically accompanied by T366S and L368A hole mutations.

[0247] In some embodiments, modifications to increase the serum half-life may be introduced. For example, in some embodiments, one or both of the Fc polypeptides present in the fusion protein described herein may contain tyrosine at position 252, threonine at position 254, and glutamic acid at position 256 (as numbered according to the EU numbering scheme). Thus, one or both of the Fc polypeptides may have M252Y, S254T, and T256E substitutions. Alternatively, one or both of the Fc polypeptides may have M428L and N434S substitutions (as numbered according to the EU numbering scheme). Alternatively, one or both of the Fc polypeptides may have N434S or N434A substitutions.

[0248] In some embodiments, one or both of the Fc polypeptides present in the fusion protein described herein are modifications that reduce operator function, chamberlain,When binding to Fc receptors expressed on effector cells that mediate effector function, they undergo modifications that reduce the ability to induce specific biological functions. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), cell-surface receptors ( chamberlain, Down-regulation of B cell receptors and B-cell activation. Operator function may vary depending on the type of antibody. For example, native human IgG1 and IgG3 antibodies can induce ADCC and CDC activity when binding to appropriate Fc receptors on immune system cells; and native human IgG1, IgG2, IgG3, and IgG4 can induce ADCP function when binding to appropriate Fc receptors present on immune cells.

[0249] In some embodiments, one or both of the Fc polypeptides present in the fusion protein described herein may also be designed to contain other modifications for heterodimerization, chamberlain, Electrostatic interaction of contact residues within the CH3-CH3 interface that are naturally charged or hydrophobic patch variants.

[0250] In some embodiments, one or both of the Fc polypeptides present in the fusion protein described herein may include additional modifications that regulate the operator function.

[0251] In some embodiments, one or both of the Fc polypeptides present in the fusion protein described herein may include modifications that reduce or eliminate operator function. An Fc polypeptide mutation for the description of reducing operator function is located within the CH2 domain, chamberlain,Substitutions at positions 234 and 235 (according to the EU numbering system) are included but not limited thereto. For example, in some embodiments, one or both of the Fc polypeptides may include alanine residues at positions 234 and 235. Thus, one or both of the Fc polypeptides may have L234A and L235A (LALA) substitutions.

[0252] Additional Fc polypeptide mutations that modulate operator function include, but are not limited to: position 329 may be mutated, wherein proline is substituted with glycine, arginine, or an amino acid residue of sufficient size to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and tryptophan residues Trp 87 and Trp 110 of Fcγ RIII. Substitutions for further description include S228P, E233P, L235E, N297A, N297D, and P331S according to the EU numbering scheme. Multiple substitutions may also be present, chamberlain, L234A and L235A of the human IgG1 Fc region; 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 (according to the EU numbering scheme) may be present. In some embodiments, one or both of the Fc polypeptides may have one or more amino acid substitutions that regulate ADCC, chamberlain, Substitutions may be found in positions 298, 333, and / or 334 (according to the EU numbering scheme).

[0253] Fc polypeptides for explanation including additional mutations

[0254] As a non-limiting example, one or both of the Fc polypeptides present in the fusion protein described herein are Knob mutations ( chamberlain, T366W when numbered according to the EU numbering system), hole mutation ( chamberlain, When numbered according to the EU numbering scheme, T366S, L368A, and Y407V), mutations that adjust actuator functions ( chamberlain, When numbered according to the EU numbering system, L234A, L235A, and / or P329G ( chamberlain, (L234A and L235A when numbered according to the EU numbering scheme), and / or mutations that increase serum stability or serum half-life ( chamberlain, (i) when numbered according to the EU numbering scheme, it may include additional mutations including M252Y, S254T, and T256E, or (ii) when numbered according to the EU numbering scheme, with or without M428L, including N434S.

[0255] In some specific examples, the Fc polypeptide is a Knob mutation ( chamberlain, When numbered according to the EU numbering scheme, it may have T366W) and has at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one of sequence numbers: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one of sequence numbers: 1, 4-90, and 124-148 may be modified to have a knob mutation.

[0256] In some specific examples, the Fc polypeptide is a Knob mutation ( chamberlain, T366W when numbered according to the EU numbering system), mutation that adjusts actuator function ( chamberlain, When numbered according to the EU numbering system, L234A, L235A, and / or P329G ( chamberlain,It may have L234A and L235A)) and may have at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one of sequence numbers: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one of sequence numbers: 1, 4-90, and 124-148 may be modified to have a knob mutation and a mutation that modulates the operator function.

[0257] In some specific examples, the Fc polypeptide is a Knob mutation ( chamberlain, T366W when numbered according to the EU numbering system), mutations that increase serum stability or serum half-life ( chamberlain, (i) when numbered according to the EU numbering scheme, may have M252Y, S254T, and T256E, or (ii) when numbered according to the EU numbering scheme, may have N434S with or without M428L, and may have at least 85% identity, at least 90% identity, or at least 95% identity for any one of sequence numbers: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one of sequence numbers: 1, 4-90, and 124-148 may be modified to have a Knob mutation and a mutation that increases serum stability or serum half-life.

[0258] In some specific examples, the Fc polypeptide is a Knob mutation ( chamberlain, T366W when numbered according to the EU numbering system), mutation that adjusts actuator function ( chamberlain, When numbered according to the EU numbering system, L234A, L235A, and / or P329G ( chamberlain, L234A and L235A), mutations that increase serum stability or serum half-life ( chamberlain,(i) when numbered according to the EU numbering scheme, may have M252Y, S254T, and T256E, or (ii) when numbered according to the EU numbering scheme, may have N434S with or without M428L, and may have at least 85% identity, at least 90% identity, or at least 95% identity for any one of sequence numbers: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one of sequence numbers: 1, 4-90, and 124-148 may be modified to have a knob mutation, a mutation that modulates the operator function, and a mutation that increases serum stability or serum half-life.

[0259] In some specific examples, the Fc polypeptide is a hole mutation ( chamberlain, When numbered according to the EU numbering scheme, it may have T366S, L368A, and Y407V) and has at least 85% identity, at least 90% identity, or at least 95% identity for any one of sequence numbers: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one of sequence numbers: 1, 4-90, and 124-148 may be modified to have a hole mutation.

[0260] In some specific examples, the Fc polypeptide is a hole mutation ( chamberlain, When numbered according to the EU numbering scheme, T366S, L368A, and Y407V), mutations that adjust actuator functions ( chamberlain, When numbered according to the EU numbering system, L234A, L235A, and / or P329G ( chamberlain,It may have L234A and L235A)) and may have at least 85% identity, at least 90% identity, or at least 95% identity with respect to any one of sequence numbers: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one of sequence numbers: 1, 4-90, and 124-148 may be modified to have a hole mutation and a mutation that modulates the operator function.

[0261] In some specific examples, the Fc polypeptide is a hole mutation ( chamberlain, Mutations that increase serum or serum half-life (when numbered according to the EU numbering scheme, T366S, L368A, and Y407V) chamberlain, (i) when numbered according to the EU numbering scheme, may have M252Y, S254T, and T256E, or (ii) when numbered according to the EU numbering scheme, may have N434S with or without M428L, and may have at least 85% identity, at least 90% identity, or at least 95% identity for any one of sequence numbers: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one of sequence numbers: 1, 4-90, and 124-148 may be modified to have a hole mutation and a mutation that increases serum stability or serum half-life.

[0262] In some specific examples, the Fc polypeptide is a hole mutation ( chamberlain, When numbered according to the EU numbering scheme, T366S, L368A, and Y407V), mutations that adjust actuator functions ( chamberlain, When numbered according to the EU numbering system, L234A, L235A, and / or P329G ( chamberlain, L234A and L235A), mutations that increase serum stability or serum half-life ( chamberlain,(i) when numbered according to the EU numbering scheme, may have M252Y, S254T, and T256E, or (ii) when numbered according to the EU numbering scheme, may have N434S with or without M428L, and may have at least 85% identity, at least 90% identity, or at least 95% identity for any one of sequence numbers: 1, 4-90, and 124-148. In some embodiments, the Fc polypeptide having any one of sequence numbers: 1, 4-90, and 124-148 may be modified to have a hole mutation, a mutation that modulates operator function, and a mutation that increases serum stability or serum half-life.

[0263] VII. Fusion protein for description containing ERT enzyme

[0264] 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 said first Fc polypeptide. In some embodiments, said first Fc polypeptide and / or said second Fc polypeptide do not contain immunoglobulin heavy chain and / or light chain variable region sequences or their antigen-binding portions. In some embodiments, said ERT enzyme is IDS, SGSH, ASM, or GBA. In some embodiments, said first Fc polypeptide is a modified Fc polypeptide and / or said second Fc polypeptide is a modified Fc polypeptide. In some embodiments, said second Fc polypeptide is a modified Fc polypeptide. In some embodiments, the modified Fc polypeptide may be a modified Fc polypeptide containing one or more modifications that promote its dimerization to another Fc polypeptide. In some embodiments, the modified Fc polypeptide contains one or more modifications that reduce operator function. In some embodiments, the modified Fc polypeptide contains one or more modifications that extend serum half-life. In some embodiments, the modified Fc polypeptide is a blood-brain barrier (BBB) ​​receptor, chamberlain, It contains one or more modifications that cause binding to the transferrin receptor (TfR).

[0265] In other aspects, the fusion protein described herein is a BBB receptor, chamberlain,It comprises a first polypeptide chain comprising a modified Fc polypeptide that specifically binds to TfR, and a second polypeptide chain comprising an Fc polypeptide that dimerizes with the modified Fc polypeptide to form an Fc dimer. An ERT enzyme may be linked to 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 the polypeptide chain. 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 the BBB receptor.

[0266] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a modified Fc polypeptide that specifically binds to TfR and a second polypeptide chain comprising an Fc polypeptide, wherein the modified Fc polypeptide and the Fc polypeptide are dimerized 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 is a BBB receptor, chamberlain, It does not specifically bind to TfR.

[0267] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a modified Fc polypeptide containing T366W (knob) substitutions that binds to TfR; and a second polypeptide chain comprising an Fc polypeptide containing T366S, L368A, and Y407V (hall) 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 polypeptide and / or the Fc polypeptide comprises human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0268] In some embodiments, the modified Fc polypeptide comprises the Knob, LALA, and YTE mutations as specified for any one of sequence numbers: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, and has at least 85% identity, at least 90% identity, or at least 95% identity for each sequence; or comprises any one of sequence numbers: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221. In some embodiments, the Fc polypeptide comprises the hole, LALA, and YTE mutations specified for any one of SEQ NOs: 101-104, having at least 85% identity, at least 90% identity, or at least 95% identity for each sequence; or comprises any one of SEQ NOs: 101-104. In some embodiments, the modified Fc polypeptide comprises any one of SEQ NOs: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, and the Fc polypeptide comprises any one of SEQ NOs: 101-104. In some embodiments, the modified Fc polypeptide and / or the N-terminus of the Fc polypeptide is a part of the IgG1 hinge region ( chamberlain, It contains DKTHTCPPCP (SEQ No.: 113). In some embodiments, the modified Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of SEQ No.: 116, 228, and 229, or comprises any one of SEQ No.: 116, 228, and 229.

[0269] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a modified Fc polypeptide containing T366S, L368A, and Y407V (Hol) substitutions, which binds to TfR; and a second polypeptide chain comprising an Fc polypeptide containing T366W (Knob) 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 polypeptide and / or the Fc polypeptide comprises human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0270] In some embodiments, the modified Fc polypeptide comprises the hole, LALA, and YTE mutations specified for any one of sequence numbers: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, having at least 85% identity, at least 90% identity, or at least 95% identity for each sequence; or comprises any one of sequence numbers: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227. In some embodiments, the Fc polypeptide comprises the Knob, LALA, and YTE mutations specified for any one of SEQ NOs: 109-112, having at least 85% identity, at least 90% identity, or at least 95% identity for each sequence; or comprises any one of SEQ NOs: 109-112. In some embodiments, the modified Fc polypeptide comprises any one of SEQ NOs: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, and the Fc polypeptide comprises any one of SEQ NOs: 109-112. In some embodiments, the modified Fc polypeptide and / or the N-terminus of the Fc polypeptide is a part of the IgG1 hinge region ( chamberlain, It contains DKTHTCPPCP; Sequence No.: 113).

[0271] In some embodiments, the ERT enzyme present in the fusion protein described herein, chamberlain, IDS, SGSH, ASM, or GBA is an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of Sequence Nos: 101-104, or Sequence Nos: 101-104 ( chamberlain,It is linked to a polypeptide chain comprising any one sequence of (as a fusion polypeptide). In some embodiments, the ERT enzyme, chamberlain, IDS, SGSH, ASM, or GBA is a linker to the Fc polypeptide, such as a soft linker, and / or a hinge region or a part thereof ( chamberlain,It is linked by DKTHTCPPCP (SEQ 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 one of SEQ Nos: 114, 230, and 234, or comprises any one of SEQ Nos: 114, 230, and 234. In some embodiments, the IDS sequence linked to the Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of SEQ Nos: 115, 117, 231, 232, 235, and 236, or comprises any one of SEQ Nos: 115, 117, 231, 232, 235, and 236. In some embodiments, the ERT enzyme comprises an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 120, or comprises the sequence of SEQ ID NO: 120. In some embodiments, the SGSH sequence associated with 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 comprises 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 respect to any one of SEQ NOs: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, or comprises any one of SEQ NOs: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221. In some embodiments, the N-terminus of the Fc polypeptide and / or the modified Fc polypeptide is a part of the IgG1 hinge region ( chamberlain,It contains DKTHTCPPCP (SEQ No.: 113). In some embodiments, the modified Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of SEQ No.: 116, 228, and 229, or comprises any one of SEQ No.: 116, 228, and 229.

[0272] 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 any one of the sequences of SEQ ID NO: 205 and 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 any one of the sequences of SEQ ID NO: 160 and 229.

[0273] 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 any one of the sequences of SEQ ID NO: 205 and 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 any one of the sequences of SEQ ID NO: 169 and 229.

[0274] 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 any one of the sequences of SEQ ID NO: 205 and 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 any one of the sequences of SEQ ID NO: 169 and 229.

[0275] In some embodiments, the ERT enzyme present in the fusion protein described herein, chamberlain, IDS, SGSH, ASM, or GBA is an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of SEQ Nos: 109-112, or SEQ Nos: 109-112 ( chamberlain, It is linked to a polypeptide chain comprising any one sequence of (as a fusion polypeptide). In some embodiments, the ERT enzyme, chamberlain, IDS, SGSH, ASM, or GBA is a linker to the Fc polypeptide, such as a soft linker, and / or a hinge region or a part thereof ( chamberlain,It is linked by DKTHTCPPCP (SEQ 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 one of SEQ Nos. 114, 230, and 234, or comprises any one of SEQ Nos. 114, 230, and 234. In some embodiments, the IDS sequence linked to the Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of SEQ Nos. 118, 233, and 237, or comprises any one of SEQ Nos. 118, 233, and 237. In some embodiments, the ERT enzyme comprises an SGSH sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 120, or comprises the sequence of SEQ ID NO: 120. In some embodiments, the SGSH sequence associated with the Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of SEQ ID NO: 152 and 153, or comprises any one 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 respect to any one of SEQ NOs: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, or comprises any one of SEQ NOs: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227. In some embodiments, the N-terminus of the Fc polypeptide and / or the modified Fc polypeptide is a part of the IgG1 hinge region ( chamberlain, It contains DKTHTCPPCP; Sequence No.: 113).

[0276] In some embodiments, the ERT enzyme present in the fusion protein described herein, chamberlain, IDS, SGSH, ASM, or GBA is a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of SEQ Nos: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221, or SEQ Nos: 97-100, 151, 156-161, 168-173, 180-185, 192-197, 204-209, and 216-221 ( chamberlain, It is linked to a polypeptide chain comprising any one sequence of (as a fusion polypeptide). In some embodiments, the ERT enzyme, chamberlain, IDS, SGSH, ASM, or GBA is a linker, such as a soft linker, and / or a hinge region or part thereof to the modified Fc polypeptide ( chamberlain,It is linked by DKTHTCPPCP (SEQ 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 one of SEQ No.: 114, 230, and 234, or comprises any one of SEQ 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 SEQ No.: 120, or comprises the sequence of SEQ No.: 120. In some embodiments, the SGSH sequence associated with the Fc polypeptide has at least 85%, at least 90%, or at least 95% identity with any one of SEQ NOs: 154 and 155, or comprises any one of SEQ NOs: 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 one of SEQ NOs: 101-104, 149 and 150, or comprises any one of SEQ NOs: 101-104, 149 and 150. In some embodiments, the modified Fc polypeptide and / or the N-terminus of the Fc polypeptide is a part of the IgG1 hinge region ( chamberlain, It contains DKTHTCPPCP; Sequence No.: 113).

[0277] In some embodiments, the ERT enzyme present in the fusion protein described herein, chamberlain,IDS, SGSH, ASM, or GBA is a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with any one of sequence numbers: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227, or sequence numbers: 105-108, 162-167, 174-179, 186-191, 198-203, 210-215, and 222-227 ( chamberlain, It is linked to a polypeptide chain comprising any one sequence of (as a fusion polypeptide). In some embodiments, the ERT enzyme, chamberlain, IDS, SGSH, ASM, or GBA is a linker, such as a soft linker, and / or a hinge region or part thereof to the modified Fc polypeptide ( chamberlain,It is linked by DKTHTCPPCP (SEQ 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 one of SEQ Nos. 114, 230, and 234, or comprises any one of SEQ Nos. 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 SEQ No. 120, or comprises the sequence of SEQ 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 one of SEQ Nos. 109-112, or comprises any one of SEQ Nos. 109-112. In some embodiments, the fusion protein comprises an SGSH sequence linked to an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity with respect to any one of SEQ NOs: 152 and 153, or comprising any one of SEQ NOs: 152 and 153. In some embodiments, the modified Fc polypeptide and / or the N-terminus of the Fc polypeptide is a part of the IgG1 hinge region ( chamberlain, It contains DKTHTCPPCP; Sequence No.: 113).

[0278] VIII. Measurement of Binding Dynamics, Affinity, Brain Concentration, and Brain Exposure

[0279] The fusion proteins and other compositions described herein may have a wide range of binding affinities. For example, in some embodiments, the protein is a blood-brain barrier (BBB) ​​receptor, chamberlain,It has an affinity for the transferrin receptor (TfR) in the range of 1 pM to 10 μM. In some embodiments, the affinity for TfR is in the range of 1 nM to 5 μM, or 10 nM to 1 μM. In some embodiments, the affinity for TfR is in the range of about 50 nM to about 250 nM.

[0280] In some embodiments, the affinity of the TfR-binding polypeptide can be measured in a monovalent format. In other embodiments, the affinity is in a bivalent format, chamberlain, It is measured as a dimer containing a polypeptide-Fab fusion protein.

[0281] BBB receptor, chamberlain, To analyze binding to TfR, methods for analyzing binding affinity, binding kinetics, and cross-linking activity are known in the art. These methods include, but are not limited to: solid-phase binding assays ( chamberlain, ELISA assay), immunoprecipitation, surface plasmon resonance ( chamberlain, Biacore™ (GE Healthcare, Piscataway, NJ)), epidemiological exclusion test( chamberlain, KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry ( chamberlain, Octet® ( ( , Inc., Menlo Park, CA), and Western blot analysis. In some embodiments, binding affinity and / or cross-linking activity are measured using ELISA. Methods for performing ELISA assays are known in the art and are also described in the Examples section below. In some embodiments, binding affinity, binding kinetics, and / or cross-linking activity are measured using surface plasmon resonance (SPR). In some embodiments, binding affinity, binding kinetics, and / or cross-linking activity are measured using kinetic exclusion assays. In some embodiments, binding affinity, binding kinetics, and / or cross-linking activity are measured using BioLayer interferometric assays.

[0282] Bonding affinity ( chamberlain, A non-limiting example measuring (for TfR) is described in Example 13 below, wherein Biacore ™ Affinity was measured by surface plasmon resonance (SPR) using an instrument. In this method, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody of interest is captured on a sensor chip, and serial dilutions of TfR are injected into the sensor chip at a specific flow rate (e.g., 30 μL / min) and temperature (e.g., room temperature). Samples are analyzed using specific coupling and dissociation times (e.g., 45 seconds and 180 seconds, respectively) and sensor chip regeneration. Steady-state affinity can be determined using software that establishes an equilibrium response to concentration after correction by subtracting the response measured from a control (e.g., using unrelated IgG at a similar density).

[0283] TfR-binding polypeptides, TfR-binding peptides, TfR-binding antibodies, or preparations engineered in brain and / or plasma ( chamberlain,The concentration of the above-mentioned engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody (linked to) can be measured, for example, using a human transferrin receptor (hTfR) knock-in mouse model. Using such a model, for example, maximum brain concentration (C max By measuring and / or comparing ) and / or brain exposure, chamberlain, C max Determines whether this will increase and / or brain exposure will be prolonged. Human peak TfR (TfR ms / hu The creation of a mouse knock-in model is described in Example 12 below. To generate a suitable model, a CRISPR / Cas9 system was used to create a murine Tfrc within the gene ( chamberlain, At this time In vivo Expression is under the control of an endogenous promoter) in humans Tfrc It is possible to generate mice expressing the apex domain. Specifically, Cas9, single guide RNAs and donor DNA ( chamberlain, Codon-optimized human apex domain coding sequence for expression in mice is into mouse embryos ( chamberlain, Pronuclear injection can be introduced. Then, the embryo can be transferred to a pseudo-pregnant woman. From the offspring of the female who received the embryo, the founder male will give birth to offspring with a wild-type female, producing F1 heterozygous mice. Homozygous mice can then be subsequently generated from the breeding of the F1 generation heterozygous mice.

[0284] The above-mentioned fabricated TfR-binding polypeptide, TfR-binding peptide, TfR-binding antibody, or preparation ( chamberlain,For the evaluation of brain and / or plasma concentrations or exposure to the associated engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody, said engineered TfR-binding polypeptide, TfR-binding peptide, TfR-binding antibody ( chamberlain, (linked to the above formulation) is the above mouse model ( chamberlain, TfR ms / hu It may be administered to ). After an appropriate time, the vascular system may be perfused with a suitable solution, and a plasma sample may be obtained from the mouse. After perfusion, the brain (or part thereof) may be extracted, homogenized, and lysed. Then, the concentration of said agent in the plasma and / or brain lysate may be determined using standard methods known to those skilled in the art. As a non-limiting example, the concentration may be measured using an ELISA-based assay such as that described in Example 3 below. Briefly, the concentration of the agent, the engineered TfR-binding polypeptide, the TfR-binding peptide, or the TfR-binding antibody (e.g., in the plasma or lysate) may be quantified using a sandwich ELISA. The capture antibody ( chamberlain, The anti-Fc capture antibody is a plate ( chamberlain, 384-well MaxiSorp™ plate) phase at a desired concentration ( chamberlain, Exposure to approximately 3 μg / mL) may occur. Block the above plate and ( chamberlain, Using 5% BSA), then diluted ( chamberlain, It was incubated with plasma (1:1,000 or 1:10,000). Then, the detection antibody was at the desired concentration ( chamberlain, It was added at approximately 0.5 μg / mL), and then a secondary antibody, such as an anti-goat-HRP antibody, was added. Subsequently, the plate was developed and ( chamberlain, Using a TMB substrate), stop ( chamberlain, With sulfuric acid), plate reader ( chamberlain, Appropriate wavelength (in BioTek plate reader) chamberlain, It has an absorbance measured at 450 nm. Appropriate ( chamberlain, A standard curve can be generated by fitting using algorithms such as a series of 4-fold dilutions and 4-parameter logistic regression analysis.

[0285] A standard curve can be generated by administering various dosages to a knock-in mouse model. Differently engineered TfR-binding polypeptides, TfR-binding peptides, or TfR-binding antibodies ( chamberlain, Preparations linked to (having different TfR affinities), or reference polypeptides or proteins ( chamberlain, By administering a preparation linked to (having a weaker affinity for TfR than the polypeptide or protein of interest), brain exposure to said preparation and / or C of said preparation in the brain max It is possible to compare the effects of engineered TfR-binding polypeptides, TfR-binding peptides, or TfR-binding antibodies in terms of value.

[0286] IX. ERT enzymes linked to FC polypeptides

[0287] In some embodiments, the fusion protein described herein comprises the two Fc polypeptides described herein, and one or both of the Fc polypeptides may further comprise a partial or full hinge region. The hinge region may be derived from any immunoglobulin subclass or isotype. For the purpose of description, the immunoglobulin hinge is an IgG hinge region, e.g., an IgG1 hinge region, chamberlain, Human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (Sequence No.: 95) or a part thereof ( chamberlain, DKTHTCPPCP; Sequence No.: 113). In some embodiments, the hinge region is located at the N-terminal region of the Fc polypeptide.

[0288] In some embodiments, the Fc polypeptide is a linker to the ERT enzyme, chamberlain, It is linked by a peptide linker. In some embodiments, the Fc polypeptide is linked to the ERT enzyme by a peptide bond or by a peptide linker, chamberlain , this becomes a fusion polypeptide. The peptide linker may be configured to allow rotation of the ERT enzyme toward the bound Fc polypeptide; and / or to be resistant to degradation by protease. The peptide linker may contain natural amino acids, non-natural amino acids, or a combination thereof. In some embodiments, the peptide linker is a soft linker, chamberlain, It may be a soft linker containing amino acids such as Gly, Asn, Ser, Thr, Ala, and similar ones. Such a linker is designed using known parameters and may be of any length and may contain any number of repetitions of units of any length (e.g., repeat units of Gly and Ser residues). For example, the linker may have repeats, such as two, three, four, five, or more Gly4-Ser (SEQ No.: 239) repeats, or a single Gly4-Ser (SEQ No.: 239). In some embodiments, the peptide linker has a protease cleavage site, chamberlain, It may contain a site that can be cleaved by enzymes present in the central nervous system.

[0289] In some embodiments, the ERT enzyme is at the N-terminus of the Fc polypeptide, chamberlain, It is linked to a Gly4-Ser linker (SEQ No.: 239) or a (Gly4-Ser)2 linker (SEQ No.: 240). In some embodiments, the Fc polypeptide may include a hinge sequence or a partial hinge sequence linked to the linker at the N-terminus, or directly linked to the ERT enzyme.

[0290] In some embodiments, the ERT enzyme is at the C-terminus of the Fc polypeptide, chamberlain, It is linked by a Gly4-Ser linker (SEQ No.: 239) or a (Gly4-Ser)2 linker (SEQ No.: 240). In some embodiments, the C-terminus of the Fc polypeptide is directly linked to the ERT enzyme.

[0291] In some embodiments, the ERT enzyme is linked to the Fc polypeptide by a chemical crosslinking agent. Such conjugates can be produced using known chemical crosslinking reagents and protocols. For example, there are numerous chemical crosslinking agents known to those skilled in the art that are useful for crosslinking the agent of interest with the polypeptide. For example, the crosslinking agent is a heterofunctional crosslinking agent, which can be used to link molecules stepwise. 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 homoprotein polymerization. Various heterogeneous functional crosslinking agents are known in the art, including N-hydroxysuccinide (NHS) or its water-soluble analogs N-hydroxysulfosuccinide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinide ester (MBS); N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propyrronate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propyrronate]hexanomate (LC-SPDP). These crosslinkers having an N-hydroxysuccimide moiety can be obtained as N-hydroxysulfosuccinimide analogs, which generally have greater water solubility. Additionally, crosslinkers having disulfide bridges within the linking chain In vivoTo reduce the amount of linker cleavage, alkyl derivatives can be synthesized instead. In addition to heterofunctional crosslinkers, there are numerous other crosslinkers, including homofunctional and photoreactive crosslinkers. Disuccinimidyl souvcrate (DSS), bismaleimidohexane (BMH), and dimethylpimelimidate (DMP) are useful homofunctional crosslinkers, and bis-[B-(4-azidosaliclamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanomate (SANPAH) are useful examples of photoreactive crosslinkers.

[0292] X. Evaluation of Protein Activity

[0293] ERT enzyme, for example, chamberlain, The activity of the fusion proteins described herein, including IDS, SGSH, ASM, or GBA, can be evaluated using various assays, including assays that measure in vitro activity using artificial substrates, such as those described in the Examples section. In a test tube An example protocol for measuring IDS activity is provided in Example 2. In a test tube An example protocol for measuring ASM activity is provided in Example 5. In a test tube An exemplary protocol for measuring SGSH activity is provided in Examples 7 and 8.

[0294] In some aspects, IDS activity is measured in samples, such as cell samples, tissue samples, or fluid samples ( chamberlain, It can be evaluated by testing CSF or urine. The amounts of heparan and dermatan sulfates are measured by cleaving the GAGs present in the sample with heparinase and chondroitinase. The resulting disaccharides are then subjected to mass spectrometry ( chamberlain,It can be verified by LC-MS / MS. Samples with high accumulation levels of heparan and dermatan sulfate will have increased amounts of heparan and dermatan sulfate-derived disaccharides. Therefore, the level of disaccharides is inversely proportional to IDS enzyme activity.

[0295] Mass spectrometry ( chamberlain, LC-MS / MS assays can be performed on any samples, including cell samples, tissue samples, and fluid samples in which GAGs accumulate. These samples may be evaluated to monitor the activity of the IDS-containing proteins described herein, for example, proteins administered to cells in vitro or, in the embodiments, administered in vivo to a subject. The subject is an animal, such as a rodent, chamberlain, It may be a mouse or a non-human primate. In some embodiments, the subject is a human patient, such as a patient with Hunter syndrome being treated with IDS therapy, and the IDS activity in said patient is monitored using the assay. In some embodiments, said human patient is being treated with the fusion protein described herein.

[0296] In the case of cell samples, such as cell or tissue samples, the assay involves destroying cells and rupturing open microvesicles. By destroying cells and rupturing open microvesicles using freeze-thaw and / or sonication, an extract containing GAGs (e.g., cell extract) can be obtained. Then, the GAGs are heparinase ( chamberlain, It is treated with any of the (described herein) and chondroitinase, which destroy heparan sulfate and dermatan sulfate GAGs. After cleavage, a supernatant containing GAG disaccharides is obtained, and said disaccharide products are subjected to mass spectrometry ( chamberlain, It is analyzed by LC-MS / MS. A protocol for explanation is provided in Example 2.

[0297] In some embodiments, cell samples to be tested for IDS activity are washed and frozen. The cell pellet is sonicated in a disaccharide digestion buffer. Then, a desired amount of total protein from the sonicated sample is added to a digestion buffer containing heparinase I, heparinase II, heparinase III, and chondroitinase B, wherein the latter enzyme is specific to dermatan sulfate. After digestion ( chamberlain, Approximately 3 hours, at 30°C), the enzyme is inactivated by EDTA and heating. Then, the sample is chamberlain, It is centrifuged at 16,000 x G, and the supernatant is transferred to a centrifugal filter and centrifuged at approximately 14,000 x G. Then, the disaccharide is resuspended in assay buffer:acetonitrile (1:1 v / v ratio), and then, chamberlain, As described in Example 2, it is analyzed by liquid chromatography and electron spray mass spectrometry. GAG-derived disaccharide products can be identified based on retention time compared to commercially available reference standards. For illustrative purposes, heparan sulfate-derived disaccharides are D0S0 and D2S0 (Lawrence et al., Nat. Methods, Includes nomenclature according to 5:291-292 (2008).

[0298] In other aspects, SGSH activity is analyzed by testing the amount of heparan sulfate glycosaminoglycans (GAGs)—whose accumulation is a result of SGSH deficiency—in a sample, e.g., a cell sample or a tissue sample. The amounts of heparan and dermatan sulfates are measured by cleaving the GAGs present in the sample with a heparinase (e.g., any of those described herein). The resulting disaccharides are then analyzed by mass spectrometry ( chamberlain,It can be verified by LC-MS / MS. Samples with high heparan sulfate accumulation levels will have increased amounts of heparan sulfate-derived disaccharides. Therefore, the level of disaccharides is inversely proportional to SGSH enzyme activity.

[0299] Mass spectrometry ( chamberlain, LC-MS / MS assays can be performed on any samples, including cell samples, tissue samples, and fluid samples in which GAGs accumulate. These samples may be evaluated to monitor the activity of the SGSH-containing proteins described herein, for example, proteins administered to cells in vitro or, in the embodiments, administered in vivo to subjects. The subjects are animals, such as rodents, chamberlain, It may be a mouse or a non-human primate. In some embodiments, the subject is a human patient, such as a patient with Sanfilippo syndrome being treated with SGSH therapy, and the SGSH activity in said patient is monitored using the assay. In some embodiments, said human patient is being treated with the fusion protein described herein.

[0300] In the case of cell samples, such as cell or tissue samples, the assay involves destroying cells and / or rupturing open microvesicles. By destroying cells and rupturing open microvesicles using freeze-thaw and / or sonication, an extract containing GAGs (e.g., cell extract) can be obtained. Then, the GAGs are heparinase ( chamberlain, They are treated by any of the methods described herein, which destroy heparan sulfate GAGs. After cutting, a supernatant containing GAG disaccharides is obtained, and said disaccharide product is subjected to mass spectrometry ( chamberlain, It is analyzed by LC-MS / MS. A protocol for explanation is provided in Example 7.

[0301] In some embodiments, cell samples to be tested for SGSH activity are washed and frozen. The cell pellet is sonicated in a disaccharide digestion buffer. Then, a desired amount of total protein from the sonicated sample is added to a digestion buffer containing heparinase I, heparinase II, and / or heparinase III. After digestion ( chamberlain, Approximately 3 hours, at 30°C), the enzyme is inactivated by EDTA and heating. Then, the sample is chamberlain, It is centrifuged at 16,000 x G, and the supernatant is transferred to a centrifugal filter and centrifuged at approximately 14,000 x G. Then, the disaccharide is resuspended in assay buffer:acetonitrile (1:1 v / v ratio), and then, chamberlain, As described in Example 7, it is analyzed by liquid chromatography and electron spray mass spectrometry. GAG-derived disaccharide products can be identified based on retention time compared to commercially available reference standards. For illustrative purposes, heparan sulfate-derived disaccharides are D0S0 and D2S0 (Lawrence et al., Nat. Methods, Includes nomenclature according to 5:291-292 (2008).

[0302] In some embodiments, a tissue sample is evaluated. The tissue sample is evaluated according to the test described above—however, multiple free-thaw cycles, chamberlain, It is evaluated using 2, 3, 4, 5, or more cycles without, typically included before the sonication step to check whether microvesicles have been destroyed and opened.

[0303] Samples that may be evaluated by the assay described herein include brain, liver, kidney, lung, spleen, plasma, serum, cerebrospinal fluid (CSF), and urine. In some embodiments, the enzyme-Fc fusion protein described herein ( chamberlain, CSF samples from patients who received IDS-Fc or SGSH-Fc fusion proteins can be evaluated.

[0304] XI. Nucleic Acids, Vectors, and Host Cells

[0305] The polypeptide chain contained in the fusion protein as described herein is typically prepared using a recombinant method. Accordingly, in some aspects, this specification provides an isolated nucleic acid comprising a nucleic acid sequence encoding any polypeptide chain including the Fc polypeptide as described herein, and a host cell into which the polypeptide-encoding nucleic acid is introduced and used to replicate and / or express them. In some embodiments, the host cell is a eukaryotic cell, chamberlain, It is a human cell.

[0306] In another aspect, a polynucleotide comprising a nucleotide sequence encoding the polypeptide chain described herein is provided. The polynucleotide is single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA. In certain embodiments, the polynucleotide is cDNA. In some embodiments, the polynucleotide is RNA.

[0307] In some embodiments, the polynucleotide is contained within a nucleic acid construct. In some embodiments, the construct is a replicable vector. In some embodiments, the vector is selected from plasmids, viral vectors, phagemids, yeast chromosome vectors, and non-episomal mammalian vectors.

[0308] In some embodiments, the polynucleotide is operably linked to one or more regulatory nucleotide sequences within the expression construct. In one series of embodiments, the nucleic acid expression constructs are adapted for use as a surface expression library. In some embodiments, the library is adapted for surface expression in yeast. In some embodiments, the library is adapted for surface expression in phages. In another series of embodiments, the nucleic acid expression constructs are adapted for the expression of the polypeptide in a system that allows for milligram or gram-sized polypeptide isolation. In some embodiments, the system is a mammalian cell expression system. In some embodiments, the system is a yeast cell expression system.

[0309] Expression vehicles for the generation of recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-induced plasmid, pEMBL-induced plasmid, pEX-induced plasmid, pBTac-induced plasmid, and pUC-induced plasmid for expression in prokaryotic cells, such as E. coli. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-induced vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Alternatively, viruses, such as bovine papillomavirus (BPV-1), or derivatives of Epstein-Barr viruses (pHEBo, pREP-induced, and p205), may be used for the transient expression of polypeptides in eukaryotic cells. In some embodiments, in some cases, it may be desirable to express the recombinant polypeptide by using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-induced vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-induced vectors (e.g., pAcUW1), and pBlueBac-induced vectors. Additional expression systems include adenoviruses, adeno-associated viruses, and other viral expression systems.

[0310] The vector can be transformed into any suitable host cell. In some embodiments, the host cell, chamberlain,Bacteria or yeast cells can be customized for use as surface expression libraries. In some cells, the vector is expressed in the host cell to express a relatively large amount of polypeptide. These host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, these cells are mammalian cells, such as Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, NS0 cells, Y0 cells, HEK293 cells, COS cells, Vero cells, or HeLa cells.

[0311] Host cells transfected with an expression vector encoding one or more of the Fc polypeptide chains described herein may be cultured under appropriate conditions that allow the expression of one or more polypeptides to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide may be retained in the cytoplasm or membrane fraction, and the cells may be collected, lysed, and the proteins isolated using a desired method.

[0312] XII. Treatment Method

[0313] A fusion protein or preparation linked to a engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody described herein ( chamberlain,LSD can be treated therapeutically using therapeutic agents. In some embodiments, patients with Hunter syndrome are treated with a fusion protein or agent linked to a engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody, including IDS. In some embodiments, patients with Sanfilippo syndrome are treated with a fusion protein or agent linked to a engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody, including SGSH. In some embodiments, patients with Niemann-Pick disease are treated with a fusion protein or agent linked to a engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody, including ASM. In some embodiments, patients with Gaucher disease or Parkinson disease are treated with a fusion protein or agent linked to a engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody, including GBA.

[0314] ERT enzyme, chamberlain,The fusion proteins described herein, including IDS, SGSH, ASM, or GBA, are administered to a subject at an effective dose or dosage for therapeutic purposes. Dosages for description include and may be used daily doses in the 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 within the range of about 500 U / mg to about 50,000 U / mg. However, the dosage may vary depending on several factors, including the selected route of administration, the formulation of the composition, patient response, severity of the condition, the subject's body 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 provided to the patient and subsequently increased to an effective dose acceptable to the patient. The determination of the effective dose is within the capabilities of those skilled in the art.

[0315] In various embodiments, the fusion protein described herein is administered parenterally. In some embodiments, the protein is administered intravenously. Intravenous administration may be made, for example, over a period of about 10 to about 30 minutes, or by an infusion over a period of at least 1 hour, 2 hours, or 3 hours. In some embodiments, the protein is administered as an intravenous bolus. A combination of infusion and bolus administration may also be used.

[0316] In some parenteral embodiments, a fusion protein or preparation linked to a engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody ( chamberlain, The therapeutic agent is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, the protein or agent linked to the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is administered intradermally or intramuscularly. In some embodiments, the protein or agent linked to the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is administered intravertebrally, e.g., by epidural administration, or into the brain.

[0317] In other embodiments, a fusion protein or preparation linked to a fabricated TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody ( chamberlain, The therapeutic agent may be administered by oral administration, pulmonary administration, intranasal administration, ocular administration, or topical administration. Pulmonary administration may also be used, for example, by using an inhaler or nebulizer, and by formulations using aerosolizing agents.

[0318] XIII. Methods for Protein Replacement

[0319] In other aspects, preparations that cross the mammalian blood-brain barrier (BBB) ​​( chamberlain,A method for delivering a agent useful for treating lysosomal storage disorders (LSD) is provided herein. 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 carries the linked agent across the BBB. In some embodiments, the maximum concentration of the agent (C) in the mammalian brain max ) is improved ( chamberlain, (Increases).

[0320] In other aspects, a method for treating LSD is provided herein. In some embodiments, the method comprises administering to a mammal a polypeptide or protein that binds to (e.g., specifically binds) TfR with an affinity of 50 nM to about 250 nM. In some embodiments, said polypeptide or protein is linked to an agent for treating LSD, and said agent is then exposed to the brain of said mammal.

[0321] In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nM (e.g., specifically bind). In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 100 nM to about 200 nM or about 110 nM to about 150 nM.

[0322] In some specific embodiments, the polypeptide or protein ( chamberlain,(associated with the above agent) binds to TfR with a weaker affinity (e.g., binds specifically) compared to the above agent associated with a reference polypeptide or protein, C of the above agent in the brain max Improves (e.g., increases)

[0323] In some specific examples, C of the above preparation in the brain max Compared to the above formulation linked to a reference polypeptide or protein (e.g., binding to TfR with weaker affinity), it is improved (e.g., increased) by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.2-fold, 2.4-fold, 2.6-fold, 2.8-fold, 3-fold, 4-fold, 5-fold, or more.

[0324] In some embodiments, mammalian brains at therapeutically effective concentrations for a shorter period compared to preparations linked to reference polypeptides or proteins ( chamberlain, Exposure to the above agent at a concentration sufficient to treat one or more signs or symptoms of LSD. In some embodiments, the duration of brain exposure is shortened by at least about 5%, 10%, 25%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, or 98%.

[0325] In some specific examples, brain exposure is brain exposure as a function of time ( chamberlain, The concentration of the agent in the brain) is plotted, and the area under the curve (AUC) is calculated and quantified. A reduced AUC may indicate reduced or shortened brain exposure. In some embodiments, the agent ( chamberlain, The brain exposure time to (therapeutically effective concentrations) is shortened.

[0326] In some embodiments, the reference polypeptide or protein binds to TfR with an affinity of about 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, or 600 nM or a weaker affinity than this ( chamberlain, (It binds specifically). In some embodiments, the reference polypeptide or protein binds to the TfR with an affinity of about 600 nM or weaker.

[0327] In some embodiments, the mammal is a primate ( chamberlain, It is a human. In some embodiments, the human is a patient requiring treatment for LSD. In some embodiments, the patient has one or more signs or symptoms of LSD.

[0328] In some embodiments, the polypeptide or protein binds to a primate TfR (e.g., specifically). In some embodiments, the primate TfR is a human TfR. In some embodiments, the polypeptide or protein binds to the apex domain of the TfR.

[0329] In some specific examples, the above formulation ( chamberlain, The therapeutic agent formulation is linked to a engineered TfR-binding polypeptide. In some embodiments, the engineered TfR-binding polypeptide comprises a CH3 or CH2 domain having a modification that allows the polypeptide to specifically bind to TfR. Suitable non-limiting examples of engineered TfR-binding polypeptides are described herein. In some embodiments, the formulation is linked to an engineered TfR-binding polypeptide described in Table 4 or Table 5. In some embodiments, the formulation is linked to an engineered TfR-binding polypeptide selected from the group consisting of CH3C.35.20.2, CH3C.35.23.2, CH3C.35.23.5, CH3C.35.21.17, and CH3C.35.21.17.2.

[0330] In some specific examples, the above formulation ( chamberlain, The therapeutic agent) is linked to a TfR-binding peptide. In some embodiments, the TfR-binding peptide is a short peptide having a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Suitable peptides ( chamberlain, Methods for generating, screening, and identifying (which bind to TfR with a desirable range of affinity) are known in the art. For example, suitable peptides can be identified using a phage display strategy in which alternating rounds of negative and positive screening are used. This strategy chamberlain, Lee et al., Eur. J. Biochem., 268:2004-2012 (2001) is described in its entirety for all purposes and is incorporated herein by reference.

[0331] In some specific examples, the above formulation ( chamberlain, The therapeutic agent) is linked to a TfR-binding antibody. Non-limiting examples of suitable TfR-binding antibodies include Thom et al., Mol. Pharm OX26 anti-TfR antibody (described in ., 15(4):1420-1431 (2018) chamberlain, (having affinities of approximately 76 nM, 108 nM, and 174 nM). In some embodiments, the formulation is linked to a protein comprising an antibody variable region that specifically binds to TfR. In some cases, the protein comprises Fab or scFv.

[0332] In some specific examples, the above formulation ( chamberlain, Therapeutic preparations) are proteins ( chamberlain, It is an enzyme). In some embodiments, the preparation is a protein replacement therapeutic. In some embodiments, the preparation is a mammalian cell or tissue ( chamberlain,It is a protein or enzyme that is deficient (e.g., underexpressed or absent) in nerve cells or tissues. In some embodiments, the preparation is a normal healthy cell or tissue of a mammal ( chamberlain, It is endogenous or expressed in nerve cells or tissues, but is deficient in mammals receiving LSD treatment ( chamberlain, It is a protein or enzyme (in the corresponding cell or tissue mentioned above).

[0333] In some embodiments, the protein replacement therapeutic agent is an enzyme. Any number of preparations ( chamberlain, Protein replacement therapies, such as enzymes, are polypeptides or proteins ( chamberlain, It may be linked to (binding to TfR). In some embodiments, the agent is an enzyme, and is an enzyme that reduces the accumulation of toxic metabolites in the brain of mammals suffering from LSD when linked to said polypeptide or protein compared to when said enzyme is linked to said polypeptide or protein. In some embodiments, said enzyme is iduronate 2-sulfatase (IDS), and said LSD is Hunter syndrome. In some cases, said toxic metabolites include heparin sulfate-derived disaccharides and / or dermatan sulfate-derived disaccharides. In some embodiments, said enzyme is N-sulfoglucosamine sulfohydrolase (SGSH), and said LSD is Sanfilippo syndrome. In some embodiments, said enzyme is acid sphingomyelinase (ASM), and said LSD is Niemann-Pick disease. In some embodiments, the enzyme is β-glucocerebrosidase (GBA), and the LSd is Gaucher disease.

[0334] In some specific examples, the above formulation ( chamberlain,The therapeutic agent formulation comprises an antibody variable region. In some embodiments, the formulation comprises an antibody fragment. In some embodiments, the formulation comprises Fab or scFv. In some embodiments, the formulation does not comprise an antibody variable region. In some cases, the formulation does not comprise anti-beta-secretase 1 (BACE1) Fab.

[0335] Additional specific examples and linkers

[0336] polypeptide ( chamberlain, The modified CH3 or CH2 domain polypeptide further described herein may be connected to another domain of the Fc region. In some embodiments, the modified CH3 domain polypeptide is connected to the CH2 domain (natural CH2 domain or variant CH2 domain), typically at the C-terminal end of the CH2 domain. In some embodiments, the modified CH2 domain polypeptide is connected to the CH3 domain (natural CH3 domain or variant CH3 domain), typically at the N-terminal end of the CH3 domain. In some embodiments, the polypeptide comprising the modified CH2 domain connected to the CH3 domain or the polypeptide comprising the modified CH3 domain connected to the CH2 domain further comprises a partial or complete hinge region of the antibody, and thus the modified CH3 domain polypeptide or the modified CH2 domain polypeptide is formatted to be part of the Fc region having a partial or complete hinge region. The above hinge region may be derived from any immunoglobulin subclass or isotype. For the purpose of explanation, the immunoglobulin hinge is an IgG hinge region, e.g., an IgG1 hinge region, chamberlain, The human IgG1 hinge amino acid sequence is EPKSCDKTHTCPPCP (sequence number: 95).

[0337] In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody is a peptide or protein useful for protein purification, chamberlain, Polyhistidine, epitope tags, e.g., FLAG, c-Myc, hemagglutinin tags and similar ones, are fused to glutathione S-transferase (GST), thioredoxin, protein A, protein G, or maltose-binding protein (MBP). In some cases, the peptide or protein fused with the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody may contain a protease cleavage site, such as a cleavage site for Factor Xa or thrombin.

[0338] In the methods of this specification, the formulation ( chamberlain, Therapeutic preparations) are polypeptides or proteins (chamberlain, It is linked to a modified TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody. The linker may be any linker suitable for linking the formulation to the polypeptide or protein. In some embodiments, the linkage is enzymatically cleavable. In certain embodiments, the linkage is cleavable by an enzyme present in the central nervous system.

[0339] In some embodiments, the linker is a peptide linker. The peptide linker is the preparation ( chamberlain, The therapeutic agent) and the polypeptide or protein may be configured to be rotatable relative to each other; and / or resistant to digestion by protease. In some embodiments, the linker is a soft linker, chamberlain, It may be a soft linker containing amino acids such as Gly, Asn, Ser, Thr, Ala, and similar ones. Such a linker is designed using known parameters. For example, the linker may have repeats, such as Gly-Ser repeats.

[0340] In various embodiments, the above formulation ( chamberlain, The therapeutic agent preparation) the above polypeptide or protein ( chamberlain, Linking to a engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody can be achieved using well-known crosslinking agents and protocols. For example, there are numerous chemical crosslinking agents known to those skilled in the art that are useful for crosslinking the agent of interest with the polypeptide or monoprotein. For example, the crosslinking agent is a heterofunctional crosslinking agent, which can be used to link molecules stepwise. Heterofunctional crosslinking agents provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrence of unwanted side reactions, such as homoprotein polymerization.

[0341] Unless the above preparation interferes with the binding of the polypeptide or protein to the transferrin receptor, the above preparation ( chamberlain, The therapeutic agent preparation) is the N-terminal or C-terminal region of the polypeptide or protein, or the polypeptide or protein ( chamberlain, It can be linked to any region of a engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody.

[0342] XIV. Pharmaceutical Composition and Kit

[0343] In other aspects, pharmaceutical compositions and kits comprising the fusion protein described herein are provided.

[0344] Pharmaceutical composition

[0345] Instructions for preparing formulations for use in the present disclosure can be found in any number of handbooks on pharmaceutical preparations and formulations known to those skilled in the art.

[0346] In some embodiments, the pharmaceutical composition comprises the fusion protein described herein and further comprises one or more pharmaceutically acceptable carriers and / or excipients. The pharmaceutically acceptable carrier comprises any solvent, dispersion medium, or coating that is physiologically compatible and does not interfere with or does not interfere with the activity of the active agent.

[0347] In some embodiments, the carrier is suitable for intravenous, intrathecal, intraocular, intraventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. A pharmaceutically acceptable carrier may contain, for example, one or more physiologically acceptable compounds that act to stabilize the composition or increase or decrease the absorption of the polypeptide. Such physiologically acceptable compounds 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 removal or hydrolysis of the active agent; excipients; or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and formulations thereof are also available in the art.

[0348] The pharmaceutical compositions described herein may be prepared by, for example, conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, capturing, or freeze-drying process means. The following methods and excipients are exemplary.

[0349] For oral administration, the fusion protein as described herein may be formulated by combining it with a pharmaceutically acceptable carrier known in the art. Such a carrier enables the fusion protein to be formulated into tablets, pills, coated tablets, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by the patient to be treated. Oral pharmaceutical preparations may be obtained by mixing the fusion protein with a solid excipient, grinding the optionally produced mixture, processing the granulated mixture, adding a suitable adjuvant if necessary, and then obtaining a tablet or coated tablet core. Suitable excipients include, for example, fillers such as sugars including lactose, sucrose, mannitol, or sorbitol; Cellulose preparations include, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragatan, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone. If desired, a disintegrant, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added.

[0350] As disclosed above, fusion proteins as described herein may be formulated for parenteral administration by injection, e.g., bolus injection or serial infusion. For injectable use, the fusion proteins may be formulated into a formulation by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol, and, if necessary, using conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the fusion proteins may be formulated into an aqueous solution such as a physiologically compatible buffer, non-limiting examples of which include Hanks solution, Ringer solution, and physiological saline buffer. Injectable formulations may be provided in unit dosage forms with added preservatives, e.g., ampoules or multi-dose containers. The above composition may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, stabilizers, and / or dispersing agents.

[0351] In some embodiments, the fusion protein as described herein is prepared for delivery in sustained-release, controlled-release, extended-release, timed-release, or delayed-release formulations, for example, in a semipermeable matrix of a solid hydrophobic polymer containing an activator. Various types of sustained-release materials have been established and are well known to those skilled in the art. Extended-release formulations include film-coated tablets, multiply particulate or pellet systems, matrix technologies using hydrophilic or lipophilic materials, and wax-based tablets with pore-forming excipients. Generally, sustained-release formulations are naturally occurring or synthetic polymers, for example, polymeric vinylpyrrolidone, e.g., polyvinylpyrrolidone; carboxyvinyl hydrophilic polymers; It can be prepared using hydrophobic and / or hydrophilic hydrocolloids, e.g., methylcellulose, ethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose; and carboxypolymethylene.

[0352] Typically, pharmaceutical compositions intended for in vivo administration are sterile. Sterilization can be achieved by methods known in the art, for example, heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0353] The dosage and desired drug concentration of the pharmaceutical compositions described herein may vary depending on the specific intended use. Suitable dosages are also described in Section XII above.

[0354] Kit

[0355] In some specific examples, LSD, chamberlain,A kit containing a fusion protein useful for treating Hunter syndrome, Sanfilippo syndrome A, Niemann-Pick disease, Gaucher disease, or Parkinson's disease is provided herein.

[0356] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit further comprises one or more additional therapeutic agents used for treating neurological symptoms of LSD, including the fusion protein described herein. In some embodiments, the kit includes instructions for carrying out the method described herein ( chamberlain, Instructional materials containing the protocol chamberlain, It further includes instructions for using a kit for administering a fusion protein containing an ERT enzyme across the blood-brain barrier. Instructional materials are generally written or printed, but are not limited to these. Any medium capable of storing instructions and communicating them to the end user is also considered herein. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROMs), and similar materials. Such media may include an address to an internet site providing such educational materials.

[0357] XV. Examples

[0358] This specification will be explained in more detail by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Those skilled in the art will readily recognize various non-critical parameters that may be changed or modified to produce essentially the same results. While efforts have been made to ensure accuracy regarding the numbers used (e.g., quantity, temperature, etc.), some experimental error and deviation may, of course, exist. Unless otherwise indicated, the practice of this disclosure will use protein chemistry, biochemistry, recombinant DNA techniques, and pharmacological methods that are conventional to those skilled in the art. These techniques are sufficiently described in the literature. Furthermore, it will be obvious to those skilled in the art that the methods applied to a specific library may also be applied to other libraries described in this specification.

[0359] Example 1. Construction of a fusion protein containing iduronate 2-sulfatase (IDS)

[0360] Planning and Cloning

[0361] The IDS-Fc fusion protein was designed to contain: (i) a fusion polypeptide, where, during maturation, a human IDS enzyme is fused to a human IgG1 fragment containing an Fc region ("IDS-Fc fusion polypeptide"); and (ii) a modified human IgG1 fragment containing an intra-Fc region mutation that is located within the Fc region and confers transferrin receptor (TfR) binding ("modified Fc polypeptide"). Specifically, IDS-Fc fusion polypeptides are produced in which the IDS fragments are fused to the N- or C-terminus of the human IgG1 Fc region. In some cases, a linker was placed between the IDS and IgG1 fragments to alleviate steric hindrance between the two fragments. In all constructs, the kappa chain V-III signal peptide, amino acids 1-20 (UniProtKB ID -P01661), was inserted upstream of the fusion protein to facilitate secretion, and the IDS was truncated to consist of amino acids S26-P550 (UniProtKB ID -P22304). The fragment of the human IgG1 Fc region used corresponds to amino acids D104-K330 of UniProtKB ID P01857 (according to position 221-447-EU numbering—containing a 10-amino acid hinge (position 221-230)). In some embodiments, a second Fc polypeptide derived from human IgG1 residue D104-K330 but without IDS fusion was co-transfected with an IDS-Fc fusion polypeptide to produce a heterodimeric fusion protein having one IDS enzyme ("monozyme"). In some constructs, the IgG1 fragments contained additional mutations to promote heterodimerization of the two Fc regions. A control IDS-Fc fusion protein lacking a TfR binding-conferring mutation was designed and constructed similarly, differing in that these proteins lack the TfR binding-conferring mutation.As an additional control, we prepared an IDS (amino acid S26-P550) with a C-terminal hexahistidine tag (sequence number: 241) to facilitate detection and purification.

[0362] The TfR-binding IDS-Fc fusion proteins used in the examples are dimers formed by an IDS-Fc fusion polypeptide and a modified Fc polypeptide that binds to TfR. In the case of a dimer in which the IDS enzyme is linked to the N-terminus of the Fc region, the IDS-Fc fusion polypeptide may have any one of the sequences SEQ NOs: 115, 231, and 235. In these sequences, the IDS sequence is indicated by an underline and contains cysteine ​​modified to formylglycine at position 59 (double underline). The IDS is linked to the Fc polypeptide by a GGGGS linker (SEQ NO: 239). A portion of the IgG1 hinge region (DKTHTCPPCP; SEQ NO: 113) is included at the N-terminus of the Fc polypeptide. The CH2 domain sequence starts at position 541 of sequence numbers 115, 231, and 235.

[0363] The IDS-Fc fusion protein ETV:IDS 35.21 used in the examples is a dimer formed by an IDS-Fc fusion polypeptide having any one of the sequences of SEQ No. 115, 231, and 235, and an Fc polypeptide having the sequence of SEQ No. 116 and modified to bind to TfR. The first 10 amino acids are part of the IgG1 hinge region. The CH2 domain sequence starts at position 11 of SEQ No. 116.

[0364] 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 any one of the sequences of SEQ Nos. 115, 231, and 235, and an Fc polypeptide having the sequence of SEQ No. 228 and modified to bind to TfR. The first 10 amino acids are part of the IgG1 hinge region. The CH2 domain sequence starts at position 11 of SEQ No. 228.

[0365] The IDS-Fc fusion protein ETV:IDS 35.23.2 used in the examples is a dimer formed by an IDS-Fc fusion ET polypeptide having any one of the sequences of SEQ No. 115, 231, and 235, and an Fc polypeptide modified to bind to TfR having the sequence of SEQ No. 229. The first 10 amino acids are part of the IgG1 hinge region. The CH2 domain sequence starts at position 11 of SEQ No. 229.

[0366] 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 any one of the sequences SEQ Nos. 115, 231, and 235, and an Fc polypeptide modified to bind to TfR having the sequence of SEQ No. 151. The N-terminus of the modified Fc polypeptide is a part of the IgG1 hinge region ( chamberlain, It may contain sequence number: 113).

[0367] Recombinant Protein Expression and Purification

[0368] To express the recombinant IDS enzyme fused to the Fc region, ExpiCHO cells (Thermo Fisher Scientific) were transfected with the associated DNA construct using the Expifectamine™ CHO Transfection Kit according to the manufacturer's instructions. Cells were grown in ExpiCHO™ expression medium in an orbital shaker (Infors HT Multitron) at 37°C, 6% CO2, and 120 rpm. In summary, 6 x 10 logarithmic-growing ExpiCHO™ cells were transfected with 0.8 μg of DNA plasmid per 1 mL of culture volume. 6 Transfection was performed at a cell / ml density. After transfection, the cells were returned to 37°C, and the transfected cultures were supplemented with feed as directed 18–22 hours after transfection. The supernatant of the transfected cell culture was collected 120 hours after transfection by centrifugation at 3,500 rpm for 20 minutes. The clarified supernatant was filtered (0.22 μM membrane) and stored at 4°C. Expression of the epitope-tagged IDS enzyme (used as a control) was performed with slight modifications to the description above. Briefly, the IDS enzyme containing the C-terminal hexahistidine tag (Sequence No. 241) was expressed in ExpiCHO cells.

[0369] IDS-Fc fusion proteins with (or without) a modified Fc region 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 an Akta Pure System). Subsequently, the column was washed with PBS by >20 column volumes (CVs). The bound proteins were eluted using 100 mM citrate / NaOH buffer pH 3.0 containing 150 mM NaCl. Immediately after elution, the fraction was neutralized using 1 M arginine-670 mM succinate buffer pH 5.0 (dilution ratio of 1:5). The homogeneity of the IDS-Fc fusion proteins in the eluted fractions was evaluated by reduced and non-reduced SDS-PAGE.

[0370] To purify the hexahistadine-tagged (SEQ No.: 241) IDS enzyme, the transfected supernatant was thoroughly dialyzed overnight in 15 L of 20 mM HEPES pH 7.4 containing 100 mM NaCl. The dialyzed supernatant was coupled to a HisTrap column (GE Healthcare Life Sciences using an Akta Pure System). After coupling, the column was washed with 20 CV PBS. The coupled protein was eluted using PBS containing 500 mM imidazole. The homogeneity of the IDS enzyme in the eluted fraction was evaluated 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 Q Sepharose High Performance (GE Healthcare). After coupling, the column was washed with 10 CV 50 mM Tris pH 7.5. The bound protein was eluted using a linear gradient of 50 mM Tris pH 7.5 and 0.5 M NaCl and collected in the 1 CV fraction. Fraction purity was evaluated by non-reducing SDS-PAGE. As shown in Figure 1, purification yielded a homogeneous IDS-Fc fusion protein and a hexahistidine-tagged (sequence number: 241) IDS enzyme.

[0371] Example 2. Characterization of IDS fusion protein.

[0372] The IDS-Fc fusion protein with a modified TfR binding site binds to human TfR.

[0373] To determine whether the IDS-Fc fusion protein with the modified TfR binding affects the ability of the modified Fc domain to interact with human TfR, the affinity of this protein for human TfR was evaluated using the Biacore™ surface plasmon resonance assay. The Biacore™ Series S CM5 sensor chip was immobilized with anti-human Fab (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, and a serial 3-fold dilution of human apical domain TfR was injected at a flow rate of 30 μL / min. Each sample was analyzed for 3 minutes of association and 3 minutes of dissociation. After each injection, the chips were regenerated using 10 mM glycine-HCl (pH 2.1). The binding reaction was modified by subtracting RU from the flow cell capturing unrelated IgG at a similar density. Steady-state affinity was obtained by fitting the reaction at equilibrium with respect to concentration using Biacore™ T200 evaluation software v3.1. As shown in Figure 2, Biacore ™ Analysis established that the IDS-Fc fusion protein, which has a TfR-binding site engineered within the Fc region, binds to human TfR. Biacore ™ Analysis also established that the IDS-Fc fusion protein ETV:IDS 35.21 binds to human TfR with an affinity of ~200 nM.

[0374] The IDS-Fc fusion protein with a modified TfR binding site is active in vitro, intracellularly, and in vivo.

[0375] of the engineered TfR-binding IDS-Fc fusion protein In vitro By evaluating cell activity, it was demonstrated that IDS maintains its enzymatic activity when fused to a human IgG fragment. In vitroActivity was measured by a two-step fluorescent enzyme assay using an artificial substrate. Specifically, 20 μL of 1 mM 4-methylumbelipril aL-idopyranosiduronic acid 2-sulfate disodium salt substrate (Carbosynth Limited, # EM03201) was diluted with assay buffer (100 mM sodium acetate, 10 mM lead acetate, 0.05% Triton X-100, pH 5.0) and mixed with 10 μL of 0.2 nM IDS. The first reaction mixture was incubated at 37°C for 4 hours and terminated with 60 μL of 0.2 M phosphate-citrate buffer, pH 5.0. Subsequently, the second reaction was performed in the presence of 15 μg of cell lysate in HEK 293T cells transiently transfected with human α-iduronidase (IDUA), incubated at 37°C for 16 hours, and stopped by the addition of 100 μL of 0.5 M sodium carbonate buffer, pH 10.5. Subsequently, the fluorescence of the reaction solution was measured (excitation at 365 nm and emission at 450 nm). The amount of product was calculated by fitting a 4-methylumbelliferone standard curve by linear regression and confirmed to be less than 10% of the total substrate cleavage. The amount of product was divided by the reaction time and the molar amount of IDS to calculate the specific activity (nmol of product / min / nmol of IDS).

[0376] examiner In the enzyme activity assay, the IDS-Fc fusion protein was active, and it was found that the fusion of the Fc region to IDS did not interfere with this enzyme activity (Fig. 3).

[0377] To provide a cell system for testing the cellular activity of engineered IDS-Fc fusion proteins, using CRISPR / CAS9 IDSKnockout (KO) cells were generated. HEK 293T cells (ATCC) were transfected with a CRISPR / CAS9 pCas-Guide-EF1a-GFP vector (Origene) containing a guide sequence targeted to the second half of exon 1 in human IDS. Single-cell clones were analyzed for the presence of indels within the genomic sequence of IDS according to the Guide-it Mutation Detection Kit (Clontech) as per the manufacturer's instructions. IDS To identify KO cells, the above-described In vitro Indel-positive clonal cell lysates were analyzed using the IDS enzyme assay. 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, 0.02% NaAzide) as previously described (Vozyni except , J. Inherit. Metab. Dis.(*, 24:675-80 (2001)). The reaction was initiated by combining 10 μL of standardized cell lysate (in water) and 1 mM substrate in 20 μL of lead acetate buffer. The first reaction mixture was incubated at 37°C for 4 hours and terminated with 60 μL of 0.2 M phosphate-citrate buffer, pH 5.0. Subsequently, the second reaction was performed with the addition of 10 μg / 10 μL of cell lysate from HEK 293T cells transiently transfected with human α-iduronidase (IDUA), allowed to proceed at 37°C for 24 hours, and stopped by the addition of 100 μL of 0.5 M sodium carbonate buffer, pH 10.3. Subsequently, the fluorescence of the reaction solution was measured (excitation at 365 nm and emission at 450 nm). IDS activity in HEK 293T CRISPR clones was compared to assay standards, HEK wild-type (WT) lysates, and recombinant IDS used in HEK cell lysates over-expressing IDS. Clones with enzymatic activity levels comparable to the background signal were sequence-validated following mini-Topo (ThermoFisher) cloning, and confirmed to be KO clones. Subsequent cell assays were performed on three distinct and validated IDS We use KO clones and three independent batches of WT HEK 293T cells.

[0378] To test the cellular activity of naked IDS enzymes or IDS-Fc fusion proteins, an LC-MS / MS-based glycomic assay was developed to monitor substrate accumulation (heparan sulfate and dermatan sulfate) as an indicator of IDS activity. Substrate accumulation was measured before and after the addition of IDS or IDS-Fc fusion proteins to the cell culture medium. IDSIt was measured in KO cells. Briefly, the cells were washed three times with PBS, pelleted, and frozen. The cell pellet was sonicated in disaccharide digestion buffer (111 mM NH4OAc, 11 mM CaOAc, pH 7.0). Protein concentration was measured using the BCA assay (Pierce). Total protein (100 μg) was added to 100 μL of digestion buffer containing 2 mM DTT, 1.25 mIU heparinase I (Galen), 1.25 mIU heparinase II (Galen), 1.25 mIU heparinase III (Galen), and 6.25 mIU chondroitinase B (Galen). After heparin sulfate and dermatan sulfate digestion was completed at 30°C for 3 hours, 20 ng of internal standard (4UA-2S-GlcNCOEt-6S HD009 [Galen]) was added to each sample. 6 μL of 250 mM EDTA was added to inactivate the enzyme, and the samples were heated at 95°C for 10 minutes. Subsequently, the samples were 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 disaccharides were concentrated by flow-through, resuspended in a mixture of [1:1, v / v] assay buffer:acetonitrile, and transferred to a mass-spectroscopic vial for further analysis.

[0379] Analysis of dirrasaccharides produced by the enzymatic digestion of heparan and dermatan sulfates was performed by electron spray mass spectrometry (Sciex 6500+ QTRAP, Sciex, Framingham, MA, USA) coupled with liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA). For each analysis, 10 μL of sample was injected into an ACQUITY UPLC BEH amide 1.7 μm, 2.1 x 150 mm column (Waters Corporation, Milford, Massachusetts, USA) using a flow rate of 0.4 mL / min at a column temperature of 50°C. Mobile phase A consisted of 10 mM ammonium formate, 0.1% formic acid, and water. Mobile phase B consisted of 0.1% formic acid and acetonitrile. The above gradient is scheduled 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 hold at 6–8.0 min at 85% B. Electrospray ionization was performed in negative-mode with the following settings applied: curtain gas at 30; collision gas set to medium; ionspray voltage -4500; temperature 450; ion source gas 1 at 50; ion source gas 2 at 60. Data acquisition was performed using Analyst 1.6.3 (Sciex) in Multi-Reaction Monitoring Mode (MRM) with a residence time of 25 msec. Collision energy -30; declustering potential -80; Entrance potential -10; collision cell exit potential -10.The GAG ​​was detected as [M--H] using the following MRM transitions: D0A0,. m / z 378.1>87.0; D0a0, m / z 378.1>175.0; D0S0, m / z 416.1>138.0; D0a4, m / z 458.1>300.0; D0A6,D2A0, D0a6, D2a0, m / z 458.1>97.0; D0S6, D2S0, m / z 496.0>416.1; D2a4, D2a6, D0a10, D2A6, m / z 538.0>458.0; D0S6 m / z 575.95>97.0 4UA-2S-GlcNCOEt-6S, m / z 472.0 (fragment ion) > 97.0 was used as the internal standard (IS). GAGs were identified based on whether their retention time and MRM transitions matched a commercially available reference standard (Iduron Ltd, Manchester, UK). Quantification was performed using MultiQuant 3.0.2 (Sciex) as the area ratio to the IS. GAGs were normalized to the total protein amount. Protein concentration was measured using the BCA assay (Pierce).

[0380] As reflected in the amount of disaccharides observed after the digestion of heparan sulfate and dermatan sulfate, compared to the control cell line, IDS Significant substrate accumulation was observed in KO cells, and this effect could be avoided by adding recombinant IDS to these cells (Fig. 4). This demonstrated that LC-MS / MS-based assays can be used to evaluate the cellular activity of IDS and IDS-Fc fusion proteins.

[0381] Using this test, the same TfR-linked Fc polypeptide ( chamberlain,N-terminal monozyme containing CH3C.35.21.17 ( chamberlain, It was firmly established that treating cells with the IDS-Fc fusion protein consisting of ETV:IDS 35.21.17) or the C-terminal monozyme reduced the levels of heparan and dermatan sulfate-derived disaccharides back to levels observed in wild-type cells (Fig. 5a). Furthermore, the activity of the N-terminal monozyme was comparable to that of IDS (Fig. 5b). Together, these data indicate that the IDS-Fc fusion protein maintains enzymatic activity, IDS It demonstrates that substrate accumulation in KO cells can be reduced.

[0382] The cellular activity of IDS-Fc fusion proteins is also 35 Fibroblasts from MPS II patients and healthy controls were examined using the S pulse-chase assay, at this time 35 As previously described, S is incorporated into newly synthesized GAGs (Lu except , Bioconjugate Chemistry , 21:151-156 (2010)). MPS II patient fibroblasts lack detectable IDS activity, resulting in approximately 10-fold accumulation of stroma and 35 This leads to a 2.5-fold accumulation of the S signal (Fig. 5c). IDS Similar to KO cells, IDS-Fc fusion proteins, such as ETV:IDS 35.23.2, are S 35 - Low picomolar levels of cellular EC to reduce the accumulation of labeled proteins 50 It indicated that it was highly effective in MPS II patient-derived cells (Fig. 5c). Furthermore, the cellular activity of IDS-Fc fusion proteins such as ETV:IDS 35.23.2 and M6P was observed in MPS II patient fibroblasts treated with the protein. 35It was proven to be M6PR-dependent because it inhibited the removal of S-labeled proteins (Fig. 5d). Collectively, these data demonstrate that M6PR-dependent trafficking and cell activity of IDS can be maintained within the IDS-Fc fusion protein format.

[0383] In vivo To measure heparan and dermatan sulfate-derived disaccharides, an LC-MS / MS-based glycomic assay was adapted for the analysis of tissues and fluids. Briefly, all tissues and fluids were collected, immediately frozen, and stored at -80°C. Samples underwent five freeze-thaw cycles and were processed as described above for cellular analysis. Compared to male wild-type littermate controls, male IDS Significant accumulation of heparan sulfate and dermatan sulfate-derived disaccharides was observed in all tissues and fluids analyzed from KO mice (Table 1). This assay In vivo It is used in the study of the effects of the above fusion protein. IDS KO mice were obtained from The Jackson Laboratories (JAX strain 024744).

[0384]

[0385] Using this method, the levels of heparan and dermatan sulfate-derived disaccharides were compared with vehicle-administered wild-type (WT) mice and IDS or IDS-Fc fusion proteins ( chamberlain, ETV:IDS 35.21) administered IDS It was evaluated in the serum of KO mice. When compared to WT mice at baseline measurements before administration, IDS Significant accumulation of heparan and dermatan sulfate-derived disaccharides in KO mouse serum was demonstrated. After administration of the IDS-Fc fusion protein, the levels of heparan and dermatan sulfate-derived disaccharides were IDSIt was significantly reduced in KO serum, which is due to the administration of IDS. IDS It shows a reduction comparable to that seen in the serum of KO mice (Fig. 6). In these data, the IDS-Fc fusion protein is In vivo It is active, IDS Substrate accumulation in KO mice can be reduced. Based on these data, at 7 days after administration of a single dose of IDS-Fc fusion protein, IDS Distribution and pharmacodynamic (PD) responses were evaluated in the tissues of KO mice. IDS KO mice were intravenously administered 40 mg / kg of IDS-Fc fusion protein or 5.3 mg / kg of IDS (25% molar equivalent) as a positive control, and GAG levels were evaluated. The distribution of the two molecules in peripheral tissues was confirmed 2 hours after administration. At 7 days after administration of IDS-Fc fusion protein, IDS Significant reduction in substrate was observed in the liver, spleen, and lungs of KO mice (Fig. 7).

[0386] To determine whether the TfR-binding IDS-Fc fusion protein exhibited improved brain delivery compared to the control IDS-Fc fusion protein, human TfR knock-in (TfR ms / hu KI) Mice were administered 50 mg / kg of the TfR-binding IDS-Fc fusion protein ETV:IDS 35.21 or a control IDS-Fc fusion protein ("IDS:Fc") lacking the mutation conferring TfR binding, and the concentration of the IDS-Fc fusion protein in the brain was measured at 4 hours post-administration using a sandwich ELISA-based assay as described in Example 3 below. TfR ms / hu The KI mouse, as described in International Patent Publication No. WO 2018 / 152285, is a murine Tfrc Humans inside the genes TfrcIt was created using CRISPR / Cas9 technology that expresses the apex domain; the resulting chimeric TfR is under the control of an endogenous promoter In vivo It was expressed. Compared to the control IDS-Fc fusion protein, significantly higher levels of the IDS-Fc fusion protein ETV:IDS 35.21 were detected, and the average brain concentration for ETV:IDS 35.21 was 23.7 nM (Fig. 8). Brain infusion of two additional TfR-binding IDS-Fc fusion proteins, ETV:IDS 35.21.17.2 and ETV:IDS 35.23.2, was the TfR ms / hu It was evaluated using KI mice. TfR ms / hu KI mice were administered 50 mg / kg of ETV:IDS 35.21.17.2, ETV:IDS 35.23.2, or a control IDS-Fc fusion protein ("IDS:Fc"), and the brain concentrations of the IDS-Fc fusion proteins were measured at 2 and 8 hours post-administration using a sandwich ELISA-based assay. Upon administration of the TfR-binding IDS-Fc fusion proteins, brain incorporation increased 5-fold at 2 hours post-administration and 10-20-fold at 8 hours post-administration compared to the control IDS-Fc fusion protein (Fig. 9a). The accumulation of intact fusion proteins in serum PK and liver was equivalent in both cases of ETV:IDS 35.21 and IDS:Fc (Fig. 9b), indicating that the IDS moiety primarily determines the distribution phase of plasma clearance. Brain levels of TfR-binding IDS-Fc fusion proteins remained elevated for 8 hours and decreased slightly with peripheral clearance. Together, these data demonstrate that the interaction of TfR-binding IDS-Fc fusion proteins with TfR generally maintains peripheral distribution while significantly improving brain exposure.

[0387] Intravenous administration of ETV:IDS reduces GAGs in the brain.

[0388] To examine whether brain exposure to the TfR-binding IDS-Fc fusion protein (referred to herein as ETV:IDS) described above and prepared according to Example 1 results in a corresponding reduction of substrate accumulated in the brain, a mouse model deficient in IDS containing a human TfR apex domain melted into a murine TfR was prepared (in this application IDS KO x TfR ms / hu (Referred to as the KI mouse). To explain briefly, TfR ms / hu By rearing male KI mice with female IDS heterozygous mice, TfR ms / hu KI homozygous background IDS KO mice were created. All mice used in this study were male, given free access to food and water, and kept under a 12-hour light-dark cycle (LabDiet JL irradiated 6F).

[0389] IDS KO x TfR ms / hu Intravenous administration of a single or four weekly active-equivalent doses of ETV:IDS or IDS (747 μmol product / min / kg or 40 mg / kg and 14.2 mg / kg, respectively) to KI mice, and pharmacokinetic and pharmacodynamic responses were evaluated. Specifically, IDS KO x TfR ms / hu The effects of peripherally administered ETV:IDS on brain and tissue GAGs in KI mice administered intravenously (iv), in saline, IDS (14.2 mg / kg body weight), or ETV:IDS (40 mg / kg body weight) once (n=8) or once weekly for 4 weeks (n=8) at 2-month-old IDS KO x TfR ms / hu It was determined using KI mice. 2-month-old TfRs that received saline injections once (n=5) or once weekly (n=5) for 4 weeks. ms / hu KI mice were used as a control group. In-life serum samples from animals administered IDS or ETV:IDS were collected via submandibular hemorrhage at various time points. All animals were sacrificed 7 days after a single dose administration or 7 days after the last 4-week administration. Urine, serum, CSF, liver, kidney, spleen, lungs, heart, and right hemicerebellum were dissected and flash-frozen on dry ice.

[0390] After a single administration, ETV:IDS exhibited a serum clearance profile similar to IDS, as evaluated using the ELISA-based assay described in Example 3 below to detect IDS concentrations, providing further support that the enzyme significantly influences peripheral clearance (Fig. 10a). Brain levels of ETV:IDS were significantly increased to average concentrations of 8.4 and 1.6 nM, respectively, compared to IDS at 2 hours post-administration, and liver and spleen levels of ETV:IDS were significantly elevated compared to IDS (Fig. 10b).

[0391] To determine whether ETV:IDS reduces substrate levels in the brain, as described in Example 2, after a single administration of this enzyme or weekly administration for 4 weeks, IDS KO x TfR ms / hu GAG levels were evaluated in KI mice. IDS reduced brain GAG levels only slightly at the initial time point; however, after 4 weeks of treatment, it was ineffective in significantly lowering GAGs (Fig. 10c). In contrast, ETV:IDS reduced brain GAG levels by approximately 58% after a single administration and by 71% after 4 weeks of treatment (Fig. 10c). Consequently, CSF GAGs decreased by approximately 75% after a single administration, a reduction that persisted even after 4 weeks of treatment (Fig. 10c). Both molecules effectively lowered GAG levels in the liver and spleen after one week, and this response was sustained by repeated administration (Fig. 10c), demonstrating that TfR binding does not negatively affect pharmacodynamic responses in these tissues. Together, these data demonstrate that ETV:IDS significantly increases brain exposure to the enzyme and potently reduces substrate accumulation in both the periphery and the CNS.

[0392] Example 3. Pharmacokinetic characteristics of IDS fusion protein.

[0393] This example describes the pharmacokinetic (PK) characteristics of an engineered IDS-Fc fusion protein in mouse plasma.

[0394] To determine the plasma half-life and clearance of TfR-binding IDS-Fc fusion proteins, 10 mg / kg of two IDS-Fc fusion protein molecules (N-terminal monozyme and C-terminal monozyme) were administered to 7-8 week old male C57BL / 6 mice via tail vein injection. The concentration of IDS-Fc fusion proteins remaining in the plasma after 24 hours was measured using an ELISA-based assay. Briefly, the concentration of IDS-Fc fusion proteins in mouse plasma was quantified using a sandwich ELISA. An anti-Fc capture antibody (Abcam #ab124055) was coated at 3 μg / mL on 384-well MaxiSorp™ plates (Thermo Scientific #464718). The plate was blocked with 5% BSA and then incubated with plasma diluted 1:1,000 or 1:10,000. Next, a polyclonal anti-IDS detection antibody (R&D Systems # AF2449) was added at 0.5 μg / mL, followed by the addition of an anti-goat-HRP antibody. The plate was developed using, for example, TMB substrate, stopped with sulfuric acid, and the absorbance was measured at 450 nm using a BioTek plate reader. Standard curves were individual constructs ranging from 200 to 0.1 ng / mL in a 4-fold dilution series and fitted using 4-parameter logistic regression.

[0395] Using this test, it was confirmed that the terminal plasma half-life of the IDS-Fc fusion protein is 7.7-10 hours (Table 2). In vivo No unexpected PK liabilities were observed in the IDS-Fc fusion protein.

[0396]

[0397] Example 4. Construction of a fusion protein containing acid sphingomyelinase (ASM)

[0398] Planning and Cloning

[0399] The ASM-Fc fusion protein was designed as a dimer of the fusion polypeptide, wherein, upon maturation, the human ASM enzyme is fused to a human IgG1 fragment containing the Fc region ("ASM-Fc fusion polypeptide"). In some embodiments, the ASM-Fc fusion polypeptide contains a modified Fc region containing a mutation that confers transferrin receptor (TfR) binding. Specifically, ASM-Fc fusion polypeptides are constructed such that the ASM fragments are fused 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 steric hindrance between the two fragments. In all constructs, the native ASM signal sequence, amino acids 1-46 (UniProtKB ID-P17405), was removed and replaced with a signal from kappa chain V-III, amino acids 1-20 (UniProtKB ID-P01661) to improve the efflux of ASM. Additionally, in the fusion protein, to prevent any undesirable cleavage between the ASM and the human IgG1 Fc region, the ASM is truncated at its C-terminus and terminated at amino acid Q620. Then, a fragment of the human IgG1 Fc region (UniProtKB ID-P01857) is positioned in the same frame as the C-terminus of the ASM starting at amino acid E99, and the cysteine ​​at position 103 is mutated to serine. In some embodiments, the IgG1 fragments contain additional mutations to promote the heterodimerization of the two Fc regions. Additionally, the ASM-Fc fusion protein was constructed to contain one or two molecules of ASM. As a control, the ASM-hexahistidine (sequence number: 241) fusion protein was designed to consist of truncated ASM amino acids 1-628 to remove C-terminal cysteine ​​and promote enzyme activation and the hexahistidine tag (sequence number: 241) fused at the C-terminus.

[0400] Recombinant Protein Expression and Purification

[0401] To express recombinant ASM fused to the Fc region, ExpiCHO-S cells (Thermo Fisher) were mixed with the Expifectamine CHO / plasmid DNA complex 6x10 times according to the manufacturer's (Thermo Fisher Scientific) instructions. 6 Transfection was performed at a cell / ml density. After transfection, cells were incubated at 32°C in an orbital shaker (Infors HT Multitron) with a humidified atmosphere of 6–8% CO2. Expifectamine enhancer and Expifectamine feed were added to the cultures 1 day after transfection. After an expression time of 48–72 hours, the supernatant was harvested by centrifugation. EDTA-free protease inhibitor (Roche) was added to the clear supernatant, and the mixture was stored at -80°C.

[0402] For the purification of the ASM-Fc fusion protein, 200 μM zinc acetate (Sigma Aldrich) was added to the supernatant of the clear medium. 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 pH 5.0 (arginine-succinate buffer). The fusion protein was eluted in 100 mM QB citrate buffer pH 3.0 supplemented with 200 μM zinc acetate. Immediately after elution, the pH was adjusted by adding arginine-succinate buffer. Protein aggregates were separated from the ASM-Fc fusion protein by size exclusion chromatography (SEC) on a Superdex 200 enlarged 10 / 300 GL column (GE Healthcare Life Sciences). The SEC mobile phase was maintained in arginine-succinate pH 5.0 buffer supplemented with 200 μM zinc acetate. All chromatographic steps were performed using an Akta Pure System or an Akta Avant System (GE Healthcare Life Sciences). Fractional purity was evaluated by non-reducing SDS-PAGE. As shown in Figure 11, homogeneous ASM-Fc fusion proteins were obtained by purification.

[0403] Example 5. Characterization of ASM fusion protein

[0404] The ASM-Fc fusion protein is active both inside the tube and within the cell.

[0405] To demonstrate whether its enzymatic activity is maintained when ASM is fused to a human IgG heavy chain, the ASM-Fc fusion protein In vitro And cell activity was evaluated. of the recombinant ASM enzyme or recombinant ASM-Fc fusion protein examinerActivity was measured using synthetic chromogenic analogs of sphingomyelin. Specifically, 2.5 mM 2-(N-hexadecanoylamino)-4-nitrophenylphosphorylcholine (EMD Millipore) was mixed with 0.75 nM ASM in 100 mM sodium acetate buffer (pH 5.3; final concentration at 100 μL reaction volume). The reaction mixture was incubated at 37°C for 16 hours and stopped by adding an equal volume of 0.2 M NaOH. Subsequently, the absorbance of the reaction solution was measured at 410 nm. The amount of product was calculated by fitting a p-nitrophenol standard curve via linear regression, and it was confirmed to be less than 10% of the total substrate cleavage. The specific activity (nmol of product / min / nmol of ASM) was calculated by dividing the amount of product by the reaction time and the molar amount of ASM. examiner In the enzyme activity assay, the ASM-Fc fusion protein was found to be active, and the fusion of the Fc region to ASM did not interfere with this enzyme activity (Fig. 12).

[0406] To provide a cell system for testing the cellular activity of ASM-Fc fusion proteins, using CRISPR / CAS9 ASM KO cells were generated. HEK 293T cells (ATCC) were transfected with a CRISPR / CAS9 pCas-Guide-EF1a-GFP vector (Origene) containing a guide sequence targeting the second half of exon 2 in human SMPD1. Single-cell clones were analyzed for the presence of indels within the ASM genomic sequence according to the manufacturer's instructions using the Guide-it Mutation Detection Kit (Clontech). Indel-positive clonal cell lysates were analyzed using the ASM chromogenic substrate 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine (EMD Millipore). In vitro It underwent ASM enzyme testing. To briefly explain, In vitroActivity assays were performed using 12.5, 25, 50, and 100 μg of cell lysates in 100 mM sodium acetate buffer (pH 5.3). The reaction was initiated by adding 2.5 mM substrate and stopped after 20 hours by adding 0.2 M NaOH. ASM activity in HEK293T CRISPR clones was compared to the assay standard, HEK wild-type (WT) lysates, and recombinant ASM used in HEK cell lysates over-expressing ASM. Clones with enzyme activity levels comparable to the background signal were sequence-validated after cloning into mini-Topo (Thermo Fisher Scientific) and confirmed to be KO clones. Subsequent cell assays were performed on three unique and validated ASM KO clones and three independent batches of WT HEK293T cells were used.

[0407] To test the cellular activity of naked ASM enzymes or ASM-Fc fusion proteins, two cell assays were developed, which ASM It is possible to monitor the amount of basic matrix accumulation (sphingomyelin) in KO cells and the amount after treatment with ASM or ASM-Fc fusion. First, ASM To monitor the amount of BODIPY-conjugated C5-sphingomyelin accumulation in KO cells, an image-based assay was developed. Briefly, HEK293T WT and ASMKO cells were plated at low density in DMEM supplemented with 10% FBS (Gibco) on PDL-coated, 96-well plates (Perkin Elmer). At 4 hours post-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 removed and replaced with fresh medium containing 1 μM BODIPY-C5-sphingomyelin (Thermo Fisher Scientific), and incubated at 37°C for 16 hours. Subsequently, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with nuclei (DAPI, Thermo Fisher) and cytoplasm (far red cell mask, Thermo Fisher Scientific). Images were acquired on an Opera Phenix confocal microscope (Perkin Elmer) equipped with a 63X objective lens, featuring multiple fields per well and three-triplicate wells per condition. Image analysis was performed using Harmony software (Perkin Elmer) to detect and analyze the average total intensity, number of punctas, and puncta intensity of BODIPY-C5-sphingomyelin per cell base. These per-cell values ​​were then averaged to per-well values ​​and used to analyze the effects of genotype and / or treatment on the accumulation of BODIPY-C5-sphingomyelin. Compared to control cell lines, ASM Significant accumulation of BODIPY-C5-sphingomyelin was observed in KO cells, and this effect can be avoided by adding recombinant ASM enzyme and ASM-Fc fusion protein (Fig. 13).

[0408] To further demonstrate that ASM-Fc fusion proteins maintain their activity in cells, ASMTo monitor the accumulation of endogenous sphingomyelin in KO cells, an LC-MS / MS-based assay was developed. HEK293T WT and ASM KO cells were cultured with the Bawak ata described above and treated with enzymes. At 68 hours post-smearing, with or without ASM or ASM-Fc fusion protein treatment, cells were thoroughly washed with PBS, lipids were spiked with appropriate internal standards, and extracted with a mixture of water:methanol [1:1, v / v]. The lipids were extracted with methyl-tert-butyl ether (MTBE), vortexed, and centrifuged at 10,000xg and 4°C for 10 minutes. Subsequently, the upper MTBE fraction containing lipids was evaporated 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 a mass spectrometry vial for further analysis.

[0409] Lipid analysis was performed by electron spray mass spectrometry (Sciex 6500+ QTRAP, Sciex, Framingham, MA, USA) coupled with liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA). For each analysis, 5 μL of sample was injected into a BEH C18 1.7 μm, 2.1×100 mm column (Waters Corporation, Milford, Massachusetts, USA) at 55°C at a flow rate of 0.25 mL / min. Mobile phase A consisted of 60:40 acetonitrile / water (v / v) with 10 mM ammonium formate and 0.1% formic acid. Mobile phase B consisted of 90:10 isopropanol / acetonitrile (v / v) with 10 mM ammonium formate and 0.1% formic acid. The above gradient is scheduled as follows: 0.0–8.0 min at 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 negative-mode with the following settings applied: curtain gas at 20; collision gas set to medium; ionspray voltage 5200; temperature 250; ion source gas 1 at 50; ion source gas 2 at 60. Data acquisition was performed using Analyst 1.6 (Sciex) in Multiple Reaction Monitoring Mode (MRM). Collision energy 40; declustering potential 80; entrance potential 10; Collision cell exit potential 12.5. Ceramide (Cer) [M-H2O+H] using the following MRM transitions +Detected as: Cer d18:1 / 16:0, m / z 538.5>264.3; Cer d18:1 / 18:0, m / z 566.6>264.3; Cer d18:1 / 20:0, m / z 594.6>264.3; Cer d18:1 / 22:0, m / z 622.6>264.3; Cer d18:1 / 24:0, m / z 650.6>264.3; Cer d18:1 / 24:1, m / z 648.6>264.3; Cer d18:1 / 17:0, m / z 552.4 > 264.3 was used as an internal standard. Sphingomyelin (SM) [M+H] using the following MRM transitions. + It was detected as: SM d18:1 / 16:0, m / z 703.7>184.1; SM d18:1 / 18:0, m / z 731.7>184.1; SM d18:1 / 20:0, m / z 759.7>184.1; SM d18:1 / 22:0, m / z 787.7>184.1; SM d18:1 / 24:0, m / z 815.7>184.1; SM d18:1 / 24:1, m / z 813.7>184.1; SM d18:1 / 18:1 (d9), m / z 738.7>184.1 was used as an internal standard. Lipids were identified based on retention times and MRM transitions of a commercially available reference standard (Iduron Ltd, Manchester, UK). Quantification was performed using MultiQuant 3.02 (Sciex). Lipids were standardized to the total protein amount. Protein concentration was measured using the BCA assay (Pierce). In LC-MS / MS analysis, the ASM-Fc fusion protein was endogenous sphingomyelin's ASM It was demonstrated that intracellular KO levels could be reduced back to levels observed in wild-type cells (Fig. 14). Furthermore, the ASM-Fc fusion protein was as potent as the naked ASM enzyme in reducing sphingomyelin in both assays (Table 3).

[0410]

[0411] Together, these data demonstrate that ASM-Fc fusion proteins maintain their activity in ASM-deficient cells and can be saved from substrate accumulation.

[0412] Example 6. Construction of a fusion protein containing N-sulfoglucosamine sulfohydrolase (SGSH)

[0413] Planning and Cloning

[0414] The SGSH-Fc fusion protein was designed to contain: (i) a fusion polypeptide, where, matured, a human SGSH enzyme is fused to a human IgG1 fragment containing an Fc region ("SGSH-Fc fusion polypeptide"); and (ii) a modified human IgG1 fragment containing an intra-Fc region mutation that is located within the Fc region and confers transferrin receptor (TfR) binding ("modified Fc polypeptide"). Specifically, SGSH-Fc fusion polypeptides are produced in which SGSH fragments are fused to the N- or C-terminus of the human IgG1 Fc region. In some cases, a linker is placed between the SGSH and IgG1 fragments to alleviate steric hindrance between the two fragments. In all constructs, the kappa chain V-III signal peptide, amino acids 1-20 (UniProtKB ID -P01661), was inserted upstream of the fusion protein to facilitate secretion, and the SGSH was truncated and composed of amino acids R21-L502 (UniProtKB ID -P51688). The fragment of the human IgG1 Fc region used corresponds to amino acids D104-K330 of UniProtKB ID P01857 (according to position 221-447-EU numbering—containing a 10-amino acid hinge (position 221-230)). In some embodiments, a second Fc polypeptide derived from human IgG1 residue D104-K330 containing a mutation that confers TfR binding but lacks SGSH fusion was co-transfected with an SGSH-Fc fusion polypeptide to produce a heterodimeric fusion protein having one SGSH enzyme ("monozyme").In other embodiments, a second Fc polypeptide derived from human IgG1 residue D104-K330 containing an SGSH-fused mutation that confers TfR binding in the Fc region was co-transfected with an SGSH-Fc fusion polypeptide to produce a heterodimeric fusion protein having two SGSH enzymes ("vizymes"). In some constructs, the IgG1 fragments contained additional mutations to promote heterodimerization of the two Fc regions. A control SGSH-Fc fusion protein lacking the TfR-conferring mutation was designed and similarly constructed. As an additional control, an SGSH (amino acid R21-L502) with a C-terminal hexahistidine tag (sequence number: 241) was prepared to facilitate detection and purification.

[0415] The SGSH-Fc fusion protein containing TfR-bonding used in the present embodiment is a dimer formed by an SGSH-Fc fusion polypeptide and a modified Fc polypeptide that binds to TfR, wherein the modified Fc polypeptide lacks an SGSH fusion ("monozyme") or is fused to a second SGSH molecule ("vizyme").

[0416] The SGSH-Fc fusion polypeptide, comprising a mature human SGSH sequence fused to the N-terminus of the IgG1 Fc polypeptide sequence along with hall and LALA mutations, has the sequence of SEQ ID NO: 149. The SGSH enzyme is linked to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 239), and the N-terminus of the Fc polypeptide contains a portion of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 113).

[0417] The SGSH-Fc fusion polypeptide, comprising a mature human SGSH sequence fused to the C-terminus of an IgG1 Fc polypeptide sequence along with hall and LALA mutations, has the sequence of SEQ ID NO: 150. The SGSH enzyme is linked to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 239), and the N-terminus of the Fc polypeptide may contain a portion of the IgG1 hinge region (e.g., SEQ ID NO: 113).

[0418] A modified Fc polypeptide that binds to TfR, comprising the sequence of clone CH3C.35.21.17 (SEQ No.: 58) having Knob and LALA mutations, has the sequence of SEQ No.: 151. The N-terminus of the modified Fc polypeptide is a part of the IgG1 hinge region ( chamberlain, It may contain sequence number: 113).

[0419] An "N-terminal monozyme" containing a single SGSH molecule at the N-terminus of the above Fc polypeptide was formed between Sequence No. 149 and Sequence No. 151. An "N-terminal monozyme" containing a single SGSH molecule at the C-terminus of the above Fc polypeptide was formed between Sequence No. 150 and Sequence No. 151.

[0420] A modified Fc polypeptide that binds to TfR contains a mature human SGSH sequence fused to the N-terminus of the sequence of clone CH3C.35.21.17 (SEQ No.: 58) having Knob and LALA mutations, and has the sequence of SEQ No.: 154. The SGSH enzyme is linked to the modified Fc polypeptide by a GGGGS linker (SEQ No.: 239), and the N-terminus of the modified Fc polypeptide contains a portion of the IgG1 hinge region (SEQ No.: 113).

[0421] The "N-terminal vizyme" containing a first SGSH molecule at the N-terminus of the above Fc polypeptide and a second SGSH molecule at the N-terminus of the above modified Fc polypeptide is formed between Sequence No. 149 and Sequence No. 154.

[0422] Recombinant Protein Expression and Purification

[0423] To express the recombinant SGSH enzyme fused to the Fc region, ExpiCHO cells (Thermo Fisher Scientific) were transfected with the associated DNA construct using the Expifectamine™ CHO Transfection Kit according to the manufacturer's instructions. Cells were grown in ExpiCHO™ expression medium in an orbital shaker (Infors HT Multitron) at 37°C, 6% CO2, and 120 rpm. In summary, 6 x 10 logarithmic-growing ExpiCHO™ cells were transfected with 0.8 μg of DNA plasmid per 1 mL of culture volume. 6 Transfection was performed at a cell / ml density. After transfection, the cells were returned to 37°C, and the transfected cultures were supplemented with feed as directed 18–22 hours after transfection. The supernatant of the transfected cell culture was collected 120 hours after transfection by centrifugation at 3,500 rpm for 20 minutes. The clear supernatant was filtered (0.22 μM membrane) and stored at 4°C. Expression of the epitope-tagged SGSH enzyme (used as a control) was performed with slight modifications to the description above. Briefly, the SGSH enzyme containing the C-terminal hexahistidine tag (SEQ No.: 241) was expressed in ExpiCHO cells.

[0424] SGSH-Fc fusion proteins with (or without) a modified Fc region 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 an Akta Pure System). Subsequently, the column was washed with PBS by >20 column volumes (CVs). The bound proteins were eluted using 100 mM citrate / NaOH buffer pH 3.0 containing 150 mM NaCl. Immediately after elution, the fraction was neutralized using 1 M arginine-670 mM succinate buffer pH 5.0 (dilution ratio of 1:5). The homogeneity of the SGSH-Fc fusions in the eluted fractions was evaluated by reduced and non-reduced SDS-PAGE.

[0425] To purify hexahistidine-tagged (SEQ No.: 241) SGSH, the transfected supernatant was thoroughly dialyzed overnight in 15 L of 20 mM HEPES pH 7.4 containing 100 mM NaCl, and 20 mM imidazole was added to the dialyzed supernatant prior to purification. The dialyzed supernatant was bound to a HisTrap column (GE Healthcare Life Sciences using Akta Pure System). After binding, the column was washed with 20 CV PBS. The bound protein was eluted using PBS containing 500 mM imidazole. The homogeneity of the SGSH enzyme in the eluted fraction was evaluated 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 Q Sepharose High Performance (GE Healthcare). After binding, this column is washed with 50 mM Tris, pH 7.5 at 10 CV. The bound protein is eluted using a linear gradient of 50 mM Tris, pH 7.5 and 0.5 M NaCl, and collected in the 1 CV fraction. Fraction purity is evaluated by non-reducing SDS-PAGE. Purification yields homogeneous SGSH-Fc fusion protein and hexahistidine-tagged (sequence number: 241) SGSH.

[0426] Example 7. Characterization of SGSH Fusion Protein

[0427] The SGSH-Fc fusion protein with a modified TfR binding site binds to human TfR.

[0428] To determine whether the SGSH-Fc fusion protein with a modified TfR binding affects the ability of the modified Fc domain to interact with human TfR, the affinity of this protein for human TfR can be evaluated using the Biacore™ surface plasmon resonance assay. The Biacore™ Series S CM5 sensor chip is immobilized with anti-human Fab (GE Healthcare’s human Fab capture kit). 5 μg / mL of SGSH-Fc fusion protein is captured in each flow cell for 1 minute, and a serial 3-fold dilution of human apical domain TfR is injected at a flow rate of 30 μL / min. Each sample is analyzed by 3-minute union and 3-minute dissociation. After each injection, the chips are regenerated using 10 mM glycine-HCl (pH 2.1). The binding reaction is modified by subtracting RU from a flow cell capturing unrelated IgG at a similar density. Steady-state affinity is obtained by fitting the reaction at equilibrium with respect to concentration using Biacore™ T200 evaluation software v3.1. Biacore ™ Through analysis, it was established that the SGSH-Fc fusion protein having a TfR binding site modified in the Fc region binds to human TfR.

[0429] The SGSH-Fc fusion protein with a modified TfR binding site is active in vitro and in cells.

[0430] of engineered TfR-binding SGSH-Fc fusion protein In vitro and by evaluating cell activity, it was demonstrated that SGSH's enzymatic activity is maintained when fused to a human IgG fragment. Recombinant SGSH In vitroActivity was measured by a two-step fluorescent enzyme assay using an artificial substrate. Specifically, 20 μL of 1 mM 4-methylumbelipril 2-deoxy-2-sulfonanido-aD-glucopyranoside sodium salt substrate (Carbosynth Limited, # EM06602) was diluted with assay buffer (0.03 M sodium acetate, 0.12 M NaCl, pH 6.5) and mixed with 10 μL of 40 nM SGSH. The first reaction mixture was incubated at 37°C for 17 hours and terminated with 10 μL of 0.2 M phosphate-citrate buffer, pH 6.7. Subsequently, 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 hours, and stopped by adding 100 μL of 0.5 M sodium carbonate buffer, pH 10.3. Subsequently, the fluorescence of the reaction solution was measured (excitation at 365 nm and emission at 450 nm). The amount of product was calculated by fitting a 4-methylumbelliferone standard curve by linear regression and confirmed to be less than 10% of the total substrate cleavage. Specific activity (fmol of product / min / pmol of SGSH) was calculated by dividing the amount of product by the reaction time and the molar amount of SGSH.

[0431] examiner In the enzyme activity assay, the SGSH-Fc fusion protein was active, and it was found that the fusion of the Fc region to SGSH did not interfere with this enzyme activity (Fig. 15).

[0432] To provide a cell system for testing the cellular activity of the engineered SGSH-Fc polypeptide, SGSH knockout (KO) cells were generated using CRISPR / CAS9. HEK 293T cells (ATCC) were transfected with a CRISPR / CAS9 pCas-Guide-EF1a-GFP vector (Origene) containing a guide sequence that targets upstream of exon 2 of reactive cysteine, which produces pomylglysine in human SGSH. SGSH To identify KO cells, a single-cell clone is grown, and the cell lysate is as described above examiner The SGSH enzyme assay was performed. To explain briefly, In vitro As previously described, the activity assay was 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). 20 μg of standardized cell lysate (in water) and 1 mM substrate were combined in 10 μL of lead acetate buffer (3X) and incubated at 37°C for 17 hours. The first reaction was stopped by adding 70 μL of 4x citrate phosphate buffer pH 6.7 + 0.5 U NAGLU (Sigma). The reaction was allowed to proceed at 37°C for 24 hours, after which it was stopped by adding 100 μL of 0.5 M sodium carbonate pH 10.3. SGSH activity in HEK293T CRISPR clones was compared to recombinant SGSH (R&D), HEK wild-type (WT) lysates, and HEK cell lysates overexpressing SGSH, which were used as assay standards. Clones with enzymatic activity levels comparable to the background signal were cloned into mini-Topo (ThermoFisher), sequence validated, and confirmed to be KO clones. Subsequent cell assays were performed on three unique and validated SGSH KO clones and three independent batches of WT HEK293T cells were used.

[0433] To test the cellular activity of naked SGSH enzymes or SGSH-Fc fusion proteins, an LC-MS / MS-based glycomic assay was developed to monitor substrate accumulation (heparan sulfate) as an indicator of SGSH activity. Substrate accumulation SGSH It was measured in KO cells and WT HEK293T cells. SGSH KO cells and WT HEK293T cells were cultured for 24 hours, and the cells were washed three times with PBS, pelleted, and frozen. The cell pellet was sonicated in disaccharide digestion buffer (111 mM NH4OAc, 11 mM CaOAc, pH 7.0). Protein concentration was measured using the BCA assay (Pierce). Total protein (100 μg) was added to 100 μL of digestion buffer containing 2 mM DTT, 1.25 mIU heparinase I (Galen), 1.25 mIU heparinase II (Galen), and 1.25 mIU heparinase III (Galen). After heparin sulfate digestion was completed at 30°C for 3 hours, 20 ng of internal standard (4UA-2S-GlcNCOEt-6S HD009 [Galen]) was added to each sample. 6 μL of 250 mM EDTA was added to inactivate the enzyme, and the samples were heated at 95°C for 10 minutes. Subsequently, the samples were 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 disaccharides were concentrated by flow-through, resuspended in a mixture of [1:1, v / v] assay buffer:acetonitrile, and transferred to a mass-spectroscopic vial for further analysis.

[0434] GAG lipid analysis was performed by electron spray mass spectrometry (Sciex 6500+ QTRAP, Sciex, Framingham, MA, USA) coupled with liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA). For each analysis, 10 μL of sample was injected into an ACQUITY UPLC BEH amide 1.7 μm, 2.1 x 150 mm column (Waters Corporation, Milford, Massachusetts, USA) using a flow rate of 0.4 mL / min at a column temperature of 50°C. Mobile phase A consisted of water containing 10 mM ammonium formate and 0.1% formic acid. Mobile phase B consisted of acetonitrile containing 0.1% formic acid. The above gradient is scheduled 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 hold at 6–8.0 min at 85% B. Electrospray ionization was performed in negative-mode with the following settings applied: curtain gas at 30; collision gas set to medium; ionspray voltage -4500; temperature 450; ion source gas 1 at 50; ion source gas 2 at 60. Data acquisition was performed using Analyst 1.6.3 (Sciex) in Multi-Reaction Monitoring Mode (MRM) with a residence time of 25 msec. Collision energy -30; declustering potential -80; Entrance potential -10; collision cell exit potential -10. The GAG ​​was detected as [M--H] using the following MRM transitions: D0A0, m / z 378.1>87.0; D0a0, m / z 378.1>175.0; D0S0, m / z 416.1>138.0; D0a4, m / z 458.1>300.0; D0A6,D2A0, D0a6, D2a0, m / z 458.1>97.0; D0S6, D2S0, m / z 496.0>416.1; D2a4, D2a6, D0a10, D2A6, m / z 538.0>458.0; D0S6 m / z 575.95>97.0 4UA-2S-GlcNCOEt-6S, m / z 472.0 (fragment ion) > 97.0 was used as the internal standard (IS). GAGs were identified based on retention time and MRM transitions matching commercially available reference standards (Iduron Ltd, Manchester, UK). Quantification was performed using MultiQuant 3.0.2 (Sciex) as the area ratio to IS. GAGs were normalized to the total protein amount. Protein concentration was measured using the BCA assay (Pierce).

[0435] As reflected in the amount of disaccharides observed after the digestion of heparan sulfate, compared to the control cell line, SGSH Significant substrate accumulation was observed in KO cells (Fig. 16). Using LC-MS / MS-based glycomic assays, it was confirmed that treating cells with the TfR-binding SGSH-Fc fusion protein reduced the levels of heparan sulfate-derived disaccharides back to levels observed in wild-type cells (Fig. 17). Together, these data indicate that the SGSH-Fc fusion protein maintains enzymatic activity, SGSH It demonstrates that substrate accumulation in KO cells can be reduced.

[0436] Example 8. In vitro assay of SGSH activity

[0437] This example is an alternative to the SGSH-Fc fusion protein In vitro It provides an active test. This test is the Karpova test. except , J. Inherit. Metab. Dis.Adapted from , 19:278-285 (1996).

[0438] The standard reaction mixture consisted of 10-15 μg of protein and 20 μL of MU-α-GlcNS (5 or 10 mmol / L in turn) in Michaelis bacteria sodium acetate buffer, pH 6.5 (29 mmol / L sodium barbiturate, 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 is available from Moscerdam Substrates. After the first incubation, 6 μl of twice-concentrated McIlvain phosphate / citrate buffer, pH 6.7, containing 0.02% sodium azide and 10 μl (0.1 U) yeast α-glucosidase (Sigma) in water was added, and a second incubation was performed at 37°C for 24 h. The long incubation at 37°C (17–24 h) was carried out in 96-well plates and hermetically sealed with wide adhesive tape to limit evaporation to <15%. Next, 200 μl of 0.5 mol / L Na2CO3 / NaHCO3, pH 10.7, was added, and the fluorescence of the released 4-methylumbelliferone (MU) was measured using a Fluoroskan (Titertek) fluorescence meter. Proteins were determined as previously described (van Diggelen except , Clin. Chim. Acta. , 187:131-139 (1990)).

[0439] Example 9. Modified Fc polypeptide binding to TfR.

[0440] This embodiment describes a modification to the Fc polypeptide for transferrin receptor (TfR) binding and transport across the blood-brain barrier (BBB).

[0441] Unless otherwise indicated, the positions of amino acid residues in this section are numbered based on the EU index numbering for the human IgG1 wild-type Fc region.

[0442] Generation and characterization of Fc polypeptide (CH3C clone) containing modifications at positions 384, 386, 387, 388, 389, 390, 413, 416, and 421

[0443] A yeast library containing an Fc region with modifications introduced into positions including amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 was generated as described below. Clones for describing binding to TfR are shown in Tables 4 and 5.

[0444] Additionally, after two rounds of classification, single clones were sequenced, and four distinct sequences were identified. These sequences contain a conserved Trp position 388 at position 388, and all of them have an aromatic residue at position 421 ( chamberlain, It has Trp, Tyr, or His). There was a lot of variation in other positions.

[0445] Four clones selected from the library were expressed as Fc fusions to the Fab fragment in CHO or 293 cells, purified by protein A and size-exclusion chromatography, and screened for binding to human TfR using ELISA in the presence or absence of holo-Tf. All of these clones bound to human TfR, and binding was unaffected by the addition of an excess amount (5 μM) of holo-Tf. The clones were also tested for binding to 293F cells endogenously expressing human TfR. These clones bound to 293F cells, but the overall binding was substantially weaker than that of the high-affinity positive control.

[0446] Next, clone CH3C.3 was used as a test clone to test whether the clone could be internalized in TfR-expressing cells. Adsorbent HEK 293 cells were grown to approximately 80% confluence in 96-well plates, the medium was removed, and samples were added at a concentration of 1 μM: clone CH3C.3, anti-TfR benchmark positive control antibody (Ab204), anti-BACE1 benchmark negative control antibody (Ab107), and human IgG isotype control (obtained from Jackson Immunoresearch). These cells were incubated at 37°C at 8% CO2 for 30 minutes, then washed, and 0.1% Triton ™ Transmit through with X-100, and anti-human-IgG-Alexa Fluor ® The cells were stained with the 488 secondary antibody. After additional washing, the cells were examined under a high-content fluorescence microscope (e.g., Opera Phenix). ™ Imaging was performed under the system, and the number of puntas per cell was quantified. At 1 μM, clone CH3C.3 showed a similar internalization tendency to the positive anti-TfR control, while the negative control did not show internalization.

[0447] Additional operations of the clone

[0448] Using a soft randomization approach, additional libraries were generated to improve the affinity of initial hits for human TfRs, from which DNA oligos were created to introduce soft mutagenesis based on each of the original four hits. Additional clones bound to TfRs were identified and selected. The selected clones belong to two generic sequence populations. Population 1 clones ( chamberlain,Clones CH3C.18, CH3C.21, CH3C.25, and CH3C.34 have Leu at position 384, Leu or His at position 386, Val at positions 387 and 389, and PTW at positions 413, 416, and 421. Cluster 2 clones have Tyr at position 384, the TXWSX motif at positions 386–390, and the S / TEF motif at positions 413, 416, and 421. Clones CH3C.18 and CH3C.35 were used for further studies as representative components of each sequence cluster.

[0449] Epitope mapping

[0450] To determine whether the above-mentioned fabricated Fc region binds to the apex domain of the TfR, the TfR apex domain was expressed on the phage surface. To properly fold and display the apex domain, one of the loops had to be truncated, and the sequence had to be permuted. Clones CH3C.18 and CH3C.35 were coated onto ELISA plates, and the phage ELISA protocol was followed. Briefly, after washing and blocking with 1% PBSA, a diluted solution of the phage display was added, and the plates were incubated at room temperature for 1 hour. Subsequently, the plates were washed, anti-M13-HRP was added, and after further washing, the plates were developed on a TMB substrate and quenched with 2N H2SO4. In this assay, both clones CH3C.18 and CH3C.35 bound to the apex domain.

[0451] Paratope mapping

[0452] To understand which residues in the Fc domain are most important for TfR binding, a series of mutant clones CH3C.18 and CH3C.35 Fc regions were generated, each containing a single mutant site in the TfR binding register that reverts to the wild type. The generated variants were recombinantly expressed as Fc-Fab fusions and tested for binding to human or cyano TfR. In the case of clone CH3C.35, sites 388 and 421 were important for binding; reverting either of them to the wild type completely eliminated binding to human TfR.

[0453] Combination characteristics of mature clones

[0454] Conjugation ELISAs were performed with purified Fc-Fab fusion variants having human or cyano TfR coated on plates as described above. The variants of the clone CH3C.18 mature library, clone CH3C.3.2-1, clone CH3C.3.2-5, and clone CH3C.3.2-19 had approximately equivalent ECs. 50 While the values ​​were bound to human and cyano TfR, parental clones CH3C.18 and CH3C.35 had superior binding to humans, which was more than 10-fold greater than that to cyano TfR.

[0455] Next, we tested whether the modified Fc polypeptide was internalized in human and monkey cells. Using the protocol described above, internalization in human HEK 293 cells and rhesus LLC-MK2 cells was tested. The variants CH3C.3.2-5 and CH3C.3.2-19, which were similarly bound to human and cyano TfR, showed significantly improved internalization in LLC-MK2 cells compared to clone CH3C.35.

[0456] Additional operations of the clone

[0457] Further modifications to the additional affinity mature clones CH3C.18 and CH3C.35 involved adding additional mutations to sites that enhance binding through direct interactions, second-shell interactions, or structural stabilization. This was achieved through generation and selection from the "NNK walk" or "NNK patch" libraries. The NNK walk library involved generating one-by-one NNK mutations on residues close to the paratop. Examining the structure of Fc bound to FcγRI (PDB ID:4W4O), 44 residues near the original modification site were identified as interrogation candidates. Specifically, the following residues targeted NNK mutagenicity: 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. 44 single-point NNK libraries were generated using Kunkel mutagenesis, and the products were pooled from other yeast libraries as described above and introduced into yeast via electroporation.

[0458] Combinations of these mini-libraries (each with one position mutated, generating 20 variants) generated small libraries selected using yeast surface libraries for random positions inducing greater affinity binding. Screening was performed as described above using TfR apex domain proteins. After three rounds of sorting, clones from the enriched yeast libraries were sequenced, and several "hot-spot" positions were identified in which specific point mutations significantly improved binding to the apex domain proteins. In the case of clone CH3C.35, these mutations included E380 (mutated to Trp, Tyr, Leu, or Gln) and S415 (mutated to Glu). Sequences of single and combination mutants of clone CH3C.35 are presented in sequence numbers 27-38. In the case of clone CH3C.18, these mutations included E380 (mutated to Trp, Tyr, or Leu) and K392 (mutated to Gln, Phe, or His). The sequences of the single mutants of clone CH3C.18 are presented in sequence numbers 21-26.

[0459] Additional maturity library to improve clone CH3C.35 affinity

[0460] An additional library for verifying mutation combinations from the NNK work library was created as previously described by adding several additional locations to the periphery of the existing yeast libraries. In this library, the YxTEWSS (sequence number: 242) and TxxExxxxF motifs were kept invariant, and the following six locations were completely randomized: E380, K392, K414, S415, S424, and S426. Locations E380 and S415 were included because they were "hot spots" in the NNK work library. Locations K392, S424, and S426 were included because they constitute part of the core where binding regions can be located, and K414 was selected due to its proximity to location 415.

[0461] As previously described, this library was classified solely by the cyano TfR apex domain. The enriched pool was sequenced after 5 rounds, and the sequences of the modified regions of the identified native clones are presented in sequence numbers 42-59.

[0462] The following library was designed to further explore acceptable diversity in major binding paratops. Two hot spots (380 and 415) at the original locations (384, 386, 387, 388, 389, 390, 413, 416, and 421) were individually randomized with NNK codons, respectively, to create a single-location saturation mutagenesis library series on EST. Additionally, each location was individually reverted to wild-type residues, and these individual clones were displayed on EST. It was noted that locations 380, 389, 390, and 415 were the only locations that substantially maintained binding to TfR upon reversion to wild-type residues (significantly reduced residual binding was observed upon reversion to wild-type at location 413).

[0463] Single-location NNK libraries were sorted for three rounds against the human TfR apex domain to collect up to ~5% of binders, and then at least 16 clones were sequenced from each library. These results indicate which amino acids are tolerated at each position with respect to clone CH3C.35 without a significant reduction in binding to the human TfR. The results are presented below:

[0464] Location 380: Trp, Leu, or Glu;

[0465] Location 384: Tyr or Phe;

[0466] Location 386: Only Thr;

[0467] Location 387: Only Glu;

[0468] Location 388: Only Trp;

[0469] Location 389: Ser, Ala, or Val (although wild-type Asn residues appear to retain some binding, they did not appear in the following library sorting);

[0470] Location 390: Ser or Asn;

[0471] Location 413: Thr or Ser;

[0472] Location 415: Glu or Ser;

[0473] Location 416: Only Glu; and

[0474] Location 421: Only Phe.

[0475] When the above residues are substituted into clone CH3C.35 by a single change or combination change, the residues exhibit paratop diversity that maintains binding to the TfR apex domain. Clones with mutations at these positions include those shown in Table 5, and the sequences of the CH3 domains of these clones are presented in sequence numbers 34-38, 58, and 60-90.

[0476] Example 10. Additional Fc positions that can be modified to impart a TfR bond.

[0477] An additional modified Fc polypeptide that binds to the transferrin receptor (TfR) at an alternative site of this Fc region, chamberlain, It was made to have a deformation at the following location:

[0478] Locations 274, 276, 283, 285, 286, 287, 288, and 290 (CH2A2 clone);

[0479] Locations 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 (CH2C clone);

[0480] Locations 268, 269, 270, 271, 272, 292, 293, 294, and 300 (CH2D clone);

[0481] Locations 272, 274, 276, 322, 324, 326, 329, 330, and 331 (CH2E3 clone); or

[0482] Locations 345, 346, 347, 349, 437, 438, 439, and 440 (CH3B clone).

[0483] CH3B clones for describing binding to TfR are presented at sequence numbers 124-128. CH2A2 clones for describing binding to TfR are presented at sequence numbers 129-133. CH2C clones for describing binding to TfR are presented at sequence numbers 134-138. CH2D clones for describing binding to TfR are presented at sequence numbers 139-143. CH2E3 clones for describing binding to TfR are presented at sequence numbers 144-148.

[0484] Example 11. Method.

[0485] Create Page-Display Library

[0486] A DNA template encoding a wild-type human Fc sequence was synthesized and incorporated into a phagemid vector. The phagemid vector contained an ompA or pelB reader sequence, an Fc insert fused to c-Myc and 6xHis (sequence number: 241) epitope tags, and an M13 coat protein pIII followed by an amber stop codon.

[0487] A private containing the "NNK" tricodon was created at the desired modification site, where N is an arbitrary DNA base ( chamberlain, A, C, G, or T), and K is G or T. Alternatively, primers for "soft" randomization were used, where a mixture of bases corresponding to 70% of the wild-type bases and 10% of each of the other three bases was used at each randomization site. A library was generated by performing PCR amplification of the Fc region fragments corresponding to the randomization sites, and then, Sfi Assemble using a terminal primer containing a restriction site, and Next SfiIt was cleaved with I and ligated into a phagemid vector. Alternatively, these primers were used to induce Kunkel mutations. The ligated product or Kunkel product was strain TG1 (Lucigen ® Electroporation-competent E. coli (obtained from) E. coli ) transformed into cells. E. coli( E. coli Cells were infected with M13K07 helper phage, harvested, and incubated overnight. The library phages were then precipitated with 5% PEG / NaCl, resuspended in 15% glycerol in PBS, and frozen until use. A typical library size is approximately 10 9 to about 10 11 It is a transformant of the range. Fc-dimers were displayed on the phage through pairing between pIII-fused Fc and soluble Fc not attached to pIII (which is generated due to the amber stop codon ahead of pIII).

[0488] Creation of the East-Display Library

[0489] A DNA template encoding a wild-type human Fc sequence was synthesized and incorporated into a yeast display vector. For the CH2 and CH3 libraries, the Fc polypeptides were displayed on Aga2p cell wall proteins. These two vectors contained prepro leader peptides with a Kex2 cleavage sequence and a c-Myc epitope fused to the Fc at the end.

[0490] A yeast display library was assembled using a method similar to that described for the phage library, except that fragment amplification was performed using primers containing homologous ends for the vector. Newly prepared electroporation-qualified yeast (e.g., strain EBY100) was electroporated using the linearized vector and the assembled library insert. Electroporation methods will be known to those skilled in the art. After recovery in selective SD-CAA medium, the yeast was normalized to confluence, split into two, and transferred to SG-CAA medium to induce protein expression. A typical library size is approximately 10 7 to about 10 9 It is a transformant of the range. The Fc dimer was formed by the pairing of adjacently displayed Fc monomers.

[0491] General method for sorting waste

[0492] The phage method was adapted from Phage Display: A Laboratory Manual (Barbas, 2001). Additional detailed protocols are available in this document.

[0493] Plate classification method

[0494] The antigen is MaxiSorp at 4℃ ® It was coated (typically 1–10 μg / mL) on microtitration plates. The phage library was added to each well and incubated overnight for binding. The microtitration wells were 0.05% Tween ®Extensive washing with PBS containing 20 (PBST) was performed, and the bound phages were eluted by incubating the wells with acid (typically 50 mM HCl + 500 mM KCl, or 100 mM glycine, pH 2.7) for 30 minutes. The eluted phages were neutralized with 1 M Tris (pH 8), 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 evaluated by comparing the titer of phages eluted from target-containing wells with the titer of phages recovered from non-target-containing wells. Selection stringency was increased by subsequently decreasing the incubation time during binding and increasing the washing time and number of washes.

[0495] Bead sorting methods

[0496] The antigen is NHS-PEG4-biotin (Pierce ™ Biotinylation was performed via free amines using (obtained from). For the biotinylation reaction, a 3- to 5-fold molar excess of biotin reagent was used in PBS. After quenching the reaction with Tris, extensive dialysis was performed in PBS. The biotinylated antigen was posed on streptavidin-coated magnetic beads ( chamberlain, It was immobilized on M280-streptavidin beads obtained from Thermo Fisher. The phage display library was incubated with the antigen-coated beads at room temperature for 1 hour. Subsequently, unbound phages were removed, and the beads were washed with PBST. The bound phages were eluted by incubation with 50 mM HCl containing 500 mM KCl (or 0.1 M glycine, pH 2.7) for 30 minutes, and then neutralized and proliferated as described above for plate sorting.

[0497] After 3 to 5 rounds of panning, monoclones were screened by expressing Fc on phages or by lysing them in E. coli protoplasm. Such expression methods will be known to those skilled in the art. Individual phage supernatants or protoplasmic extracts were exposed to blocked ELISA plates coated with antigens or negative controls, and subsequently detected using HRP-conjugated goat anti-Fc (obtained from Jackson Immunoresearch) or anti-M13 (GE Healthcare) for phages, and then developed with TMB reagent (purchased from Thermo Fisher). OD approximately 5-fold larger than background values 450 Wells with values ​​were considered positive clones, and after sequencing, some clones were expressed as fused to the soluble Fc fragment or Fab fragment.

[0498] Common method for selecting yeast

[0499] Bead sorting (self-assisted cell sorting (MACS)) method

[0500] MACS and FACS screening is Ackerman except , Biotechnol. Prog It was carried out similarly to that described in ., 25(3):774 (2009). Streptavidin magnetic beads ( chamberlain ThermoFisher's M-280 streptavidin beads were labeled with biotinylated antigens and incubated with yeast (typically 5-10x library varieties). Unbound yeast was removed, the beads were washed, and the bound yeast was grown on selection medium and induced for subsequent selection rounds.

[0501] Fluorescence-activated cell sorting (FACS) method

[0502] Yeast was labeled with anti-c-Myc antibodies to monitor the expression and biotinylated antigens (concentrations varied depending on the classification round). In some experiments, the antigen was streptavidin-Alexa Fluor ® It was pre-mixed with 647 to enhance the antibody binding ability of the interaction. In other experiments, the biotinylated antigen was streptavidin-Alexa Fluor ® It was detected after binding with 647 and washing. Singlet yeast with binding was sorted using a FACS Aria III cell sorter. The sorted yeast was grown in selection medium and then induced for a subsequent selection round.

[0503] After obtaining an enriched yeast population, the yeast was plated on SD-CAA agar plates, a single colony was grown, and expression was induced, and then labeled as described above to measure the tendency to bind to the target. Subsequently, positive monoclones were sequenced for antigen binding, and some clones were expressed as fused to a soluble Fc fragment or Fab fragment.

[0504] General methods for selection

[0505] Screening by ELISA

[0506] Clones were selected from the Panning yield and grown in individual wells of 96-well deep-well plates. Clones were induced for protoplasmic expression using self-induction medium (obtained from EMD Millipore) or infected with helper phages for phage-display of individual Fc variants on the phage. ELISA plates were typically coated overnight with 0.5 mg / mL of antigen and then blocked with 1% BSA before adding phage or protoplasmic extracts. After 1-hour incubation and washing away unbound proteins, HRP-conjugated secondary antibodies (i.e., anti-Fc or anti-M13 against soluble Fc or phage-displayed Fc) were added and incubated for 30 minutes. The plates were washed again, developed with TMB reagent, and quenched with 2N sulfuric acid. Plate reader (BioTek ® Absorbance at 450 nm was quantified using ), and binding curves were polled using Prism software where available. In some assays, soluble transferrin or other competitors were typically added in significant molar excess during the binding step.

[0507] Screening by flow cytometry

[0508] Fc variant polypeptides (expressed on phages, in protoplasmic extracts, or as soluble fusions to Fab fragments) were added to cells in 96-well V-bottom plates (approximately 100,000 cells per well in PBS+1% BSA (PBSA)) and incubated at 4°C for 1 hour. Subsequently, the plates were spun, the medium was removed, and the cells were washed once with PBSA. These cells were then treated with a secondary antibody (typically goat anti-human IgG-Alexa Fluoride). ®They were resuspended in PBSA containing 647 (obtained from Thermo Fisher). After 30 minutes, these plates were spun, and after removing the medium, the cells were washed 1-2 times with PBSA, and then these plates were subjected to a flow cytometer ( chamberlain The readings were taken on a FACSCanto™ II flow cytometer. The median fluorescence value for each condition was calculated using FlowJo software, and the coupling curve was plotted using Prism software.

[0509] Example 12. Selection of TfR-binding polypeptide affinities.

[0510] This embodiment describes the correlation between the affinity of a TfR-binding polypeptide for a transferrin receptor (TfR) and the resulting brain exposure to a therapeutic agent associated with said TfR-binding polypeptide.

[0511] Figure 18 shows that when the therapeutic agent is linked to a polypeptide having a relatively strong affinity for TfR, brain exposure to the therapeutic agent (evaluated by determining the area under the curve of brain concentration over time (AUC)) is shortened. In particular, when the therapeutic agent is linked to a polypeptide having a TfR affinity stronger than about 250 nM, brain exposure is substantially shortened.

[0512] As shown...

Claims

Claim 1 (a) a first Fc polypeptide linked to an enzyme replacement therapy (ERT) enzyme, wherein the first Fc polypeptide is an enzyme deficient in lysosomal storage disorder; and (b) a protein comprising a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the first Fc polypeptide and / or the second Fc polypeptide is a modified Fc polypeptide comprising (1) specificly binding to a transferrin receptor (TfR) and (2) amino acids at positions 380-390 and 413-421 of any one of SEQ ID NOs: 34-38, 58 and 60-90 according to EU numbering; wherein the protein is a protein that does not contain an immunoglobulin heavy chain and / or light chain variable region sequence or an antigen-binding portion thereof. Claim 2 The protein of claim 1, wherein the ERT enzyme is i) iduroneate 2-sulfatase (IDS), ii) N-sulfoglucosamine sulfohydrolase (SGSH), iii) acid sphingomyelinase (ASM), or iv) β-glucocerebrosidase (GBA). Claim 3 A protein according to claim 1, wherein the ERT enzyme is i) IDS and comprises any one of the amino acid sequences of SEQ Nos. 91, 92, 114, 230, and 234; ii) SGSH and comprises any one of the amino acid sequences of SEQ Nos. 119 and 120; iii) ASM and comprises any one of the amino acid sequences of SEQ Nos. 121, 122, and 123; or iv) GBA and comprises any one of the amino acid sequences of SEQ Nos. 93 and 94. Claim 4 A protein according to claim 1, wherein the first Fc polypeptide is connected to an ERT enzyme by a peptide bond or a polypeptide linker. Claim 5 The protein of claim 4, wherein the protein comprises a single ERT enzyme, and the N-terminus or C-terminus of the first Fc polypeptide is connected to the ERT enzyme. Claim 6 A protein according to claim 1, wherein the second Fc polypeptide is linked to an ERT enzyme, and the first Fc polypeptide and / or the second Fc polypeptide is linked to the ERT enzyme by a peptide bond or a polypeptide linker. Claim 7 A protein according to claim 6, wherein i) the N-terminus of the first Fc polypeptide and / or the N-terminus of the second Fc polypeptide is connected to an ERT enzyme, or ii) the C-terminus of the first Fc polypeptide and / or the C-terminus of the second Fc polypeptide is connected to an ERT enzyme. Claim 8 A protein according to claim 1, wherein, according to EU numbering, the first Fc polypeptide contains T366S, L368A, and Y407V substitutions and the second Fc polypeptide contains T366W substitutions; or, according to EU numbering, the first Fc polypeptide contains T366W substitutions and the second Fc polypeptide contains T366S, L368A, and Y407V substitutions. Claim 9 A protein according to claim 8, wherein the first Fc polypeptide connected to the ERT enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 117, 118, 232, 233, 236, and 237. Claim 10 In claim 1, the first Fc polypeptide and / or the second Fc polypeptide comprises a modification that reduces the effector function, wherein the modification that reduces the effector function is a protein that is a substitution of Ala at position 234 and Ala at position 235 according to EU numbering. Claim 11 A protein according to claim 10, wherein the first Fc polypeptide connected to the ERT enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 115, 149, 150, 152, 153, 231, and 235. Claim 12 A protein according to claim 1, wherein the first Fc polypeptide and / or the second Fc polypeptide has a CH3 domain having amino acids 111-217 of any one of SEQ ID NOs: 34-38, 58, 60-90, 151, and 156-227. Claim 13 A protein according to claim 1, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 156-229. Claim 14 A protein according to claim 1, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 98, 169, 181, 193, 205, and 151. Claim 15 A protein according to claim 1, wherein the second Fc polypeptide can specifically bind to TfR and has an amino acid sequence of any one of SEQ ID NOs: 97-100, 116, 151 and 156-229; and wherein the ERT enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 91, 92, 114, 230 and 234. Claim 16 A protein according to claim 15, wherein the first Fc polypeptide is connected to an ERT enzyme by a peptide bond or a polypeptide linker. Claim 17 A protein according to claim 15, wherein the first Fc polypeptide is connected to an ERT enzyme by a polypeptide linker, and the polypeptide linker is a glycine-rich linker. Claim 18 The protein of claim 17, wherein the glycine-rich linker is G4S (SEQ No.: 239) or (G4S)2 (SEQ No.: 240). Claim 19 A protein according to claim 15, wherein the protein comprises a single ERT enzyme, and the N-terminus of the first Fc polypeptide is connected to the ERT enzyme. Claim 20 In claim 15, (a) according to EU numbering, the first Fc polypeptide contains T366S, L368A, and Y407V substitutions and the second Fc polypeptide contains T366W substitutions and / or; (b) according to EU numbering, the first Fc polypeptide and the second Fc polypeptide contain mutants L234A and L235A and / or; (c) according to EU numbering, the second Fc polypeptide contains mutants T366W, L234A, and L235A, and has an amino acid sequence of any one of SEQ Nos. 157, 158, 169, 170, 181, 182, 193, 194, 205, 206, 217, 218, 228, and 229. protein. Claim 21 A protein according to claim 15, wherein the second Fc polypeptide has an amino acid sequence of any one of SEQ ID NOs: 97-100, 116, 151, 156-161, 168-173, 180-185, 192-197, 204-209, 216-221, 228 and 229. Claim 22 A protein according to claim 21, wherein the second Fc polypeptide has an amino acid sequence of any one of SEQ ID NOs: 116, 228, and 229. Claim 23 A protein according to claim 15, wherein the first Fc polypeptide has an amino acid sequence of any one of SEQ ID NOs: 101-104. Claim 24 A protein according to claim 15, wherein the first Fc polypeptide connected to the ERT enzyme has an amino acid sequence of any one of SEQ Nos. 115, 117, 231, 232, 235, and 236. Claim 25 A protein according to claim 24, wherein the first Fc polypeptide connected to the ERT enzyme has an amino acid sequence of any one of SEQ ID NOs: 115, 231, and 235. Claim 26 A protein according to claim 1, wherein i) the first Fc polypeptide connected to the ERT enzyme comprises the amino acid sequence of SEQ ID NO: 235 and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 205 and 228; or ii) the first Fc polypeptide connected to the ERT enzyme comprises the amino acid sequence of SEQ ID NO: 235 and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 169 and 229. Claim 27 A protein according to claim 1, wherein the first Fc polypeptide connected to the ERT enzyme comprises the amino acid sequence of SEQ ID NO: 235 and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:

229. Claim 28 A protein according to claim 6, wherein the first Fc polypeptide connected to the ERT enzyme comprises the amino acid sequence of SEQ ID NO: 149, and the second Fc polypeptide connected to the ERT enzyme comprises the amino acid sequence of SEQ ID NO:

154. Claim 29 A protein according to claim 6, wherein the first Fc polypeptide connected to the ERT enzyme comprises the amino acid sequence of SEQ ID NO: 150, and the second Fc polypeptide connected to the ERT enzyme comprises the amino acid sequence of SEQ ID NO:

155. Claim 30 The protein of claim 1, wherein the protein binds to TfR with an affinity of 10 nM to 1 μM. Claim 31 The protein of claim 1, wherein the protein binds to TfR with an affinity of 50 nM to 250 nM. Claim 32 A pair of polynucleotides comprising a polynucleotide comprising a nucleic acid sequence encoding a first Fc polypeptide as described in any one of claims 1 to 31 and a polynucleotide comprising a nucleic acid sequence encoding a second Fc polypeptide as described in any one of claims 1 to 31. Claim 33 One or more vectors comprising a pair of polynucleotides of claim 32. Claim 34 An isolated host cell comprising a pair of polynucleotides of claim 32. Claim 35 A method for producing a polypeptide comprising culturing an isolated host cell containing a pair of polynucleotides of claim 32 under conditions in which said pair of polynucleotides is expressed. Claim 36 A pharmaceutical composition for use in treating lysosomal storage disorders (LSD), comprising a protein of any one of claims 1 to 31 and a pharmaceutically acceptable excipient. Claim 37 A pharmaceutical composition comprising a protein of any one of claims 1 to 31 for use in treating LSD in patients suffering from lysosomal storage disorder (LSD). Claim 38 A pharmaceutical composition according to claim 37, wherein i) the LSD is Hunter syndrome and the ERT enzyme is IDS; ii) the LSD is Sanfilippo syndrome A and the ERT enzyme is SGSH; iii) the LSD is Niemann-Pick disease and the ERT enzyme is ASM; or iv) the LSD is Gaucher disease or Parkinson disease and the ERT enzyme is GBA. Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete Claim 70 delete Claim 71 delete Claim 72 delete Claim 73 delete Claim 74 delete Claim 75 delete Claim 76 delete Claim 77 delete Claim 78 delete Claim 79 delete Claim 80 delete Claim 81 delete Claim 82 delete Claim 83 delete Claim 84 delete Claim 85 delete Claim 86 delete Claim 87 delete Claim 88 delete Claim 89 delete Claim 90 delete Claim 91 delete Claim 92 delete Claim 93 delete Claim 94 delete Claim 95 delete Claim 96 delete Claim 97 delete Claim 98 delete Claim 99 delete Claim 100 delete Claim 101 delete Claim 102 delete Claim 103 delete Claim 104 delete Claim 105 delete Claim 106 delete Claim 107 delete Claim 108 delete Claim 109 delete Claim 110 delete Claim 111 delete Claim 112 delete Claim 113 delete Claim 114 delete Claim 115 delete Claim 116 delete Claim 117 delete Claim 118 delete Claim 119 delete Claim 120 delete Claim 121 delete Claim 122 delete Claim 123 delete Claim 124 delete Claim 125 delete Claim 126 delete Claim 127 delete Claim 128 delete Claim 129 delete Claim 130 delete Claim 131 delete Claim 132 delete Claim 133 delete Claim 134 delete Claim 135 delete Claim 136 delete Claim 137 delete Claim 138 delete Claim 139 delete Claim 140 delete Claim 141 delete Claim 142 delete

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